METHODS FOR ANALYZING LARIAT RNA

Abstract

The present invention relates to compositions and methods useful for analyzing lariat RNA, which plays a role in the regulation of gene expression. A sample of RNA is specifically treated to remove linear mRNA and enrich for lariat RNA. The enriched lariat RNA sample may be analyzed further to identify introns, branch point sequences, alternative splicing patters, and gene transcription levels. The enriched lariat RNA sample may also be exploited as a detection or compound screening tool, as well as other uses.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to compositions, methods, and kits for analyzing lariat RNA. In particular, the invention relates to enriching an RNA population for lariat RNA and then analyzing the lariat RNA population.

BACKGROUND OF THE INVENTION

[0002] Pre-mRNA introns play an important role in the regulation of gene expression for many eukaryotes because their presence allows for the occurrence of alternative splicing. Such alternative splicing results in the creation of multiple proteins from a single gene, many of which are expressed in cell- or tissue-specific patterns. The pre-mRNA introns are excised in a lariat conformation to produce mRNA. Following excision, the 3' tails of the lariats are subject to exonucleolytic degradation up to the lariat branch point. The predominant pathway for further exonucleolytic degradation requires cleavage of the 2'-5' bond located at the branch point. This cleavage event occurs via a RNA debranching enzyme, a 2'-5' phosphodiesterase.

[0003] Although intron RNA sequences contain information necessary for their removal from pre-mRNAs, some introns contain additional information. In most eukaryotes microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) are encoded within introns. In studies with human cells it has been found that the vast majority of intronic miRNAs are excised from pre-mRNAs. Intronic snoRNAs, on the other hand, are processed from excised introns, as determined in baker's yeast, humans, and other eukaryotes.

[0004] Debranching and subsequent degradation of most intron RNAs are rapid, resulting in low steady state levels of intron RNAs relative levels of the corresponding mRNAs. The exceptions are intron sequences corresponding to RNAs with additional functions (e.g. snoRNAs). Studies in many different organisms have determined that cleavage of the 2'-5' bond by an RNA debranching enzyme is important for the maturation of intron-encoded snoRNAs and mirtrons, which is another class of miRNAs that are processed from excised introns.

[0005] Genome-wide studies analyzing excised intron RNAs in fruit flies and yeast have identified new introns and alternative splicing patterns. These analyses relied on creating cell populations that accumulate excised intron RNAs at elevated levels due to either mutation of the gene encoding debranching enzyme or knock down of debranching enzyme expression with siRNA. Analysis of RNA samples with elevated levels of RNA lariats increases the detectability of rare splicing variants. Cells defective for RNA debranching activity accumulate excised introns in their lariat forms with shorted 3' tails. Without the full length 3' tail, information for the 3' intron-exon junction is not obtainable from the intron lariat RNA sequences. However, studies have shown that the positions of RNA branch points may be deduced from analyzing intron RNA lariats. Direct information on branch points is only obtainable from analysis of RNA lariats. Therefore, there is a need to provide new compositions and methods for the analysis of RNA lariats that allow analysis of rare splicing variants and branch point sequences.

REFERENCE TO COLOR FIGURES

[0006] The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0008] FIG. 1 illustrates reverse transcriptase polymerase chain reaction (RT-PCR) detection of lariat RNA. FIG. 1A shows the annealing positions of primers for RT-PCR detection of ACT1 gene intron lariat RNA and mRNA. The intron lariat RNA is detected using primers oligo 146 and oligo 363 (depicted by arrows within the intron lariat RNA loop). The linear mRNA is detected using primers oliog 215 and oligo 216 (depicted by small arrows below the mRNA arrow). FIG. 1B shows an agarose gel analysis of RT-PCRs for ACT1 RNA detected using the primers illustrated in FIG. 1A (mRNA=oligos 215/216; intron=oligos 146/363). Lanes 1-4 contain reactions run 15 cycles after the touchdown phase of polymerase chain reaction (PCR); lanes 5-8 contain reactions run 11 cycles after the touchdown phase of PCR; Lanes 1, 2, 5, and 6 contain reactions using wild type (DBR1) RNA samples; and, lanes 3, 4, 7, and 8 contain reactions using dbr1 mutant RNA samples. The different numbers of cycles were run to show the linearity of the PCRs.

[0009] FIG. 2 illustrates selective degradation of linear RNAs and not lariat RNAs. FIG. 2A shows an agarose gel analysis of RT-PCRs for ACT1 intron lariat RNA following a series of enzyme treatments (PNPase) at decreasing amounts (indicated by the wedge at the tope of the gel image, highest amount of enzyme used in lane 1 and lowest in lane 7). RT-PCRs for ACT1 intron lariat RNA were performed with primers 146 and 363 (see FIG. 1) and run for 15 cycles after the touchdown phase of the reaction.

[0010] FIG. 2B shows an agarose gel analysis of RT-PCRs for ACT1 linear mRNA from the same series of enzyme treatments of FIG. 2A. Lanes 15 and 16 contain ACT1 intron lariat and ACT1 linear mRNA RT-PCRs, respectively, of RNA samples that did not undergo PNPase treatment. The products in these lanes serve as size markers for the intron lariat and mRNA products in lanes 1-14. RT-PCRs for ACT1 mRNA were performed with primers 215 and 216 (see FIG. 1) and run for 24 cycles after the touchdown phase of the reaction.

[0011] FIG. 3 illustrates processivity of PNPase on FLO8 mRNA. FIG. 3A shows primer pairs for amplifying different segments along the length of FLO8 mRNA: 1/2=primers 372 and 373; 3/4=primers 374 and 375; =primers 376 and 377; 7/8=primers 378/and 379; 9/10=primers 380 and 381; 11/12=primers 382 and 383. FIG. 3B shows a PAGE analysis of RT-PCRs for FLO8 mRNA segments following enzyme treatment (PNPase, + lanes) and mock treatment (- lanes) of a total cellular RNA sample that had been pretreated with DNase I. Lanes containing the various FLO8 RT-PCRs are indicated below the gel image; the FLO8 primer pairs are indicated above the gel image. RT-PCRs for ACT1 RNAs are in the four lanes under the ACT1 title and serve as controls that indicate the PNPase reactions preceded as expected. The RT-PCRs for ACT1 mRNA and intron RNA are indicated below the corresponding lanes. These reactions used primer pairs 215/216 and 146/363, respectively. The lane marked "M" contains a DNA molecular weight standard (50 base pair (bp) ladder). FIG. 3c shows a PAGE analysis of RT-PCRs as described for FIG. 3B except that the total cellular nucleic acid samples were not treated with DNase I prior to PNPase enzyme treatment and RT-PCRs. For all RT-PCRs in FIGS. 3B and 3C, reactions were performed with 24 cycles after the touchdown phase.

[0012] FIG. 4 illustrates the purification of Dbr1p. FIG. 4A shows the elution profile of histidine-tagged yeast Dbr1p, purified from E. coli, collected for 100 mM and 200 mM concentrations of imidazole. Dbr1p bound to a nickel-nitrilotriacetic acid (nickel-NTA) column was eluted with increasing concentrations of imidazole. Six, ˜1.5 mL fractions were collected for each imidazole concentration. FIG. 4B shows the elution profile of histidine-tagged yeast Dbr1p collected for 300 mM and 500 mM concentrations of imidazole. Key: "M" is the protein molecular weight standard; and, 1-6 are the fractions collected from the nickel-nitrilotriacetic column. FIG. 4c shows the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis to assess the molecular mass of the main elution product in fractions 2-6 of the 100 mM imidazole elution.

[0013] FIG. 5 illustrates an in vitro debranching reaction. Specifically, FIG. 5 shows an agarose gel analysis of RT-PCRs for ACT1 RNAs following treatment with Dbr1p (+ lanes) and mock treatment (- lanes) of total cellular RNA sample. Key: lanes 2 and 4 contain RT-PCRs for ACT1 intron lariat RNA; lanes 1 and 3 are RT-PCRs for ACT1 mRNA; lane M contains a DNA molecular weight standard (50 bp ladder). RT-PCRs for ACT1 intron lariat RNA were run for 19 cycles after the touchdown phase of the reaction; RT-PCRs for ACT1 mRNA were run for 24 cycles after the touchdown phase of the reaction.

[0014] FIG. 6 shows combinations of PNPase and Dbr1p enzyme treatments. FIG. 6A shows an agarose gel analysis of RT-PCRs for ACT1 RNAs following treatment of a total cellular RNA sample from a dbr1 strain with Dbr1p (+Dbr1p) and PNPase (+PNPase) as well as mock treatment (- treatment). In this experiment, PNPase treatment preceded Dbr1p treatment for samples that were treated with both enzymes. Lanes 1, 3, 5, and 7 contain RT-PCRs for ACT1 mRNA of a total cellular RNA sample. Lanes 2, 4, 6, and 8 contain parallel RT-PCRs for ACT1 intron lariat RNA. FIG. 6B shows an agarose gel analysis of RT-PCRs for ACT1 RNAs following treatment of a total cellular RNA sample from a dbr1 strain with Dbr1p and PNPase as well as mock treatment. In this experiment, Dbr1p treatment preceded PNPase treatment for samples that were treated with both enzymes. Lanes 1-4 contain RT-PCRs for ACT1 mRNA of a total cellular RNA sample. Lanes 5-8 contain parallel RT-PCRs for ACT1 intron lariat RNA. For both FIGS. 6A and 6B, RT-PCRs for ACT1 intron lariat RNA were run for 19 cycles after the touchdown phase of the reaction and RT-PCRs for ACT1 mRNA were run for 24 cycles after the touchdown phase of the reaction. The lanes marked "M" and "m" contain DNA molecular weight standards ("M"=A phage DNA cut with HinDIII+EcoRI; "m"=50 bp ladder).

[0015] FIG. 7 shows real-time quantitative RT-PCR (qRT-PCR) measurement of lariat RNA levels. FIG. 7A shows the annealing positions of primers for RT-PCR detection of mRNA (FWDm primer and REVm primer) and intron lariat RNA species (FWDi primer and REVi primer). A TaqMan probe is designed to span the same exon-exon junction. The star and the triangle at opposite ends of the TaqMan probes represent the fluorescent reporter molecule and the quencher that are bound to the 5' and 3' ends, respectively. The TaqMan probes that anneal to a particular mRNA and lariat RNA pair contain different fluorescent reporter molecules, indicated by solid and stippled stars. Note that lariat RNA detection does not involve annealing of PCR primers or TaqMan probes across lariat branch points. FIG. 7B graphically illustrates the relative quantification of ACT1 intron lariat RNA in total RNA samples from different yeast strains. RQ, the relative quantification, is the ratio of intron RNA to mRNA for a particular sample relative to the ratio of intron RNA to mRNA for the DBR1 (wild-type) sample at the left end of the bar graph (which sets the RQ for DBR1 itself to 1). Quantification experiments were repeated three times and the qPCRs were performed in triplicate each time. The standard error bars display the calculated maximum (RQmax) and minimum (RQMin) expression levels that represent standard error of the mean expression level (RQ value). FIG. 7C graphically illustrates the relative quantification of RPP1B intron lariat RNA for the same RNA samples presented in FIG. 7B. FIG. 7D graphically illustrates the relative quantification of YRA1 intron lariat RNA for the same RNA samples presented in FIG. 7B.

[0016] FIG. 8 graphically illustrates a time course of in vitro debranching reaction.

[0017] FIG. 9 illustrates the RNA lariat enrichment following treatment of an RNA sample with a 3' exonuclease. The parentheses at the left end of the linear RNA mean that these RNAs include both 5' capped and 5' uncapped species. The circular dot within the parentheses represents the cap. The arrow on the right side of the linear RNA represents the 3' end. Dashed lines represent degradation.

[0018] FIG. 10 illustrates the RNA lariat enrichment following treatment of a decapped RNA sample with a 5' exonuclease. Linear RNAs at the top, below the lariat RNA, are a mixture of 5' capped and 5' uncapped species. The circular dot at the left of the 5' capped RNA represents the cap. The arrows on the right side of the linear RNAs represent the 3' ends. Dashed lines represent degradation.

[0019] FIG. 11 illustrates RT-PCR detection of ACT1 mRNA (linear RNA) and intron (lariat RNA) in a total RNA sample from Saccharomyces cerevisiae cells following treatment with the 3' exonuclease polynucleotide phosphorylase (PNPase) (lanes 1 and 2), debranching enzyme (Dbr1p) followed by PNPase (lanes 3 and 4), and no treatment (lanes 5 and 6).

[0020] FIG. 12 illustrates the RT-PCR detection of ACT1 mRNA (linear RNA) and intron (lariat RNA) in total RNA samples from dbr1 mutant yeast cells following Dbr1p treatment (lanes 3 and 4) or no treatment (lanes 1 and 2).

[0021] FIG. 13 illustrates the high-throughput sequencing of cDNAs representing PNPase-treated S. cerevisiae RNA. FIG. 13A shows chromosome 6 is depicted at the top, below which a 20 kilo-base pair (kbp) segment is highlighted (black bar), along with a detailed map of the genes that lie within this segment. Gene open reading frames (ORFs) are indicated by red or blue bars, depending on which DNA strand of the chromosome encodes the sense strand for each ORF (red for the upper strand, blue for the lower strand). FIG. 13B graphically illustrates the number of sequence reads that map within the 20 kbp segment. The ACT1 gene is the only gene in this 20 kbp segment that contains an intron, which is depicted as a white box within the blue ACT1 ORF.

[0022] FIG. 14 shows the conserved amino acid conservation among RNA debranching enzymes using the sequence of Saccharomyces cerevisiae Dbr1 (405 total amino acid residues) as a representative example. (Key: green numbers=amino acid residue number of first and last amino acid in each line (out of 405 total amino acids residues); highlighted yellow=identical among all RNA debranching enzymes; red=conserved among all RNA debranching enzymes; blue=not conserved; [X].=gaps in sequence between conserved regions (number of amino acid residues).

SUMMARY OF THE INVENTION

[0023] The present invention is directed to compositions and methods for analyzing lariat RNA. The compositions of the invention include isolated enzymes and supportive buffers for efficient use of the isolated enzymes. The methods of the invention include methods of enriching an RNA population for lariat RNA and analyzing lariat RNA. The compositions and methods of the invention may be provided in a kit.

[0024] The enzymes of the invention include linear RNA degrading enzymes, 5' cap removing enzymes and debranching enzymes. Suitable linear RNA degrading enzymes include those capable of degrading linear RNA or mRNA. Such linear RNA degrading enzymes include, without limitation, exonucleases, 3' exonucleases, 5' exonucleases, those with both 5' and 3' exonuclease activity, those known in the art or yet to be discovered, and combinations thereof.

[0025] Suitable 5' cap removing enzymes include those capable of degrading or excising the 5' cap of linear RNA or mRNA. Such enzymes include those commonly known in the art, such as Dcp1 or Dcp2, as well as those yet to be discovered, and combinations thereof.

[0026] Suitable debranching enzymes include those capable of degrading, excising, or cleaving the 2'-5' bond at the branch point of lariat RNA. Such enzymes include 2'-5' phosphodiesterases, such as Dbr1, all those known in the art or yet to be discovered, and combinations thereof. Also, such enzymes include those encoding an amino acid sequence having at least 35% sequence identity to at least one of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66. The sequence identity may be about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. In another embodiment, the nucleic acid sequence may have at least 35% sequence identity to the metallophosphatase domain of at least one of SEQ ID NO: 46-66. The sequence identity may be about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. In S. cerevisiae Dbr1 (SEQ ID NO: 47), the metallophosphatase domain is located at amino acid residues 6 to 238 (FIG. 14).

[0027] The invention also includes methods of enriching an RNA population for lariat RNA. Such methods include providing an RNA population and contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population. Suitable methods may further include contacting the RNA population with a debranching enzyme.

[0028] The invention also includes methods of analyzing the lariat RNA in an RNA sample or population. Such methods include providing an RNA population and contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population. The lariat RNA enriched population may be used to create a cDNA library. In one embodiment the cDNA library is created by reverse transcribing the lariat RNA enriched population. Methods known in the art for creating a cDNA library may be used. Suitable methods may also further include sequencing the cDNA library created using the lariat RNA enriched population.

[0029] The invention includes kits for practicing the methods of the invention. Suitable kits contain at least one linear RNA degrading enzyme and instructions. Kits may also include a linear RNA degrading enzyme buffer, debranching enzyme, debranching enzyme buffer, 5' decapping enzyme, 5' decapping enzyme buffer, and combinations thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In accordance with the present invention, processes of comprehensively analyzing lariat RNA have been discovered. In particular, the present invention provides compositions, methods, and kits useful for analyzing lariat RNA. The compositions and methods are directed to enriching an RNA population for lariat RNA and analyzing the lariat RNA.

[0031] Various aspects of the invention are described in further detail in the following subsections.

I. Compositions

[0032] A. Enzymes

[0033] One aspect of the invention pertains to isolated enzymes that are used in the methods described herein. Suitable enzymes include those capable of degrading linear RNA, linearizing lariat RNA, removing the 5' cap from linear RNA (mRNA), or combinations thereof.

[0034] Enzymes capable of degrading linear RNA are used to remove the linear RNA from the RNA population, enriching the population for lariat RNA. Suitable linear RNA degrading enzymes include, without limitation, 3' exonucleases, 5' exonucleases, 5'/3' exonucleases, and combinations thereof. Any enzyme capable of degrading linear RNA is contemplated herein, as well as those not yet discovered. For example, the polynucleotide phosphorylases of Bacillus stearothermophilus (BsPNPase) and Thermus thermophilus (TtPNPase), as well as the RNase of E. coli (RNase R) are suitable linear RNA degrading enzymes.

[0035] Enzymes capable of removing the 5' cap from linear RNA or mRNA are used to allow linear RNA degrading enzymes to work, where the 5' cap may inhibit degradation. Suitable 5' cap removing enzymes include those capable of cleaving or degrading the 5' cap from linear RNA or mRNA. Any enzyme capable of 5' cap removal is contemplated herein, as well as those not yet discovered. For example, the 5' cap removing enzymes Dcp1 and Dcp2 are suitable for the invention. The invention also includes 5' cap removal treatments known in the art or yet to be discovered.

[0036] Enzymes capable of linearizing lariat RNA are debranching enzymes, which are used to unfold the lariat structure of the RNA to allow further analysis. Suitable debranching enzymes are those capable of cleaving the 2'-5' bond at the branch point of lariat RNA. Such debranching enzymes include, without limitation, debranching enzymes having sequence homology to SEQ ID NO: 46-66.

[0037] Preferably, the nucleic acid sequence of debranching enzymes have at least 35% sequence identity to the nucleic acid sequence that encodes the amino acid sequence of at least one of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66. The sequence identity may be about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. In another embodiment, the nucleic acid sequence may have at least 35% sequence identity to the metallophosphatase domain of the nucleic acid sequence that encodes at least one of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66. The sequence identity may be about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. In S. cerevisiae Dbr1 (SEQ ID NO: 47), the metallophosphatase domain is located at amino acid residues 6 to 238 (FIG. 14).

[0038] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 46-66, or a complement of any of these nucleotide sequences, may be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequences of SEQ ID NO:46-66, debranching enzyme nucleic acid molecules may be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0039] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding debranching enzymes that contain changes in amino acid residues that may or may not be essential for activity. Such debranching enzymes proteins differ in amino acid sequence from SEQ ID NO: 46-66. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the amino acid sequence of SEQ ID NO: 46-66. An isolated nucleic acid molecule encoding a debranching enzymes having a sequence which differs from that of SEQ ID NO: 46-66, may be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of debranching enzymes (SEQ ID NO: 46-66) such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0040] The present invention encompasses antisense nucleic acid molecules. Antisense molecules are complementary to a sense nucleic acid encoding a protein, complementary to the coding strand of a double-stranded cDNA molecule, or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid hydrogen bonds to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire debranching enzyme coding strand, or to only a portion thereof, such as all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to a non-coding region of the coding strand of a nucleotide sequence encoding a debranching enzyme. The non-coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences that flank the coding region and are not translated into amino acids. Given the coding strand sequences encoding debranching enzymes disclosed herein, antisense nucleic acids of the invention may be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule may be complementary to the entire coding region of debranching enzyme mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or non-coding region of a debranching enzyme mRNA. For example, the antisense oligonucleotide may be complementary to the region surrounding the translation start site of a debranching enzyme mRNA. An antisense oligonucleotide may be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which may be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-aino-3-N2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid may be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

[0041] The antisense nucleic acid molecules of the invention are generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a debranching enzyme to thereby inhibit expression of the enzyme, e.g., by inhibiting transcription and/or translation. The hybridization may be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.

[0042] The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) may be used to catalytically cleave debranching enzyme mRNA transcripts to thereby inhibit translation of debranching enzyme mRNA. A ribozyme having specificity for a debranching enzyme-encoding nucleic acid may be designed based upon the nucleotide sequence of the debranching enzyme cDNA. For example, debranching enzyme mRNA may be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.

[0043] The invention also encompasses nucleic acid molecules which form triple helical structures. For example, debranching enzyme gene expression may be inhibited by targeting nucleotide sequences complementary to the regulatory region of the debranching enzyme gene (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the debranching enzyme gene in target cells. See generally, Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.

[0044] A useful debranching enzyme protein is a protein which includes an amino acid sequence at least about 45%, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 46-66, and retains the functional activity of a debranching protein of SEQ ID NO:46-66.

[0045] To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100).

[0046] The determination of percent homology between two sequences may be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Nat'l Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences similar or homologous to nucleic acid sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0047] One useful fusion protein is a fusion protein in which the debranching enzyme sequences are fused to tag or marker sequences. Such fusion proteins can facilitate the purification of recombinant debranching enzymes. Suitable tag and marker sequences are well known in the prior art and include all those known in the art or yet to be discovered. Exemplary tags or markers include, without limitation, HIS tag, GST, MYC tag, fluorescent proteins, fluorophores, and others that are too numerous to include herein.

[0048] One skilled in the art will recognize that activity of enzymes depends upon conditions that are specific to each enzyme. Some enzymes are active at higher temperatures, such at 65° C., while others are active at lower temperatures, such at 37° C. Other conditions include pH and salt content. As such conditions depend upon the enzyme; the invention includes all conditions for which the enzymes useful for the invention are active.

II. Methods

[0049] The present invention includes methods of preparing and analyzing lariat RNA populations. Methods of the invention also include using the compositions described herein to modulate the proportion of lariat RNA in an RNA population.

[0050] Methods of preparing lariat RNA populations or enriched lariat RNA populations include providing an RNA population and contacting it with a linear RNA degrading enzyme to form a lariat RNA enriched population. In some embodiments, methods may further include contacting the RNA population with a debranching enzyme. The order with which the RNA population is contacted with the linear RNA degrading enzyme and debranching enzyme determines the composition of the resulting enriched RNA population. If the RNA population is contacted with the linear RNA degrading enzyme before the debranching enzyme, then the resulting enriched RNA population will be enriched for lariat RNA. If the RNA population is contacted with the debranching enzyme before the linear RNA degrading enzyme, then the resulting enriched RNA population will not be enriched for lariat RNA or linear RNA.

[0051] In some embodiments, methods may further include contacting the RNA population with a 5' cap removing enzyme or be subjected to a 5' cap removal treatment. Preferably, the 5' cap removing enzyme or treatment is contacted or used on the RNA population before the linear RNA degrading enzyme.

[0052] In some embodiments, methods may include inhibiting the RNA debranching enzyme in a population of cells prior to the methods of enriching for lariat RNA. Inhibiting the RNA debranching enzyme in a population of cells would allow the proportion of lariat RNA in a population of cells to increase, thereby allowing the enriched lariat RNA population to increase. The RNA debranching enzyme may be inhibited using methods known in the art. Such methods may include, without limitation, siRNA technology, ribozymes, knockout cell lines, knock down cell lines, and other methods known in the art.

[0053] The invention also includes methods of analyzing the lariat RNA in an RNA sample or population. In some embodiments, methods include providing an RNA population and contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population. The lariat RNA enriched population is contacted with a debranching enzyme and then subsequently with a linear RNA degrading enzyme to confirm true lariat RNAs are present.

[0054] In other embodiments, methods include providing an RNA population and contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population. The lariat RNA enriched population is then used to create a cDNA library. In one embodiment, the cDNA library is created by reverse transcribing the lariat RNA enriched population. Methods known in the art for creating a cDNA library may be used. Suitable methods may also further include sequencing the cDNA library created using the lariat RNA enriched population. Methods known in the art for sequencing may be used.

III. Kits

[0055] The present invention includes articles of manufacture and kits containing materials useful for preparing enriched lariat RNA populations as described herein. The article of manufacture may include a container of a composition as described herein with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic.

[0056] In one embodiment, containers hold a composition having an active agent which is effective for degrading linear RNA or linearizing lariat RNA. The active agent may be an enzyme. Suitable enzymes include 3' exonucleases, 5' exonucleases, 5'/3' exonucleases, debranching enzymes, decapping enzymes, or combinations thereof. Active agents may be combined into a single container or provided in separate containers. Preferably, the active agents are provided in separate containers.

[0057] In another embodiment, containers may hold a composition having a supportive agent, which is supportive of the active agent. Such supportive agents may be buffers. The supportive agent will depend upon the active agent. Exemplary supportive agents include, without limitation, exonuclease reaction buffer, debranching enzyme reaction buffer, decapping enzyme reaction buffer, siRNA reaction buffer, RT-PCR reaction buffer, or combinations thereof. Supportive agents may be combined into a single container or provided in separate containers. Preferably, the active agents are provided in separate containers.

[0058] In another embodiment, containers may contain siRNAs or sources for producing siRNA. The siRNA may be species specific. Any siRNA known in the art or yet to be discovered may be provided with the kit.

[0059] In another embodiment, containers may contain total RNA for control RT-PCRs to assess lariat purification. The total RNA may be from any species.

[0060] In another embodiment, containers may contain oligonucleotides, or primers, for control RT-PCRs. Such primers will amplify a well characterized linear RNA, lariat RNA, or combinations thereof, depending upon the control desired. One skilled in the art will recognize that the primers may be species specific and may depend upon the source species of the total RNA. For example, if the source of the total RNA is Saccharomyces cervisiae, then the control primers could be those that would amplify ACT1 mRNA and the ACT1 intron lariat RNA.

[0061] The article of manufacture may also contain instructs of use.

DEFINITIONS

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0063] As used herein, the phrase "metallophosphatase domain" refers to the amino acids that are conserved among debranching enzymes isolated from various species.

[0064] As used herein, the term "enrich" or forms thereof refer to increasing the amount of a substance found in a heterogeneous population. For example, enriching for lariat RNA in an RNA population refers to increasing the proportion of lariat RNA in an RNA population to a proportion above the other types of RNA found in the RNA population. The enrichment includes purifying an RNA population to only include a specific type of RNA, such as lariat RNA.

[0065] As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2.×SSC, 0.1% SDS at 50-65° C. (e.g., 50° C. or 60° C. or 65° C.) Preferably, the isolated nucleic acid molecule of the invention that hybridizes under stringent conditions corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to RNA or DNA molecules having a nucleotide sequence that occurs in a human cell in nature (e.g., encodes a natural protein).

[0066] As used herein, the phrase "lariat RNA" refers to the pre-mRNA that is excised during the formation of mRNA. This excised pre-mRNA forms a lariat structure.

[0067] As used herein, the phrase "linear RNA" refers to RNA that does not form a lariat structure and that can be degraded by exonucleases.

[0068] As used herein, the phrase "linear RNA degrading enzyme" refers to any enzyme capable of degrading linear RNA. Such enzymes include, without limitation, 3' exonucleases, 5' exonucleases, exonucleases with 3' and 5' activity, as well as others known in the art or yet to be discovered.

[0069] As used herein, the term "nucleic acid sequence" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA or lariat) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded.

[0070] As used herein, the phrase "RNA population" refers to a sample containing ribonucleic acid. The RNA population may or may not be purified RNA.

[0071] The following examples are simply intended to further illustrate and explain the present invention. The invention, therefore, should not be limited to any of the details in these examples.

EXAMPLES

Example 1

Materials and Methods

[0072] Yeast and Bacterial Strains, Plasmids, and General Procedures.

[0073] The following yeast strains were used: TMY30 (MATα ura3-52 lys2-801 ade2-101 trpl-Δ63 h is 3-Δ 200 leu2-Δ1), TMY60 (TMY30 dbr1::neo^r), TMY497 [=TMY30 mutated to dbr1 (D180Y allele)], TMY498 [TMY30 mutated to dbr1 (G84A allele)], TMY499 [=TMY30 mutated to dbr1 (Y68S allele)}. TMY453, a dbrl1Δ::hisG version of sigma strain 10560-23C, was used for FLO8 RT-PCR experiments (sigma strain 10560-23C=MATalpha ura3-52 his3::hisG leu2::hisG). The dbr1Δ::hisG allele was created using pTM513, a DBR1 gene blaster plasmid containing dbr1 Δ::hisG-URA3-hisG, and targeted to replace DBR1 chromosomal sequences by digestion with PvuII.

[0074] The following E. coli strains were used: Rosetta DE3 [F ompT hsdS_B(r_B.sup.- m_B.sup.-) gal dcm (DE3) pLysSRARE (Cam^R)]; XL1 Blue [F'::Tn10 proA⁺B⁺ lacI^q Δ(lacZ)M15/recA1 endA1 gyrA96 (Nal^r) thi hsdR17 (r_k^m_k⁺) supE44 relA1 lac]; JM109 [F' traD36 lacI^q- Δ(lacZ)M15 proA⁺ B⁺/e14.sup.- (McrA.sup.-) Δ(lac-proAB) thi gyrA96 (Na1^r) endA1 hsdR17 (r_k^m_k⁺) relA1 supE44 recA1], ES1301 [lacZ53 thyA36 rha-5 metB1 deoC IN(rrnD-rrnE) mutS201::Tn5]; and TOP10 (F-mcrA Δ (mrr-hsdRMS-mcrBC) φ80lacZ Δ M15 ΔlacX74 recA1 deoR araD139 D(ara-leu)7697 galU galK rpsL (Str^R) endA1 nupG).

[0075] The following plasmids were used for this study: pET16b-DBR1 was used to express Dbr1p in E. coli. pRS306 was used as a URA3 template for making a PCR fragment to create a dbr1Δ::URA3 allele at the DBR1 locus. YEp351 (LEU2) was used in co-transformations with the PCR fragment that resulted in the creation of a dbr1Δ::URA3 strain. This strain was an intermediate in the creation of dbr1 point mutants. pTM431, pTM432, and pTM435 were all created by random mutagenesis of pYES2/GS-DBR1 and encode Dbr1p D180Y, Dbr1p G84A, and Dbr1p Y68S, respectively. The DBR1 gene blaster plasmid pTM513 was created in three steps. First, the 3.8 kbp BamHI-BglII fragment from pNKY51, containing hisG-URA3-hisG, was ligated into the BamHI site of pBluescript to create pTM509. Second, the 5' UTR of DBR1 was amplified from genomic DNA using oligonucleotide primers 331 and 332, then the PCR product was trimmed with EcoRI and BamHI and ligated into EcoRI and BamHI sites of pTM509 to create pTM511. Third, the 3' UTR of DBR1 was amplified from genomic DNA using oligonucleotide primers 333 and 336, then the PCR product was trimmed with XbaI and NotI and ligated into XbaI and NotI sites of pTM511 to create pTM513.

[0076] When not specifically described, general molecular techniques (Ausubel et al. 2003) as well as standard yeast media and general procedures (Kaiser et al. 1994) were used. Oligonucleotides are listed in Tables 1 and 2.

[0077] RNA Extraction.

[0078] Yeast strains were grown to mid-logarithmic phase prior to isolating total cellular RNA. In some cases yeast cells were used directly for RNA preparation after cell growth was complete. In other cases, yeast cells were pelleted and flash frozen in a dry ice ethanol bath and stored at -80° C. prior to RNA preparation. No difference was found in results for RNAs prepared from cells processed in these two ways. Total yeast RNA was prepared by the hot acid phenol method (Ausubel et al. 2003) or by a column purification method (RNeasy kit, Qiagen) from small cultures (10 ml) grown to mid-logarithmic phase (OD₆₀₀=˜1). RNA samples were treated with RNase free DNase I to remove DNA contamination. RNA concentration was measured spectrophotometrically by reading OD₂₆₀. The OD₂₆₀/OD₂₈₀ ratio was used as an RNA quality assessment.

[0079] Preparation of Dbr1p Enzyme from E. Coli.

[0080] The pET16b-DBR1 expression plasmid encodes yeast Dbr1p as an N-terminal 10×-histidine-tagged protein. Expression and purification of the histidine-tagged Dbr1p were performed as described in Martin et al. 2002. Rosetta DE3 E. coli cells were used for expression of Dbr1p instead of E. coli strain BL21-Codon Plus(DE3)RIL. Sonication of cells was performed on ice for 60 sec., in 1 sec. pulses, with a large probe at 50% power. Triton X-100 was added after sonication to a final concentration of 0.1%. The tagged Dbr1p was purified from E. coli extracts by binding to and eluting from Nickel-nitrilotriacetic acid-agarose columns and fractions were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Peak fractions from the elution were dialyzed against debranching buffer (20 mM HEPES KOH, pH 7.9; 125 mM KCl; 0.5 mM MgCl₂; 1 mM DTT; 10% glycerol). In some cases, Dbr1p was concentrated by spinning through a Microcon YM-30 spin concentrator at 14,000×g for 40 min. at 4° C. in a Beckman Allegra 25R centrifuge (TA-15-1.5 rotor). The concentrations of Dbr1p preparations were 50-100 ng/μl. Mass spectrometry of purified Dbr1p was performed.

[0081] Enzymatic Treatments of RNA.

[0082] Bacillus stearothermophilus PNPase was acquired (Sigma, St. Louis, Mo.) and a stock of 3.5 units/ml was prepared by dissolving the protein in water, then adding Tris HCl, pH 8.5, to a final concentration of 50 mM. PNPase reactions were performed in PNPase buffer (50 mM Tris HCl, pH 8.5; 1 mM 2-mercaptoethanol; 1 mM EDTA; 20 mM KCl; 15 mM MgCl₂; 10 mM Na₂HPO₄, pH 8.3) on 20-1000 ng of total yeast RNA in 20 μl reactions for 1 h at 60° C., using 1 μl of the PNPase stock. Upon completion of reactions, samples were heated to 85° C. for 10 min, then either used directly in RT-PCRs or ethanol precipitated. Mock treatments were performed in the same way, minus PNPase.

[0083] Approximately 50-100 ng of yeast Dbr1p prepared from E. coli was used for in vitro debranching reactions of 20-200 ng of RNA. Reactions were performed at 30° C. for 45 min. in a 20 μl volume containing lx debranching buffer (20 mM HEPES-KOH pH 7.9, 125 mM KCl, 0.5 mM MgCl₂, 1 mM DTT and 10% glycerol). Reactions were stopped by heating at 65° C. for 10 minutes (min.). Mock treatments were performed in the same way, minus Dbr1p.

[0084] For sequential enzymatic treatments, RNA samples were phenol/chloroform extracted and ethanol precipitated after the first treatment (PNPase or Dbr1p) then resuspended and treated with the second enzyme.

[0085] RT-PCR Methods.

[0086] RT-PCRs of lariat and linear RNAs were performed with QIAGEN one-step RT-PCR kit (Valencia, Calif.) under the following general conditions: 50° C., 30 min; 95° C., 15 min; 9 cycles of 94° C. for 30 sec, 54° C. for 30-60 sec [touchdown to 46° C. (-1° C. per cycle)], 72° C. for 30 sec; X cycles (see below) of 94° C. for 30 sec, 46° C. for 30 sec, 72° C. for 3045 sec; 72° C. for 5-10 min; 4° C. hold. The number of cycles in the post-touchdown phase of different RT-PCRs (X cycles above) varied with the experiment and are reflected in the following reaction profile names: ACT1-1, 29 cycles, post-touchdown; ACT1-2, 24 cycles, post-touchdown; ACT1-3, 19 cycles, post-touchdown; ACT1-4, 15 cycles, post-touchdown; and ACT1-5, 11 cycles, post-touchdown. RNA amounts between 2 ng and 50 ng were used in RT-PCRs. RT-PCRs were analyzed by either PAGE or agarose gel electrophoresis.

[0087] Real-Time RT-PCR (qRT-PCR) of Lariat and Linear RNAs.

[0088] Primers and probes for qPCR were designed using Sequence Detection Systems software from Applied Biosystems (Carlsbad, Calif.) and are listed in Table 1. All probes and primers for qRT-PCR were purchased from Applied Biosystems. Validation experiments were performed that demonstrated that the efficiencies of target and reference PCRs are approximately equal.

TABLE-US-00001 TABLE 1 Primers and probes for qRT-PCR. Target SEQ and ID Primers NO: Probe Sequence Position^a 28 FWD TCCCAAGATCGAAAATTTACTGAAT -30 to 6 primer 29 REV TTTACACATACCAGAACCGTTATCAAT 54 to 28 primer 30 TaqMan VIC-TGAATTAACAAGGTTGCTGCT- -4 to 26 probe MGBNFQ ACT1 intron: 31 FWD ATTTTTCACTCTCCCATAACCTCCTATA 94 to 121 primer 32 REV TTTCAAGCCCCTATTTATTCCAAT 173 to 150 primer 33 TaqMan 6FAM-TGACTGATCTGTAATAACCA- 123 to 142 probe MGBNFQ RPP1B mRNA: 34 FWD AGGCCGCTGGTGCTAATG 89 to 106 primer 35 REV TCCAAAGCCTTAGCGTAAACATC 146 to 124 primer 36 TaqMan VIC-CGACAACGTCTGGGC- 108 to 122 probe MGBNFQ RPP1B intron: 37 FWD AATGCAACCTAAAACGACTTTGTG 12 to 35 primer 38 REV TTTCTCGGGACGATTGTTGTC 77 to 57 primer 39 TaqMan 6FAM-ACTACGAAGAGAAAGATT- 38 to 55 probe MGBNFQ YRA1 mRNA: 40 FWD AGGTTTGCCAAGGGACATTAAG 249 to 270 primer 41 REV ACACCACCTACTTGAGATGCAAAA 314 to 291 primer 42 TaqMan VIC-AGGATGCTGTAAGAGAAT- 272 to 289 probe MGBNFQ YRA1 intron: 43 FWD CGCATCGTCTCGTGTGGAT 42 to 60 primer 44 REV GATCAAAAGCGTGTGCCATATC 107 to 86 primer 45 TaqMan 6FAM- 62 to 84 probe CGAGAAATATTCTTTGTAAGGAA- MGBNFQ ^aRelative to start of coding sequence for mRNA primers and probes. Relative to start of intron sequence for intron primers and probes.

[0089] For total RNA samples (untreated or treated with Dbr1p/PNPase, as described above), 20-1000 ng of RNA was reverse transcribed into cDNA using random hexamers in a 100 μl reaction at 45° C. for 60 min.

[0090] PCR MasterMix reagents from Applied Biosystems were used for qPCR reactions, which were performed in triplicate for each sample. Reactions were prepared and run according to a standard protocol established by Applied Biosystems on an ABI 7500 real-time PCR machine. Briefly, reactions contained 2×PCR MasterMix, 900 nM forward primer, 900 nM reverse primer, 250 nM TaqMan probe, and cDNA (˜20 ng). Reactions were incubated for 2 minutes at 50° C. and then 10 minutes at 95° C. and before proceeding through 40 cycles of a 30 second (sec) incubation at 95° C. and a 60 second incubation at 60° C. Completed reactions were held at 4° C.

[0091] Relative quantification (RQ) of results was performed using the comparative CT method (ΔΔC_T) (Schmittgen and Livak 2008). The amplification of each target intron sequence was compared to amplification of the corresponding mRNA sequence and a ΔC_T was determined. To compare the different samples to each other, the wild-type sample was used as the calibrator sample. Therefore, the ΔC_T of the wild-type sample was subtracted from the ΔC_T for each sample to determine -ΔΔC_T values. In FIG. 7, RQ 2.sup.-ΔΔCT for each -ΔΔC_T and represents the fold-difference in intron levels between a given sample and the wild-type sample (DBR1).

[0092] In Vitro Debranching Time Course.

[0093] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, the exogenous control for qPCR in these experiments, was generated by reverse transcribing 600 ng of human RNA at 45° C. for 1 hour (hr) using the reverse transcriptase (RT) kit from Applied Biosystems. A debranching reaction mix was set up on ice and contained 5600 ng of total RNA from TMY60 (dbr1) cells, about 6 ng GAPDH cDNA, 140 μl of purified Dbr1p, and 350 μl 2× debranching buffer in a final volume of 700 μl. Seven 100 μl aliquots of this mix were distributed to 0.2 mL PCR tubes. The debranching reaction was directly inactivated in one tube (0 min reaction time) by raising the temperature to 95° C., followed by phenol/chloroform extraction and ethanol precipitation. The remaining six tubes were incubated at 30° C. and individual reactions were stopped after 2.5 min, 5 min, 10 min, 15 min, 30 min, and 60 min. Reactions were stopped by raising the temperature to 95° C., followed by phenol/chloroform extraction and ethanol precipitation. RNAs were then treated with PNPase, as described above, to degrade intron lariats linearized by Dbr1p. Reverse transcription of the RNAs remaining from the different debranching reactions was performed using the RT kit from Applied Biosystems and random hexamer primers. qPCRs using these cDNAs were performed as described above, amplifying a volume of cDNA roughly corresponding to about 20 ng of starting total RNA, using primers and probes for yeast ACT1, YRA1, and RPP1B introns as well as human GAPDH. GAPDH cDNA was the exogenous control because it is insensitive to PNPase and remained at a constant level in each reaction.

[0094] Creation of dbr1 Point Mutant Strains.

[0095] Mutants were created using modifications of the delitto perfetto method (Storici et al. 2001) and the site specific genomic (SSG) method (Gray et al. 2004). Initially, a dbr1Δ::URA3 strain was created to facilitate the introduction of point mutant alleles of dbr1 into the DBR1 locus. Yeast strain TMY490, containing a URA3-marked deletion of 1090 bp of the 1215 bp DBR1 coding sequence (nts 71-1160 deleted), was constructed by transformation of TMY30 with a PCR fragment containing the URA3 gene from pRS306 flanked by ends corresponding to 5' and 3' segments of the DBR1 coding region.

[0096] The fragment used for making the dbr1Δ::URA3 allele was created by PCR of pRS306 with oligonucleotides 443 and 444, the 3' 20 nucleotide (nt) of which anneal to the ends of the URA3 gene on pRS306 and the 5' 40 nt of which correspond to DBR1 sequences (see Table 2).

TABLE-US-00002 TABLE 2 Oligonucleotides. SEQ ID NO: Primer Sequence Position^a 1 146 cactctcccataacctccta ACT1 intron nt 100-119 2 215 ctcaaaccaagaagaaaaagaa ACT1 nt -128 to -107 3 216 tgataccttggtgtcttggtct ACT1 nt 130 to 109 4 331 aggatgtttccgtctttagaa -761 to -741 upstream of DBR1 ORF 5 332 gaggatcctgataaatgtctgcccatctt -10 to -30 upstream of DBR1 ORF; EcoRI site added at 5' end 6 333 gctctagaacgaatgcagacggaattaga 16 to 30 after of DBR1 stop codon; XbaI site added at 5' end 7 336 ataagaatgcggccgcaaagggatccaatgtggtga 779 to 760 after of DBR1 stop codon; NotI site added at 5' end 8 363 gcaagcgctagaacatacttag ACT1 intron nt 18-1, 265-262 9 372 agtgaatagttcgtatccagattc FLO8 nt 12-35 10 373 catacaaaaagccttgaggtg FLO8 nt 418-398 11 374 ggtagcaaatattctgggacatct FLO8 nt 422-445 12 375 attctgggttggccctacattt FLO8 nt 837-816 13 376 agtcaaaacgttactggctgg FLO8 nt 841-861 14 377 tgcttgattgcggaagttag FLO8 nt 1260-1241 15 378 ttggcgaggaagatatttattc FLO8 nt 1268-1289 16 379 aagataatggactggatacagccg FLO8 nt 1675-1652 17 380 ttcgatccagaaagtggcaa FLO8 nt 1693-1712 18 381 ttttcctctggagtagataatgtg FLO8 nt 2036-2013 19 382 atcaaggatatgattttgacgc FLO8 nt 2054-2075 20 383 cagccttcccaattaataaaattg FLO8 nt 2399-2376 21 408 taaatagcttggcagcaacagg URA3 nt 67-46 22 417 ttgcgaattgctgtacaagg DBR1 nt 10-29 23 418 caagtcatgaatttagagataaatgc DBR1 nt 1217-1192 24 443 gctgtcatggtcagctaaaccaaatttataaagaagtgt 5' 40 nt = DBR1 nt 31- . . . 70 25 443cont . . . taactatgcggcatcagagc 3' 20 nt = URA3 flank in pRS306 26 444 gataaatgctttagtttgtcgtacttcatctttctgaata 5' 40 nt = DBR1 nt . . . 1200-1161 27 444cont . . . cctgatgcggtattttctcc 3' 20 nt = URA3 flank in pRS306 ^aFor the ACT1, FLO8, URA3 and DBR1 genes, the nucleotide positions are relative to the first nucleotide of the coding sequence, except for the ACT1 intron, where positions are relative to the first nucleotide of the intron.

[0097] The dbr1Δ::URA3 disruption on yeast chromosome XI was created by homologous recombination between the DBR1 locus and the dbr1Δ:: URA3 PCR fragment. Briefly, TMY30 was transformed with the dbr1Δ:: URA3 PCR fragment and transformants were selected on SD-Uracil plates. Transformants were screened by PCR with primer pairs 401/402, which anneal within the DBR1 sequences that are deleted in the dbr1Δ:: URA3 allele, and 417/418, which anneal outside the DBR1 sequences that are deleted in the dbr1Δ:: URA3 allele. Transformants containing the dbr1Δ:: URA3 allele template a 417/418 PCR product but not a 401/402 PCR product. DNA sequencing of PCR products was performed to verify the presence of the dbr1Δ:: URA3 allele.

[0098] Replacement of the chromosomal dbr1Δ:: URA3 allele with dbr1 point mutations was accomplished by transformation. TMY490 (dbr1Δ:: URA3 strain) was co-transformed with YEp351(LEU2) and PCR fragments of dbr1 point mutants. The PCR fragments were generated from plasmids pTM431, pTM432, and pTM435 with PCR primer pairs 417/418. Transformants (with YEp351) were selected in SD-leucine liquid media during a 48 hr incubation period at 30° C. (with shaking). After this selection period, cells were spread onto 5-fluoroorotic acid plates to select for cells that lost function of the URA3 gene within the DBR1 locus. Recombinants within the FOA^r population that have replaced the dbr1Δ:: URA3 allele with a dbr1 point mutant allele were identified by PCR screening. Positive clones were identified as those that template a 417/418 PCR product but not a 417/408 PCR product (specific for the dbr1Δ:: URA3 allele). DNA sequencing of PCR products was performed to verify the presence of a dbr1 point mutant allele.

Example 2

RT-PCR Detection of Lariat RNAs

[0099] S. cerevisiae ACT1, which encodes actin, is a robustly expressed gene that contains an intron of 308 nt. The first example of a spliceosomal intron discovered in yeast, the ACT1 intron contains all the canonical features of yeast introns and is efficiently spliced from pre-mRNA, producing an excised lariat with a 265 nt circle. This well-characterized gene was chosen to assess intron levels as tools were developed and tested for detecting and enriching excised intron lariats. Primers were designed for use in RT-PCR to detect the lariat form of the ACT1 intron RNA and, as a control, ACT1 mRNA (FIG. 1A). RT-PCR of total yeast RNA using primers that flank the ACT1 exon-exon junction (primers 215 and 216) amplifies a 285 bp product from ACT1 mRNA. Primer 363 spans the ACT1 intron lariat branch point and is used in combination with primer 146, which anneals to sequences complementary to the ACT1 intron upstream of the lariat branch point, in an RT-PCR that amplifies a 184 bp product from the lariat form of the ACT1 intron RNA. As expected, when RT-PCRs are performed using total RNA samples from wild-type (TMY30) and dbr1 mutant yeast cells (TMY60), the amounts of ACT1 mRNA products are similar when using equivalent amounts of RNA from the two cell types (FIG. 1B, lanes 1 and 3 as well as lanes 5 and 7). However, the ACT1 intron RNA lariat product is much more readily produced from dbr1 cells (FIG. 1B, lane 4 vs. 2 and lane 8 vs. 6). These data clearly show that a dbr1 mutant strain or, where appropriate, a Dbr1p knock-down strain contains a rich source of expressed intron sequences. It is also evident that the use of intron-specific RT-PCR could be used to detect excised introns from genes expressed at very low levels. For studies on alternative splicing, the use of RT-PCR on RNA from Dbr1p-deficient cells can allow detection of rare splice variants.

[0100] A previous report described the use of radiolabeled primers spanning intron RNA branch points for analyzing intron populations by primer extension (Spingola et al. 1999). The RT-PCR method we describe could be modified to survey intron lariats containing specific sequences at intron 5' ends and branch points. RT-PCR has added utility because the products can be cloned and sequenced to identify the individual introns represented in a lariat population.

Example 3

Insensitivity of Lariat RNAs to the 3' Exonuclease PNPase

[0101] Linear and lariat RNAs have different sensitivities to 3' exonucleases, including PNPase, a component of bacterial RNA degradation systems. PNPase degrades linear RNAs but does not proceed past the 2' branch present in intron RNA lariats. Therefore, treatment of RNA samples with an enzyme like PNPase should result in a vast enrichment of excised intron lariats in the RNA that remains intact after treatment. This difference should be evident in the results of the RT-PCR assay described above when amplifying PNPase-treated RNA samples. Since RNA secondary structures reduce the efficiency of PNPases, reactions were performed at elevated temperature (60° C.) using PNPase from Bacillus strearothermophilus to circumvent this problem. Total RNA samples from a dbr1 mutant strain (TMY60) were treated with a range of PNPase concentrations and then subjected to RT-PCR to detect ACT1 intron RNA lariats as well as the linear mRNA (FIG. 2). Results are consistent with expectations that the use of PNPase selectively preserves RNA lariats.

[0102] The high temperature reaction using PNPase from a thermophile appears to be much more efficient than the reported reaction with the E. coli PNPase at 37° C. In order to eliminate the RT-PCR product from the ACT1 mRNA, PNPase must degrade, at the very least, the RNA corresponding to the binding site for the downstream primer (oligonucleotide 216). To accomplish this, PNPase must degrade all the RNA that lies to the 3' side of the oligonucleotide 216 binding site, which includes 998 nt of the ACT1 coding sequence plus the 3' UTR and the polyA tail. To further examine the processivity of Bacillus strearothermophilus PNPase, the degradation of FLO8 mRNA was assessed. FLO8 mRNA is >2.4 kb in length. Primer pairs were designed to amplify different portions of this mRNA along its length (FIG. 3A). Total nucleic acid samples and RNA samples (DNased total nucleic acid samples) were treated with PNPase and subjected to RT-PCR to detect the various segments of FLO8. As shown in FIG. 3B, PNPase readily degrades every segment of FLO8 mRNA assayed. As expected, PNPase has no effect on FLO8 DNA present in the total nucleic acid samples (FIG. 3c). Other enzymes that worked as well as Bacillus strearothermophilus PNPase in our studies are Thermus thermophilus PNPase at 65° C. and Escherichia coli RNase R at 37° C.

Example 4

Sensitivity of Lariat RNAs to Dbr1p

[0103] Linear and lariat RNAs also have different sensitivities to RNA debranching enzyme, which can be exploited to confirm that an RNA species have a lariat conformation. The RT-PCR strategy employing a primer that spans a lariat branch point, as described above for the ACT1 intron, can be used to demonstrate the cleavage of the 2'-5' bond. This is due to the fact that after Dbr1p treatment the binding site for the primer that spans the ACT1 intron branch point (oligonucleotide 363) is split into two non-contiguous sections, with the section that anneals to the 3' end of the primer being only 3 base pairs (bp) in length. After debranching of the lariat, the critical 3' end of the primer will not effectively anneal to the intron RNA to prime RT-PCR. Dbr1p treatment has no effect on ACT1 mRNA, which should still be readily detected by RT-PCR.

[0104] In order to perform Dbr1p treatments, S. cerevisiae Dbr1p was expressed in E. coli and purified by metal affinity chromatography (FIGS. 4A and 4B). Although histidine-tagged Dbr1p is expected to have a mass of about 50 kilo-dalton (kDa), the mobility of the main product in SDS-PAGE is about 45 kDa. Others have observed this anomalous mobility for histidine-tagged Dbr1p and have speculated that the protein may undergo limited proteolysis in E. coli. However, mass spectrometric analysis of the main band in the stained gel shows it to be the expected molecular mass of the histidine-tagged Dbr1p (50062 Dalton (Da)) (FIG. 4c), indicating that the protein is intact and must run anomalously in SDS-PAGE because of its physical properties.

[0105] Using the Dbr1p enzyme preparation, debranching reactions were carried out on total RNA samples from a dbr1 mutant strain. RT-PCR analysis reflects the differential sensitivity of linear and lariat RNAs to Dbr1p. After Dbr1p treatment, RT-PCR detection of ACT1 RNA lariat is greatly decreased (FIG. 5, lane 4 vs. 2). On the other hand, the product indicative of ACT1 linear mRNA is still readily detectable after Dbr1p treatment (FIG. 5, lane 3 vs. 1).

Example 5

Combinations of PNPase and Dbr1p Treatments

[0106] PNPase and Dbr1p treatments can be used in combination when exploring the properties of a particular RNA species. Sequential enzymatic treatments can also be used to enrich for RNA lariats and then linearize them for further manipulations. To demonstrate this, ACT1 RNA species present within a total RNA sample from a dbr1 mutant strain were analyzed by RT-PCR following sequential PNPase and Dbr1p treatments. As shown in FIG. 6A (lanes 1-4), initial treatment of the RNA sample with PNPase degrades the linear mRNA (lanes 1 and 3), but leaves lariat RNA intact (lane 2). Subsequent treatment with Dbr1p shows that the resistant RNA is a lariat (lane 4). As shown in FIG. 6A (lanes 5-8), skipping the initial PNPase treatment leaves the linear mRNA intact (lanes 5 and 7) as well as the lariat RNA (lane 6). The lariat RNA is then distinguished by its sensitivity to cutting with Dbr1p (lane 8). The order of the PNPase and Dbr1p reactions can be switched to generate a complementary set of predictable results (FIG. 6B).

Example 6

Real-Time RT-PCR Measurement of Lariat RNA Levels

[0107] A real-time RT-PCR method (qRT-PCR), using the TaqMan detection system (Applied Biosystems), was developed to quantitatively compare the intron RNA lariat levels of different samples. The study included not only the ACT1 intron but also the YRA1 and RPP1B introns to investigate the generality of the methods. YRA1 encodes an RNA binding protein involved in mRNA export from the nucleus and is moderately expressed, although less than ACT1. The YRA1 intron is 765 nt in length, which is larger than the 300 nt average for yeast introns, and contains a non-canonical branch point sequence. Furthermore, the intron is inefficiently spliced from pre-mRNA, which is important for the auto-regulation of Yra1p protein levels. RPP1B encodes a ribosomal protein and is even more highly expressed than ACT1. The RPP1B intron is typical for yeast, 301 nt in length, with canonical sequences.

[0108] Initially, a strategy similar to the one used for RT-PCR of ACT1 intron lariats described above, with one primer spanning the lariat branch point and serving as both the RT primer and the reverse primer for PCR was used. However, a different strategy using random primers for the RT step was also used to allow amplification of the different target sequences from a common pool of cDNA. Consequently, both PCR primers anneal upstream of the branch point for each target gene, with a TaqMan probe annealing between them (FIG. 7A). Since these types of primers will also prime amplification of genomic DNA we ran control PCRs for each sample without a prior RT step to ensure that DNA contamination was not contributing to the PCR product. The mRNA for each target gene served as the endogenous control for qRT-PCR (FIG. 7A, top). Using this strategy, intron sequences for ACT1, RPP1B, and YRA1 were amplified from dbr1 and wild-type yeast strains (TMY60 and TMY30). As shown in FIGS. 7B, 7C and 7D [DBR1 (wild type) vs. dbr1 null mutant), the real-time method generated the expected results: the different intron RNAs accumulate at higher levels in the dbr1 null mutant strain than in wild type.

[0109] qRT-PCR was also used to analyze mutant variants of Dbr1p. Previously, a set of point mutants had been created by random PCR mutagenesis and analyzed for intron RNA levels by an RNase protection assay. In these experiments, the dbr1 mutant alleles were under the control of a strong, inducible promoter (pGAL1) and carried on a high copy plasmid. The yeast strain carried a dbr1Δ mutation [open reading frame (ORF) deletion] at the DBR1 locus so the plasmid-borne dbr1 mutant alleles were the only sources of Dbr1p. For the current study, three dbr1 point mutants (D180Y, G84A, and Y68S) were analyzed by qRT-PCR to determine their levels of intron lariat RNA relative to wild-type (DBR1) and dbr1Δ. To make the analysis more biologically relevant, each of the dbr1 mutant alleles was placed at the DBR1 locus, replacing the wild-type allele, and was under the control of the native DBR1 promoter. After log-phase growth of cells, RNA samples from wild-type and mutant strains were harvested and subjected to qRT-PCR to amplify intron and messenger RNA sequences from ACT1, RPP1B, and YRA1. The three dbr1 alleles tested show strong intron RNA accumulation phenotypes, comparable to the dbr1Δ knockout allele (FIGS. 7B, 7C, and 7D).

Example 7

qRT-PCR Analysis of a Debranching Time Course

[0110] Using a combination of Dbr1p and PNPase treatments, in vitro debranching reactions of total cellular RNA from a dbr1 strain were followed over time courses of thirty minutes. Debranching reactions were stopped at different times and the reaction products were treated with PNPase to degrade linearized intron RNAs. The remaining intron lariats were detected by qRT-PCR as described herein. Because the PNPase treatment step degrades all linear RNAs, human GAPDH cDNA was added to the yeast RNA samples as an exogenous control. The GAPDH cDNA is insensitive to both Dbr1p and PNPase, remaining at the same level in the various samples. Debranching of the ACT1 and RPP1B intron lariats was almost complete within the first 5 minutes of the reactions (FIG. 8). However, the debranching rate of the ACT1 intron lariat appeared to be only two-thirds the initial rate of the RPP1B intron lariat.

[0111] The results observed from using qRT-PCR to follow in vitro debranching, show that the debranching rates can vary from one intron lariat to another. The ACT1 intron is debranched at only two-thirds the initial rate at which the RPP1B intron lariat is debranched. These data suggest that different intron lariats are debranched at different rates in vivo, which may be of functional significance. Slower rates of debranching may occur for introns that contain snoRNAs or mirtrons, reflecting the binding of additional factors to intron sequences or specific folding properties of the RNA. Thus, the rate of the debranching of introns can be used to predict which introns may contain additional information. Relative debranching rates can be inferred from quantitative analysis of intron RNA levels relative to mature mRNA levels for a given gene compared to a standard, rapidly debranching intron RNA. For these types of experiments, RNA samples could be taken from a wild-type strain (DBR1), where lariat RNAs are not stabilized. Inefficient splicing would have to be ruled out before further study of candidate slow debranchers. As described above, YRA1 is an example of a gene that uses splicing inefficiency to regulate protein levels.

[0112] Quantitative RT-PCR of lariat RNAs can be used to determine the relative rates of transcription for different intron-containing genes. The use of intron RNA lariats as a novel data source for estimating relative levels of transcription for pre-mRNAs limits the utility to intron-containing genes, a notable limitation for S. cerevisiae. Furthermore, a Dbr1p-deficient strain would have to be used for intron lariats to be a stable record of transcription. Work with yeast dbr1 mutants over the years has not found any significant perturbation of cellular physiology other than the accumulation of intron RNA lariats. In the experiments shown in FIG. 7B-D, the level of RPP1B intron RNA in a dbr1 strain relative to the level in wild type is much greater (about 330-fold) than the corresponding levels of ACT1 and YRA1 intron RNAs (about 13-fold). These data indicate that the transcription rate for RPP1B is almost 30-fold greater than the rates for ACT1 and YRA1 (summarized in Table 3). These relative transcription rates are very different from estimates based on nuclear run-on assays, mRNA steady state levels plus half-lives, and DTA (Table 3).

TABLE-US-00003 TABLE 3 ACT1, YRA1, and RPP1B mRNA expression. Relative intron Gene Transcriptional frequency^a DTA^b levels^c ACT1 45.5^d (1) 7.2^e (1) 63.2 (1) 1.0 (YFL039C) YRA1 16.2^d (0.4) 80.6^e (11.2) 88.9 (1.4) 1.1 (YDR381W) RPP1B 120.0^d (2.6) 23.0^e (3.2) 192.7 (3) 28.1 (YDL130W) ^amRNAs/cell/hr; numbers in parentheses are levels normalized to ACT1 level; ^bDTA = dynamic transcriptome analysis, measured as mRNAs/cell/cell cycle time (150 min); numbers in parentheses are levels normalized to ACT1 level; ^cDerived from data in FIG. 7 for the dbr1 null strain versus wild type for each gene and normalized to ACT1 level; ^dEstimated from RNA expression levels and mRNA half-lives; ^eEstimated from genomic run on experiments.

[0113] An area where the utility of excised introns is clearer is in determining relative rates of alternative splicing for a particular gene. Variable stabilities of different mRNAs confound estimates of their rate of synthesis, whether the synthesis that produces the mRNAs in question is transcription or alternative splicing. The use of a Dbr1p-deficient strain, which stabilizes the alternatively excised intron lariats equivalently, results in intron RNA lariat levels that directly reflect the rate of alternative splicing.

[0114] The methods described herein can also be applied to genome-wide analysis of introns themselves and are an improvement on previous analyses that also directly analyzed intron RNA lariats. RNA-seq of intron RNA lariat populations prepared using PNPase can provide complementary information to RNA-seq of whole transcriptomes and may reveal new lariat sequences not evident from transcriptome analysis alone. Intron RNA lariat levels can be greatly enhanced by blocking the RNA debranching reaction, which increases the likelihood of detecting even rare splicing events. Because cells defective for RNA debranching activity accumulate excised introns in their lariat forms, with shorted 3' tails, information on the 3' intron-exon junction is not obtainable from intron lariat RNA sequences. Nevertheless, lariat sequences provide information about branch points that is not obtainable from whole transcriptome sequencing. Such information is especially useful for studies of introns in organisms whose branch point sequences are not as highly conserved as those in S. cerevisiae [e.g. humans]. Finally, the absence of known intron sequences from an RNA population enriched-for RNA lariats can indicate that a gene is not expressed under the growth regimen employed. However, if an intron-containing gene is known to be expressed during the experiment, absence of intron sequences from the RNA lariat population could be an indication that the intron is removed by the hydrolytic splicing pathway observed for self splicing group II introns rather than the predominant branching pathway. High-throughput sequencing of enriched lariat RNAs from human cells is useful for much more detailed analysis of human branch point sequences.

Example 8

Amino Acid Conservation Among RNA Debranching Enzymes

[0115] Dbr1 is an RNA lariat debranching enzyme that hydrolyzes 2'-5' phosphodiester bonds at the branch points of excised intron lariats. The alignment model shown in FIG. 14 represents the N-terminal metallophosphatase domain of Dbr1. This domain belongs to the metallophosphatase (MPP) superfamily. MPPs are functionally diverse, but all share a conserved domain with an active site consisting of two metal ions (usually manganese, iron, or zinc) coordinated with octahedral geometry by a cage of histidine, aspartate, and asparagine residues. The MPP superfamily includes: Mre11/SbcD-like exonucleases, Dbr1-like RNA lariat debranching enzymes, YfcE-like phosphodiesterases, purple acid phosphatases (PAPs), YbbF-like UDP-2,3-diacylglucosamine hydrolases, and acid sphingomyelinases (ASMases). The conserved domain is a double beta-sheet sandwich with a di-metal active site made up of residues located at the C-terminal side of the sheets. This domain allows for productive metal coordination.

Example 9

Creation of an RNA Sample that is Highly Enriched for RNA Lariats

[0116] Linear and lariat RNAs have different sensitivities to exonucleolytic enzymes. Almost all linear RNAs are susceptible to complete or nearly complete degradation by enzymes that have 3' exonucleolytic activity. The key is to use an enzyme that is blocked by the lariat branch point and cannot degrade past the branch point. Such enzymes leave the lariat loop intact. Since lariat RNAs lack a 5' end, they are insensitive to 5' exonucleolytic activity; however, many linear RNAs are also sensitive to 5' exonucleolytic activity. Linear RNAs with 5' cap structures, which protect RNAs from 5' exonucleolytic activity, are made sensitive to 5' exonucleolytic activity by removing their caps. Cap removal treatments do not make lariat RNAs sensitive to exonucleases with 5' exonucleolytic activity. FIG. 9 and FIG. 10 illustrate the 3' and 5' exonucleolytic strategies, respectively, to create an RNA population that is highly enriched for RNA lariats.

[0117] Several methods may be employed to create an RNA population that is highly enriched for RNA lariats. Such methods include: 1) treat the RNA sample with a nuclease that has 3' exonucleolytic activity, a combination of nucleases with 3' exonucleolytic activity can also be used; 2) treat the RNA sample to remove the 5' cap structure from mRNAs, then treat with a nuclease that has 5' exonucleolytic activity, a combination of nucleases with 5' exonucleolytic activity can also be used; 3) treat the RNA sample with a nuclease that has both 5' and 3' exonucleolytic activity, with or without prior treatment to remove the 5' cap structure from mRNAs, a combination of nucleases, one or more with 5' exonucleolytic activity plus one or more with 3' exonucleolytic activity, can also be used.

[0118] To increase the proportion of lariat RNAs in the RNA population, RNA samples can be obtained from cells in which RNA debranching enzyme activity has been lowered or eliminated. Because the enhancement of RNA lariat levels in these cells is so dramatic, the resulting sample is useful for identifying RNA lariat species that are normally present at very low levels (i.e. in cells that have wild-type levels of RNA debranching enzyme activity). Another method that can be employed to increase the proportion of lariat RNAs in the RNA population being studied is to selectively remove rRNA species from the RNA sample prior to lariat RNA enrichment.

[0119] A control RNA sample that is depleted of lariat RNAs can be created and processed in parallel to the exonuclease-treated RNA sample to identify which RNAs are lariats in the exonuclease-resistant RNA population. The lariat depleted RNA sample is created by treatment of an RNA sample with RNA debranching enzyme prior to the exonucleolytic treatment protocol.

[0120] Following acquisition and treatment of RNA samples as outlined above, RNAs are processed for high-throughput sequencing. Although different platforms for high-throughput sequencing have been developed and continue to be developed, all of platforms involve parallel sequencing of large numbers of DNA fragments. All of these platforms are used for RNA sequencing by incorporating cDNA production protocols. The lariat-seq technique requires conversion of lariat-enriched RNA samples into cDNA populations, which are then processed for high-throughput sequencing according to the methods developed for the individual high-throughput sequencing platforms.

[0121] To aid in determining which cDNA sequences represent lariat RNAs, sequencing data resulting from experimental and control RNA samples are compared. RNAs originally in a lariat conformation will be represented at lower levels (proportionally and absolutely) in the control sample, resulting in a proportional (and absolute) reduction in the number of their corresponding cDNA sequences relative to the experimental sample. Some RNAs that are not in a lariat conformation in samples extracted from cells will survive the treatments to create an RNA population enriched for RNA lariats, for example RNAs with covalent modifications, other than a 2'-5' branch, that block the exonuclease used to create the experimental sample. These RNAs will be represented approximately equally (in absolute terms) in experimental and control samples. Selective removal of rRNA species from the RNA sample prior to lariat RNA enrichment, as stated above, will remove many RNAs that contain non-lariat covalent modifications that block the exonuclease.

[0122] Further evidence that a nuclease-resistant RNA identified by lariat-seq has a lariat conformation comes from signature cDNA products unique to lariat RNAs. Reverse transcriptase (RT) used for creating cDNA for sequencing is blocked by the presence of a 2' branch in an RNA substrate. However, when traveling along the branch segment itself, RT will read across the 2'-5' bond, creating cDNAs that juxtapose sequences that are not contiguous in the reference genome. Furthermore, when RT reads across the 2'-5' bond it inserts a nucleotide that is not expected according to Watson-Crick base pairing rules. Typically, for an intron lariat branch point, RT inserts an A opposite the branch point A instead of a T. Sequence reads that contain discontinuous genome segments with an unexpected nucleotide at the junction of the two segments are evidence that the cDNA was created from a lariat RNA.

[0123] The different sensitivities of linear and lariat RNAs to the 3' exonuclease polynucleotide phosphorylase (PNPase) are shown in FIG. 11. Using RT-PCR to measure RNA levels, it is apparent that exonuclease treatment degrades a linear RNA down to the limit of detection while a lariat RNA remains virtually untouched (comparing lanes 1 and 2 (PNPase treatment) to lanes 5 and 6 (untreated)).

[0124] Linear and lariat RNAs also have different sensitivities to RNA debranching enzyme, which can be exploited to confirm that an RNA species has a lariat conformation. In vitro cleavage of intron RNA lariats with purified S. cerevisiae Dbr1p is readily detectable with an RT-PCR assay, as shown in FIG. 12, lanes 3 and 4 (Dbr1p treatment) versus lanes 1 and 2 (untreated). For the RT-PCR in FIG. 12, a primer that spans the branch point was used for RT-PCR, which is why the intron signal is reduced upon Dbr1p treatment.

[0125] The use of sequential Dbr1p and PNPase treatments to explore the properties of a particular RNA species (control described above) is depicted in FIG. 11. As shown in FIG. 11, lanes 3 and 4 show the loss of a known lariat RNA when Dbr1p treatment precedes PNPase treatment. Compare to lanes 1 and 2 (PNPase treatment only) as well as lanes 5 and 6 (no treatment).

[0126] Data from high-throughput sequencing of cDNAs created from PNPase-treated RNA samples support the feasibility and operability of lariat-seq. Total S. cerevisiae RNA from a strain lacking a functioning RNA debranching enzyme was converted into cDNA after PNPase treatment and subjected to a high-throughput sequencing protocol (Illumina platform). An example of a small portion of the results obtained is depicted in FIG. 13, which shows the sequence reads that match to a segment of chromosome 6 (FIG. 13A). What is striking about the results is that the only sequence reads that map to this 20 kbp segment of the S. cerevisiae genome are from cDNAs that represent the intron region of the ACT1 gene (FIG. 13B). Furthermore, all these reads map within the sequences corresponding to the lariat loop of the intron; none of the sequence reads represent the 43 by that lie within the intron downstream of the lariat branch point. The fact that no sequence reads mapped to the ACT1 coding region or any other gene in the 20 kbp segment depicted in FIG. 13 indicates how efficiently the 3' exonuclease degraded the linear RNAs in the sample.

Example 10

RNA Lariat Enrichment Kit

[0127] The components necessary for RNA lariat enrichment can be provided in a kit for ease of use. An example of such a kit is described below. Variations of the kit are also contemplated.

[0128] Components of RNA lariat purification and analysis kit include the following: Bacillus stearothermophilus polynucleotide phosphorylase (BsPNPase); 2× BsPNPase reaction buffer: 100 mM Tris HCl, pH 8.5; 2 mM 2-mercaptoethanol; 2 mM EDTA; 40 mM KCl; 3 mM MgCl₂; 20 mM Na₂HPO₄, pH 8.3; Saccharomyces cerevisiae RNA debranching enzyme (ScDbr1); 10× ScDbr1 reaction buffer: 200 mM HEPES KOH (pH 7.9), 1.25 M KCl, 5 mM MgCl₂, 10 mM dithiothreitol; siRNAs (or siRNA sources) targeting mRNA for RNA debranching enzyme (different siRNA resources are packaged, depending on the organism for which the kit is specified); Saccharomyces cerevisiae total RNA samples (from dbr1 mutant and wild-type cells) for control RT-PCRs to assess lariat purification; primers for control RT-PCRs [to amplify ACT1 mRNA (linear RNA) and the ACT1 intron lariat RNA from Saccharomyces cerevisiae total RNA samples]; and, primers for control RT-PCRs for the organism for which the kit is specified [to amplify a known linear RNA and a known lariat RNA].

[0129] The kit also includes instructions of use. An example of such instructions includes the following:

[0130] 1. Grow cells for RNA preparation. Two growth conditions can be used, one in which expression of endogenous RNA debranching enzyme is reduced, causing intron lariats to accumulate, and one in which the endogenous RNA debranching enzyme expression is unperturbed. If applicable, deploy the supplied siRNA resources to create cells with enhanced RNA lariat levels.

[0131] 2. Harvest cells and purify total cellular RNA. Alternatively, store cells after harvesting for future RNA purification.

[0132] 3. Treat 1 nanogram-10 micrograms of total RNA with 10 units of BsPNPase in 1×BsPNPase reaction buffer for 60 minutes at 60° C.

[0133] 4. Incubate completed BsPNPase reactions at 85° C. for 10 minutes to inactivate the enzyme.

[0134] 5. Phenol/chloroform extract RNA samples and ethanol precipitate them.

[0135] 6. BsPNPase-treated RNA samples can be used for RT-PCRs of specific target RNAs (e.g. known linear and lariat RNAs) or for creation of cDNA libraries for Lariat-seq.

[0136] The kit may also include a control sample that is not enriched for RNA lariats. A control RNA sample that reflects the total RNA sample purified from cells is created by performing the above procedure but without BsPNPase in step 3.

[0137] The kit may also include a control sample that contains debranched RNA lariats. True lariat RNAs present in the BsPNPase-resistant RNA population will be sensitive to BsPNPase in RNA samples pretreated with ScDbr1.

[0138] 1. Treat 1 nanogram-10 micrograms of total RNA with 10 units of ScDbr1 in 1×ScDbr1 reaction buffer in a 20 microliter reaction volume for 45 minutes at 30° C.

[0139] 2. Incubate completed ScDbr1 reactions at 65° C. for 10 minutes to inactivate the enzyme.

[0140] 3. Phenol/chloroform extract RNA samples and ethanol precipitate them. Resuspend RNAs in 1× BsPNPase reaction buffer.

[0141] 4. Continue with BsPNPase treatment as described in steps 3-6 above (Procedure for creating purified RNA lariats).

[0142] In order to Confirm the enrichment of RNA lariats, control RT-PCRs for known linear and lariat RNAs are performed on treated RNA samples (both the samples enriched for RNA lariats and the control samples). Primers are provided for use with Saccharomyces cerevisiae RNA as well as for the organism for which the kit is specified.

[0143] Following treatment of RNA samples as outlined above (and confirmation of lariat-enrichment), RNAs are processed for high-throughput sequencing. The next step is to create a cDNA library from each treated RNA sample using procedures established for the high-throughput sequencing platform to be used (Illumina, SOLiD, etc). Materials for creating cDNA libraries are available from several different manufacturers.

[0144] Bacillus stearothermophilus polynucleotide phosphorylase (BsPNPase) storage buffer: 50% glycerol, 50 mM Tris-HCl (pH 8.5), 100 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100 and 1 mM dithiothreitol.

[0145] Saccharomyces cerevisiae RNA debranching enzyme (ScDbr1) storage buffer: 50% glycerol, 20 mM HEPES KOH (pH 7.9), 125 mM KCl, 0.5 mM MgCl2, 1 mM dithiothreitol.

[0146] One unit of BsPNPase activity is defined as the amount of PNPase that forms 1 μmol of ADP per hour at 60° C. by depolymerizing of Poly A.

[0147] One unit of ScDbr1 activity is defined as the amount of ScDbr1 that debranches 50% of the ACT1 intron present in 1 microgram of a total Saccharomyces cerevisiae RNA preparation (from mid-log phase cells) from a dbr1 mutant strain per hour at 30° C.

[0148] The invention illustratively disclosed herein suitably may be practiced in the absence of any element, which is not specifically disclosed herein. It is apparent to those skilled in the art, however, that many changes, variations, modifications, other uses, and applications to the method are possible, and also changes, variations, modifications, other uses, and applications which do not depart from the spirit and scope of the invention are deemed to be covered by the invention, which is limited only by the claims which follow.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Sequence CWU 1

1

66120DNAArtificial SequencePCR Primer, ACT1 intron nt 100-119 1cactctccca taacctccta 20222DNAArtificial SequencePCR Primer, ACT1 nt -128 - -107 2ctcaaaccaa gaagaaaaag aa 22322DNAArtificial SequencePCR Primer, ACT1 nt 130-109 3tgataccttg gtgtcttggt ct 22421DNAArtificial SequencePCR Primer, -761 - -741 upstream of DBR1 ORF 4aggatgtttc cgtctttaga a 21529DNAArtificial SequencePCR Primer, -10 - -30 upstream of DBR1 ORF, EcoRI site added at 5' end 5gaggatcctg ataaatgtct gcccatctt 29629DNAArtificial SequencePCR Primer, 16-30 after of DBR1 stop codon, XbaI site added at 5' end 6gctctagaac gaatgcagac ggaattaga 29736DNAArtificial SequencePCR Primer, 779-760 after of DBR1 stop codon, NotI site added at 5' end 7ataagaatgc ggccgcaaag ggatccaatg tggtga 36822DNAArtificial SequencePCR Primer, ACT1 intron nt 18-1, 265-262 8gcaagcgcta gaacatactt ag 22924DNAArtificial SequencePCR Primer, FLO8 nt 12-35 9agtgaatagt tcgtatccag attc 241021DNAArtificial SequencePCR Primer, FLO8 nt 418-398 10catacaaaaa gccttgaggt g 211124DNAArtificial SequencePCR Primer, FLO8 nt 422-445 11ggtagcaaat attctgggac atct 241222DNAArtificial SequencePCR Primer, FLO8 nt 837-816 12attctgggtt ggccctacat tt 221321DNAArtificial SequencePCR Primer, FLO8 nt 841-861 13agtcaaaacg ttactggctg g 211420DNAArtificial SequencePCR Primer, FLO8 nt 1260-1241 14tgcttgattg cggaagttag 201522DNAArtificial SequencePCR Primers, FLO8 nt 1268-1289 15ttggcgagga agatatttat tc 221625DNAArtificial SequencePCR Primers, FLO8 nt 1675-1652 16aagataatgg actggataca gcscg 251720DNAArtificial SequencePCR Primers, FLO8 nt 1693-1712 17ttcgatccag aaagtggcaa 201824DNAArtificial SequencePCR Primers, FLO8 nt 2036-2013 18ttttcctctg gagtagataa tgtg 241922DNAArtificial SequencePCR Primers, FLO8 nt 2054-2075 19atcaaggata tgattttgac gc 222024DNAArtificial SequencePCR Primers, FLO8 nt 2399-2376 20cagccttccc aattaataaa attg 242122DNAArtificial SequencePCR Primers, URA3 nt 67-46 21taaatagctt ggcagcaaca gg 222220DNAArtificial SequencePCR Primers, DBR1 nt 10-29 22ttgcgaattg ctgtacaagg 202326DNAArtificial SequencePCR Primers, DBR1 nt 1217-1192 23caagtcatga atttagagat aaatgc 262440DNAArtificial SequencePCR Primers, 5' 40 nt = DBR1 nt 31-70 24tgctgtcatg gtcagctaaa ccaaatttat aaagaagtgt 402520DNAArtificial SequencePCR Primers, 3' 20 nt = URA3 flank in pRS306 25taactatgcg gcatcagagc 202640DNAArtificial SequencePCR Primers, 5' 40 nt = DBR1 nt 1200-1161 26gataaatgct ttagtttgtc gtacttcatc tttctgaata 402720DNAArtificial SequencePCR Primers, 3' 20 nt = URA3 flank in pRS306 27cctgatgcgg tattttctcc 202825DNAArtificial SequenceqRT-PCR Primers, FWD Primer 28tcccaagatc gaaaatttac tgaat 252927DNAArtificial SequenceqRT-PCR Primers, Reverse primer at position 54-28 29tttacacata ccagaaccgt tatcaat 273021DNAArtificial SequenceqRT-PCR Probe, TaqMan probe at position -4 to 26 30tgaattaaca aggttgctgc t 213128DNAArtificial SequenceqRT-PCR Primers, FWD Primer at position 94-121 31atttttcact ctcccataac ctcctata 283224DNAArtificial SequenceqRT-PCR Primers, Reverse primer at position 173-150 32tttcaagccc ctatttattc caat 243320DNAArtificial SequenceqRT-PCR Probe, TaqMan probe at position 123-142 33tgactgatct gtaataacca 203418DNAArtificial SequenceqRT-PCR Primers, FWD Primer at position 89-106 34aggccgctgg tgctaatg 183523DNAArtificial SequenceqRT-PCR Primers, Reverse primer at position 146-124 35tccaaagcct tagcgtaaac atc 233615DNAArtificial SequenceqRT-PCR Probe, TaqMan probe at position 108-122 36cgacaacgtc tgggc 153724DNAArtificial SequenceqRT-PCR Primers, FWD Primer at position 12-35 37aatgcaacct aaaacgactt tgtg 243821DNAArtificial SequenceqRT-PCR Primers, Reverse primer at position 77-57 38tttctcggga cgattgttgt c 213918DNAArtificial SequenceqRT-PCT Primer, TaqMan probe at position 38-55 39actacgaaga gaaagatt 184022DNAArtificial SequenceqRT-PCR Primers, FWD Primer at position 249-270 40aggtttgcca agggacatta ag 224124DNAArtificial SequenceqRT-PCR Primers, Reverse primer at position 314-291 41acaccaccta cttgagatgc aaaa 244218DNAArtificial SequenceqRT-PCT Primer, TaqMan probe at position 272-289 42aggatgctgt aagagaat 184319DNAArtificial SequenceqRT-PCR Primers, FWD Primer at position 42-60 43cgcatcgtct cgtgtggat 194422DNAArtificial SequenceqRT-PCR Primers, Reverse primer at position 107-86 44gatcaaaagc gtgtgccata tc 224523DNAArtificial SequenceqRT-PCT Primer, TaqMan probe at position 62-84 45cgagaaatat tctttgtaag gaa 2346544PRTHomo sapiens 46Met Arg Val Ala Val Ala Gly Cys Cys His Gly Glu Leu Asp Lys Ile 1 5 10 15 Tyr Glu Thr Leu Ala Leu Ala Glu Arg Arg Gly Pro Gly Pro Val Asp 20 25 30 Leu Leu Leu Cys Cys Gly Asp Phe Gln Ala Val Arg Asn Glu Ala Asp 35 40 45 Leu Arg Cys Met Ala Val Pro Pro Lys Tyr Arg His Met Gln Thr Phe 50 55 60 Tyr Arg Tyr Tyr Ser Gly Glu Lys Lys Ala Pro Val Leu Thr Leu Phe 65 70 75 80 Ile Gly Gly Asn His Glu Ala Ser Asn His Leu Gln Glu Leu Pro Tyr 85 90 95 Gly Gly Trp Val Ala Pro Asn Ile Tyr Tyr Leu Gly Leu Ala Gly Val 100 105 110 Val Lys Tyr Arg Gly Val Arg Ile Gly Gly Ile Ser Gly Ile Phe Lys 115 120 125 Ser His Asp Tyr Arg Lys Gly His Phe Glu Cys Pro Pro Tyr Asn Ser 130 135 140 Ser Thr Ile Arg Ser Ile Tyr His Val Arg Asn Ile Glu Val Tyr Lys 145 150 155 160 Leu Lys Gln Leu Lys Gln Pro Ile Asp Ile Phe Leu Ser His Asp Trp 165 170 175 Pro Arg Ser Ile Tyr His Tyr Gly Asn Lys Lys Gln Leu Leu Lys Thr 180 185 190 Lys Ser Phe Phe Arg Gln Glu Val Glu Asn Asn Thr Leu Gly Ser Pro 195 200 205 Ala Ala Ser Glu Leu Leu Glu His Leu Lys Pro Thr Tyr Trp Phe Ser 210 215 220 Ala His Leu His Val Lys Phe Ala Ala Leu Met Gln His Gln Ala Lys 225 230 235 240 Asp Lys Gly Gln Thr Ala Arg Ala Thr Lys Phe Leu Ala Leu Asp Lys 245 250 255 Cys Leu Pro His Arg Asp Phe Leu Gln Ile Leu Glu Ile Glu His Asp 260 265 270 Pro Ser Ala Pro Asp Tyr Leu Glu Tyr Asp Ile Glu Trp Leu Thr Ile 275 280 285 Leu Arg Ala Thr Asp Asp Leu Ile Asn Val Thr Gly Arg Leu Trp Asn 290 295 300 Met Pro Glu Asn Asn Gly Leu His Ala Arg Trp Asp Tyr Ser Ala Thr 305 310 315 320 Glu Glu Gly Met Lys Glu Val Leu Glu Lys Leu Asn His Asp Leu Lys 325 330 335 Val Pro Cys Asn Phe Ser Val Thr Ala Ala Cys Tyr Asp Pro Ser Lys 340 345 350 Pro Gln Thr Gln Met Gln Leu Ile His Arg Ile Asn Pro Gln Thr Thr 355 360 365 Glu Phe Cys Ala Gln Leu Gly Ile Ile Asp Ile Asn Val Arg Leu Gln 370 375 380 Lys Ser Lys Glu Glu His His Val Cys Gly Glu Tyr Glu Glu Gln Asp 385 390 395 400 Asp Val Glu Ser Asn Asp Ser Gly Glu Asp Gln Ser Glu Tyr Asn Thr 405 410 415 Asp Thr Ser Ala Leu Ser Ser Ile Asn Pro Asp Glu Ile Met Leu Asp 420 425 430 Glu Glu Glu Asp Glu Asp Ser Ile Val Ser Ala His Ser Gly Met Asn 435 440 445 Thr Thr Ile Gly Arg Ser Leu Leu Ile Lys Leu Leu Ser Phe Cys Ser 450 455 460 Phe Ser Asp Val Arg Ile Leu Pro Gly Ser Met Ile Val Ser Ser Asp 465 470 475 480 Asp Thr Val Asp Ser Thr Ile Asp Arg Glu Gly Lys Pro Gly Gly Leu 485 490 495 Val Glu Ser Gly Asn Gly Glu Asp Leu Thr Lys Val Pro Leu Lys Arg 500 505 510 Leu Ser Asp Glu His Glu Pro Glu Gln Arg Lys Lys Ile Lys Arg Arg 515 520 525 Asn Gln Ala Ile Tyr Ala Ala Val Asp Asp Asp Asp Asp Asp Ala Ala 530 535 540 47405PRTSaccharomyces cerevisiae 47Met Thr Lys Leu Arg Ile Ala Val Gln Gly Cys Cys His Gly Gln Leu 1 5 10 15 Asn Gln Ile Tyr Lys Glu Val Ser Arg Ile His Ala Lys Thr Pro Ile 20 25 30 Asp Leu Leu Ile Ile Leu Gly Asp Phe Gln Ser Ile Arg Asp Gly Gln 35 40 45 Asp Phe Lys Ser Ile Ala Ile Pro Pro Lys Tyr Gln Arg Leu Gly Asp 50 55 60 Phe Ile Ser Tyr Tyr Asn Asn Glu Ile Glu Ala Pro Val Pro Thr Ile 65 70 75 80 Phe Ile Gly Gly Asn His Glu Ser Met Arg His Leu Met Leu Leu Pro 85 90 95 His Gly Gly Tyr Val Ala Lys Asn Ile Phe Tyr Met Gly Tyr Ser Asn 100 105 110 Val Ile Trp Phe Lys Gly Ile Arg Ile Gly Ser Leu Ser Gly Ile Trp 115 120 125 Lys Glu Trp Asp Phe Asn Lys Gln Arg Pro Asp Trp Asn Asp Leu Glu 130 135 140 Asn Asn Asn Trp Lys Ala Asn Ile Arg Asn Leu Tyr His Val Arg Ile 145 150 155 160 Ser Asp Ile Ala Pro Leu Phe Met Ile Lys His Arg Ile Asp Ile Met 165 170 175 Leu Ser His Asp Trp Pro Asn Gly Val Val Tyr His Gly Asp Thr Lys 180 185 190 His Leu Leu Lys Leu Lys Pro Phe Phe Glu Gln Asp Ile Lys Glu Gly 195 200 205 Lys Leu Gly Ser Pro Val Thr Trp Gln Leu Leu Arg Asp Leu Arg Pro 210 215 220 Gln Trp Trp Leu Ser Ala His Leu His Val Arg Phe Met Ala Ser Ile 225 230 235 240 Lys His Asn Lys Arg Ser His Glu Pro Pro Asn Lys Ser Thr Ser Lys 245 250 255 Thr Lys Lys Asn Asn Asn Glu Ile Asp Leu Asp Leu Ser Ser Asp Glu 260 265 270 Asp Glu Arg Ser Gly Ile Met Asn Cys Gln Glu Glu Asn Glu Tyr Asp 275 280 285 Ser Lys Tyr Gly Glu Thr Arg Phe Leu Ala Leu Asp Lys Cys Leu Pro 290 295 300 Arg Arg Arg Trp Leu Glu Ile Leu Glu Ile Glu Pro Asp Thr Ser His 305 310 315 320 Ala Ser Trp Lys Asp Glu Asn His Arg Met Phe Trp Asp Pro Glu Phe 325 330 335 Ile Asn Asn Leu Val Ile Cys Gln Lys Asn Lys Asn Leu Leu Ser Asn 340 345 350 Lys Pro Phe Asn Ser Val Asn Trp Ile Glu Leu Ser Gln Ser Asn Arg 355 360 365 Glu Glu Gly Arg Asp Ile Asp Trp Glu Asn Tyr Ala Ile Pro Ala Tyr 370 375 380 Thr Leu Asp Ile Gln Lys Asp Glu Val Arg Gln Thr Lys Ala Phe Ile 385 390 395 400 Ser Lys Phe Met Thr 405 48479PRTCandida albicans 48Met Arg Asn Thr Ile Tyr Lys His Ser Ser Ile Val Thr Ala Met Ser 1 5 10 15 Asn Thr Leu Lys Ile Ala Ile Glu Gly Cys Cys His Gly Glu Leu Asn 20 25 30 Asp Ile Tyr Asn Ser Ile Pro Asp Ile Glu Ser Leu Asp Leu Leu Leu 35 40 45 Ile Cys Gly Asp Phe Gln Ser Leu Arg Asn Lys Cys Asp Leu Gln Ser 50 55 60 Leu Asn Val Pro Leu Lys Tyr Gln Arg Met Ala Asp Phe His Glu Tyr 65 70 75 80 Tyr Ser Gly Lys Arg Lys Ala Pro Val Leu Thr Ile Phe Ile Gly Gly 85 90 95 Asn His Glu Cys Ser Ser Tyr Leu Gln Glu Leu Lys Tyr Gly Gly Trp 100 105 110 Val Ala Pro Asn Ile Tyr Tyr Leu Gly Glu Phe Gly Ser Ile Trp Tyr 115 120 125 Lys Gly Leu Gln Ile Thr Gly Trp Ser Gly Ile Phe Asn Tyr His Thr 130 135 140 Phe Ile Ala Asn Asn Ile Glu Met Glu Lys Leu Pro Phe Asp Ser Arg 145 150 155 160 Thr Ile Arg Ser Val Tyr His Gln Lys Leu Ala Asn Phe Leu Lys Met 165 170 175 Tyr Met Met Asn His Asp Met Asp Ile Val Leu Ser His Asp Trp Pro 180 185 190 Val Gly Ile Glu Lys Tyr Gly Asn Val Lys Arg Leu Leu Lys Leu Lys 195 200 205 Pro Phe Phe Arg Asp Asp Ile Gln Arg Gly Gln Leu Gly Ser Pro Leu 210 215 220 Asn Lys Phe Leu Ile His Tyr Leu Arg Pro Arg Tyr Trp Phe Ser Gly 225 230 235 240 His Leu His Val Lys Phe Glu Ala Arg Ile Val Asp Leu Val Arg Ser 245 250 255 Thr Asp Lys Lys Lys Ser Ala Thr Thr Val Asp Leu Ile Thr Glu Ser 260 265 270 Asn Lys Glu Glu Ile Ser Leu Asp Met Asp Asp Glu Glu Glu Glu Glu 275 280 285 Gly Gly Asn Val Arg Glu Val Ser Phe Glu Glu Lys Phe Tyr Phe Lys 290 295 300 Gln His Ser Asn Pro Ala Lys Arg Pro Lys Asn Asp Leu Thr Pro Glu 305 310 315 320 Arg Asp Val Cys Glu His Ala Thr Glu Phe Leu Ala Leu Asp Lys Cys 325 330 335 Gly Lys Arg Arg Gln Phe Leu Asp Ile Lys Thr Ile Glu Val His Asn 340 345 350 Thr Ser His Pro Ser Phe Ile Asn Ala Gly Lys Leu Tyr Tyr Ser Lys 355 360 365 Arg Ser Ile Ala Ile Asn Lys Val Val Glu Lys Tyr Leu Asn Asp Asn 370 375 380 Arg Gln Asp Phe Thr Glu Leu Asn Thr Lys Gln Ile Leu Ser Asn Pro 385 390 395 400 Gln Gln Phe Pro Leu Val Asn Glu Leu Met Pro Ile Ile Glu Asn Asp 405 410 415 Phe Lys Ser Met Gln Lys Asn Ile Thr Asp Glu Asp Phe Phe Met Val 420 425 430 Pro Glu Asn Phe Gln Thr Ile Ala Pro Thr Asp Asp Glu His Thr Glu 435 440 445 Ser Lys Leu Lys Tyr Tyr Pro Asn Asn Gln Thr Ser Glu Tyr Cys Glu 450 455 460 Lys Phe Gly Ile Pro Lys Leu Val Leu Ser Lys Glu Ser Asp Gln 465 470 475 49405PRTCandida glabrata 49Met Asn Glu Arg Lys Leu Arg Ile Ala Val Gln Gly Cys Cys His Gly 1 5

10 15 Glu Leu Asn Lys Val Phe Ala Thr Val Ala Asp Met His Lys Arg Thr 20 25 30 Pro Ile Asp Leu Leu Ile Ile Leu Gly Asp Phe Gln Ser Ile Arg Asp 35 40 45 Asn Ser Asp Phe Gln Ser Ile Ser Ile Pro Pro Lys Tyr Gln Lys Leu 50 55 60 Gly Asp Phe His Ala Tyr Tyr Glu Asn Asp Tyr Tyr Arg Ala Pro Val 65 70 75 80 Phe Thr Ile Val Ile Gly Gly Asn His Glu Ser Met Arg His Leu Met 85 90 95 Gln Leu Pro Tyr Gly Gly Tyr Leu Ala Asn Asn Ile Tyr Tyr Met Gly 100 105 110 Tyr Ser Gly Val Val Trp Phe Lys Gly Phe Arg Ile Ala Ala Leu Ser 115 120 125 Gly Ile Trp Lys Glu Trp Asp Phe Glu Lys Lys Arg Pro Ser Trp Lys 130 135 140 Phe Leu Glu Glu Asn Asn Lys Trp Lys Asp Ser Val Arg Gln Leu Tyr 145 150 155 160 His Ile Arg Lys Asp Asp Val Ala Pro Leu Phe Ala Leu Ser Asp Asn 165 170 175 Ile Asp Ile Cys Leu Ser His Asp Trp Pro Ser Gly Val Val His Tyr 180 185 190 Gly Asn Val Lys Gln Leu Leu Lys Tyr Lys Pro Phe Phe Glu Lys Asp 195 200 205 Ile Lys Ser Gly Lys Leu Gly Asn Pro Ile Ala Trp Lys Leu Leu Thr 210 215 220 Asn Leu Lys Pro Arg Trp Trp Phe Ser Ala His Leu His Val Lys Tyr 225 230 235 240 Glu Ala Glu Ile Thr His Asn Lys Arg Arg Leu Ala Asp Ser Lys Gly 245 250 255 Ala Lys Lys Leu Lys Ser Asn Ser Asp Glu Ile Glu Leu Asn Leu Asp 260 265 270 Asp Glu Ser Ser Leu Asp Leu Ser Cys His Asp Asp Ser Leu Asp Ser 275 280 285 Ala Glu His Thr Arg Phe Leu Ser Leu Asp Lys Cys Met Pro Arg Arg 290 295 300 Lys Trp Leu Glu Ile Val Glu Ile Glu Lys Arg Tyr Asp Ser Ile Pro 305 310 315 320 Gln Gly Leu Asp Cys Asp Lys Met Tyr Trp Asp Pro Ser Tyr Ile Ile 325 330 335 Ala Leu Gln Asn Leu Glu Lys Gln Ser Arg Leu Val Ala Asp Thr Pro 340 345 350 Phe Asn Glu Ile Ile Trp Ser Arg Phe Ser Ser Gly His Ile Asp Asp 355 360 365 Ile Asn Trp His Lys Tyr Glu Ile Pro Lys Tyr Glu Ser Gly Leu Gln 370 375 380 Arg Asp Glu Ala Ser Gln Thr Asn Tyr Phe Leu Ser Lys His Met Leu 385 390 395 400 Ser Lys Gly Ser Arg 405 50417PRTCandida parapsilosis 50Met Lys Thr Leu Lys Val Ala Ile Glu Gly Cys Cys His Gly Asp Leu 1 5 10 15 Asn Lys Ile Tyr Lys Gly Ile Pro Ser Ser Thr Glu Leu Leu Leu Ile 20 25 30 Cys Gly Asp Phe Gln Ala Leu Arg Asn Thr Ser Asp Tyr Gln Ala Leu 35 40 45 Ser Val Pro Glu Lys Tyr Arg Arg Leu Gly Asp Phe Gln Ser Tyr Tyr 50 55 60 Thr Ser Lys Lys Lys Ala Pro Val Leu Thr Ile Phe Ile Gly Gly Asn 65 70 75 80 His Glu Ser Ser Ser Tyr Leu Gln Glu Leu Lys Tyr Gly Gly Trp Val 85 90 95 Ala Pro Asn Ile Tyr Tyr Leu Gly Glu Phe Gly Ser Val His Tyr Lys 100 105 110 Gly Leu Ser Ile Cys Gly Trp Ser Gly Ile Tyr Asn Pro His Thr Tyr 115 120 125 Met Asn Lys Ser Phe Asn Val Glu Arg Leu Pro Phe Asp Ser Asn Ser 130 135 140 Ile Arg Ser Val Tyr His Gln Lys Leu Ser Asn Phe Leu Lys Met Tyr 145 150 155 160 Leu Gln Arg Asp Met Asp Ile Val Leu Ser His Asp Trp Pro Val Gly 165 170 175 Ile Glu Lys Phe Gly Asp Lys Tyr Arg Leu Leu Lys Gln Lys Pro Phe 180 185 190 Phe Thr Gln Asp Ile Lys Lys Gly Gln Leu Gly Ser Pro Leu Asn Asn 195 200 205 Val Leu Leu His His Leu Lys Pro Arg Tyr Trp Phe Ser Gly His Leu 210 215 220 His Val Lys Phe Lys Ala Asn Val Asn His Asn Ile Ser Lys Pro Lys 225 230 235 240 Gln Val Lys Asn Ala Asn Glu Ile Leu Leu Asp Met Glu Ser Ser Asp 245 250 255 Glu Ala Ser Asp Gly Glu Asn Gln Pro Gln Lys Lys Met Lys Pro Asn 260 265 270 Gly His Val Val His Asp Thr Gln Phe Leu Ala Leu Asp Lys Tyr Gly 275 280 285 Pro Arg Arg Ser Tyr Phe Glu Val Ile Asn Ile Pro Ile Leu Glu Asn 290 295 300 Asn His Pro Ser Val His Asp Asp Gly Leu Tyr Tyr Asp Lys Arg Ala 305 310 315 320 Ile Ala Ile Asn Arg Val Val Glu Lys Tyr Arg Ile Asp Gln Lys Thr 325 330 335 Glu Phe Glu Ser Met Ser Pro Arg Glu Ile Leu Arg Asp Pro Arg Lys 340 345 350 Leu Glu Lys Phe Ile Pro Leu Val Ala Lys Glu Ser Glu Glu Ile Asn 355 360 365 Gln Ile Asp Asp Asn Gln Phe Val Ile Pro Lys Asn Phe Glu Val Val 370 375 380 Ala Pro Ala Asp Tyr Asp Gly Glu Leu Lys Tyr Tyr Pro Asn Ala Gln 385 390 395 400 Thr Glu Glu Tyr Cys Arg Lys Phe Gly Ile Pro Gln Gln Asp Tyr Gln 405 410 415 Leu 51746PRTHistoplasma capsulatum 51Met Gln His Ile Pro Ala Ser Pro Ser Arg Asn Leu Arg Val Ala Leu 1 5 10 15 Glu Gly Cys Lys Tyr Lys Gln Ile Gly Asp Phe His Glu Tyr Tyr Ser 20 25 30 Gly Ala Arg Val Ala Pro Tyr Leu Thr Ile Phe Val Gly Gly Asn His 35 40 45 Glu Ala Ser Asn His Leu Phe Glu Leu Tyr Tyr Gly Gly Trp Val Ala 50 55 60 Pro Asn Ile Tyr Tyr Leu Gly Ala Ala Asn Val Ile Arg Cys Gly Pro 65 70 75 80 Leu Arg Ile Ala Gly Ile Ser Gly Ile Trp Lys Gly Tyr Asp Tyr Arg 85 90 95 Lys Ser His Phe Glu Arg Leu Pro Tyr Asn Arg Ala Asp Met Gln Ser 100 105 110 Ile Tyr His Val Arg Glu Leu Asp Val Arg Lys Leu Leu Gln Ile Arg 115 120 125 Thr Gln Val Asp Leu Gly Leu Ser His Asp Trp Pro Gln Gly Ile Glu 130 135 140 Trp His Gly Asp Phe Gln Lys Leu Phe Gln Lys Lys Pro Leu Phe Glu 145 150 155 160 Pro Asp Ala Asn Ser Gly Arg Leu Gly Ser Val Ala Ala Arg Tyr Ile 165 170 175 Met Asp Arg Leu Arg Pro Ala Phe Trp Phe Ser Ala His Leu His Cys 180 185 190 Lys Tyr Ala Ala Ser Leu Thr His Gly Asp Tyr Lys Pro Ala Glu Leu 195 200 205 Lys Asn Arg Phe Asn Pro Asn Pro Gln Pro His His Gln Pro Gln Gln 210 215 220 Pro Ser Leu Gly Asp Asp Val Leu Ser Ser Pro Ser Leu Val Thr Asn 225 230 235 240 Glu Gln Pro Glu Val Pro Gly Ser Thr Asn Glu Gly Val Thr Ala Thr 245 250 255 Arg Ser Val Glu Val Gly Ser Asp Ala Ala His Ile Ser Ser Lys Glu 260 265 270 Val Ser Thr Thr Ile Val Asp Thr Ala Met Ser Glu Glu Ile Ile Met 275 280 285 Ser Thr Leu Gly Gly Asp Asp Glu Ala Thr Thr Arg Ala Ala Glu Ser 290 295 300 Ala Lys Asn Ala Pro Gln Pro Gln Pro Ala Gln Gly Ala Glu Arg Asp 305 310 315 320 Arg Ala Gln Leu Ser Ala Trp Gln Asn Phe His Ser Val Ala Thr Lys 325 330 335 Asn Asp Ala Glu Glu Asn Val Arg Leu Met Lys Glu Ala Ala Glu Tyr 340 345 350 Glu Lys Gln Ile Glu Ala Gly Leu Ile Ser Arg Pro Glu Val Asn Tyr 355 360 365 Gln Leu Thr Trp Lys Lys Val Ala Val Lys Asp Asp Asn Leu Gly Arg 370 375 380 Glu Ile Ala Gly Val Ala Lys Ile Gly Tyr Asn Ala Gln Gln Glu Leu 385 390 395 400 Arg Glu Ile Thr Glu Gln Glu Ile Arg Asp Gly Gly Thr Glu Val Lys 405 410 415 Asn Pro Asp Glu Ile Asp Ile Cys Leu Asp Ser Ser Ser Asp Thr Ser 420 425 430 Glu Lys Leu Glu Gln Lys Asp Thr Ile Ser Thr Lys Thr Arg Ser Thr 435 440 445 Asp Lys Met Glu Ile Glu His Ser Ile Ser Ser Gly Lys Ala Glu Lys 450 455 460 Thr Ala Thr Ala Ala Val Asp Ala Ala Glu Ala Ser Gln Ser Gly Asp 465 470 475 480 Ile Pro Lys Glu Ile Arg Asp Gln Leu Pro Ala Ser Phe Arg Lys Pro 485 490 495 Glu Thr Ile Leu Asp Asp Ala Pro Val Phe Glu Ser Thr Leu Pro Glu 500 505 510 Ala Ile Ser Asn Thr Glu Thr Asn Phe Leu Ala Leu Asp Lys Cys Asp 515 520 525 Arg His Arg Gln Phe Ile Glu Leu Val Glu Tyr Pro Ala Ile Ser Ser 530 535 540 Pro Glu Glu Gly Glu Thr Gly Glu Glu Ser Arg Pro Tyr Gln Leu Lys 545 550 555 560 Tyr Asp Lys Glu Trp Leu Ala Ile Thr Arg Ala Phe Ala Asp Glu Leu 565 570 575 Thr Leu Gly Asp Pro Asn Ala Ser Val Pro Thr Asn Lys Gly Asp Ala 580 585 590 Arg Tyr Lys Pro Ser Ile Leu Ala Ala Glu Gln Trp Val Glu Glu Asn 595 600 605 Val Val Lys Pro Gly Arg Met Thr Ile Pro His Asn Phe Ser Ile Thr 610 615 620 Ala Pro Val Tyr Asp Pro Ala Val Pro Ile Thr Thr Thr Glu Met Pro 625 630 635 640 Pro Glu Tyr Thr Asn Pro Gln Thr Ala Gln Phe Cys Asp Leu Ile Gly 645 650 655 Ile Glu Asn Lys Phe His Ala Ser Asp Glu Glu Arg Phe Ala Arg Ala 660 665 670 Asp Ala Gly Pro His Pro Glu Pro Leu Gln Gln Arg His Gly Gln Arg 675 680 685 Phe Arg Gly His Gln Asp Arg Ser Phe Asn Ser Leu Gly Arg Gly Arg 690 695 700 Gly Arg Gly Phe Gly Arg Asp Gly Gly Arg Trp Gln Gly Gly Arg Gly 705 710 715 720 Gly Arg Gly Gly Gly Gly Arg Asn Arg Ala Gly Arg Gly Gly Arg Gly 725 730 735 Gly Arg Gly Glu Tyr Tyr Gly Ala Pro Ile 740 745 52795PRTBlastomyces dermatitidis 52Met Gln His Ile Pro Thr Pro Pro Ser Arg Ser Leu Arg Val Ala Leu 1 5 10 15 Glu Gly Cys Gly His Gly Lys Leu Asn Asp Ile Tyr Thr Ser Val Thr 20 25 30 Arg Ala Ala Glu Ile Lys Gly Trp Asp Gly Val Asp Leu Leu Ile Ile 35 40 45 Gly Gly Asp Phe Gln Ala Val Arg Asn Ser Tyr Asp Leu Ser Cys Met 50 55 60 Ser Val Pro Gln Lys Tyr Arg Gln Ile Gly Asp Phe His Glu Tyr Tyr 65 70 75 80 Ser Gly Ala Arg Val Ala Pro Tyr Leu Thr Ile Phe Val Gly Gly Asn 85 90 95 His Glu Ala Ser Asn His Leu Phe Glu Leu Tyr Tyr Gly Gly Trp Val 100 105 110 Ala Pro Asn Ile Tyr Tyr Leu Gly Ala Ala Asn Val Ile Arg Cys Gly 115 120 125 Pro Leu Arg Ile Ala Gly Ile Ser Gly Ile Trp Lys Gly Tyr Asp Tyr 130 135 140 Arg Lys Pro His Phe Glu Arg Leu Pro Tyr Asn Arg Ser Asp Ile Gln 145 150 155 160 Ser Ile Tyr His Val Arg Glu Leu Asp Val Arg Lys Leu Leu Gln Ile 165 170 175 Arg Thr Gln Val Asp Leu Gly Leu Ser His Asp Trp Pro Gln Gly Ile 180 185 190 Glu Trp His Gly Asp Phe Gln Lys Leu Phe Gln Lys Lys Pro Leu Phe 195 200 205 Glu Pro Asp Ala Asn Ser Gly Arg Leu Gly Ser Val Ala Ala Arg Tyr 210 215 220 Val Leu Asp Arg Leu Arg Pro Pro Tyr Trp Phe Ser Ala His Leu His 225 230 235 240 Cys Lys Tyr Thr Ala Asn Leu Ile His Gly Asp Tyr Lys Pro Ala Gly 245 250 255 Leu Lys Asp Arg Phe Ala Ser Asn Gln Gln Pro His Gln Pro Gln Leu 260 265 270 Gly Gly Asp Ala Gly Ala Ser Pro Ser Ile Ile Ala Asp Glu Pro Ala 275 280 285 Glu Val Ser Gly Ser Thr Asn Glu Gly Ile Arg Ser Thr Pro Val Val 290 295 300 Glu Glu Val Ser Asn Ala Leu Arg Ile Ser Pro Asn Glu Pro Ser Thr 305 310 315 320 Thr Ile Thr Gly Thr Ala Met Arg Glu Glu Thr Ile Leu Ser Thr Pro 325 330 335 Gly Gly Glu Thr Glu Pro Thr Thr Leu Ser Thr Glu Gly Ile Glu Ser 340 345 350 Ile Pro Gln Gln Gln Asn Pro Thr Gln Arg Ala Glu Arg Glu Arg Ala 355 360 365 Gln Leu Ser Ala Trp Gln Asn Phe His Ser Val Ala Thr Lys Asn Asp 370 375 380 Ala Glu Glu Gly Ala Arg Leu Met Glu Glu Ala Ala Asp Tyr Glu Lys 385 390 395 400 Gln Val Glu Ala Gly Phe Ile Ser Arg Pro Glu Val Asn Tyr Gln Leu 405 410 415 Thr Trp Lys Lys Ile Gly Val Lys Asp Asp Gly Leu Gln Arg Glu Ile 420 425 430 Glu Asp Val Ala Lys Ile Gly Tyr Asp Ser Gln Glu Pro Gly Lys Val 435 440 445 Thr Glu Gln Glu Glu Arg Gly Gly Val Thr Thr Val Arg Asn Thr Asp 450 455 460 Glu Ile Asp Ile Ser Leu Asp Ser Ser Ser Glu Thr Ser Glu Lys Leu 465 470 475 480 Glu Gln Glu Asp Thr Thr Ser Ala Ile Pro Lys Asn Thr Asp Val Met 485 490 495 Glu Ile Asp Ser Gly Thr Ser Phe Glu Lys Ala Glu Val Ala His Ile 500 505 510 Gln Thr Pro Thr Ala Thr Ala Asn Ala Thr Gly Ala Leu Gln Leu Asp 515 520 525 Asp Ile Pro Ala Asp Ile Leu Asp Gln Leu Pro Ala Ser Phe Arg Lys 530 535 540 Pro Gln Pro Val Pro Gly His Thr Pro Ile Phe Glu Pro Thr Leu Pro 545 550 555 560 Glu Ala Ile Lys Asn Thr Val Thr Glu Phe Leu Ala Leu Asp Lys Cys 565 570 575 Glu Thr Arg Arg Gln Phe Ile Glu Leu Val Glu Tyr Ser Ala Ile Ser 580 585 590 Ser Pro Glu Glu Glu Glu Ile Gly Glu Glu Ser Arg Pro Tyr Gln Leu 595 600 605 Lys Tyr Asp Lys Glu Trp Leu Ala Ile Thr Arg Ala Phe Ala Asp Glu 610 615 620 Leu Ile Leu Gly Asp Pro Asn Ala Ser Val Pro Pro Asn Lys Gly Asp 625 630 635 640 Ala Gly Tyr Lys Pro Asp Ile Leu Ala Ala Ala Gln Trp Val Glu Glu 645 650 655 Asn Ile Val Lys Pro Gly Arg Met Thr Ile Pro His Asn Phe Ser Ile 660 665 670 Thr Ala Pro Val Tyr Asp Pro Ala Ile Ser Ile Met Thr Thr Glu Met 675 680 685 Pro Pro Glu Cys Thr Asn Pro Gln Thr Ala Arg Phe Cys Glu Leu Val 690 695 700 Gly Ile Glu Asn Lys Phe His Ala Ser Asp Glu Glu Arg Phe Ala Arg 705 710 715 720 Ala Asp Ala Gly Pro Arg Pro Glu Pro Pro Gln Ser Arg Tyr Gly Gln 725 730

735 Arg Ser Arg Gly His Gln Asp Gly Ser Val His Ser Phe Gly Arg Gly 740 745 750 Glu Gly Glu Gly Gly Glu Gly Gly Gly Gly Arg Val Gly Val Gly Lys 755 760 765 Val Val Val Glu Gly Gly Glu Val Glu Ala Gly Glu Gly Val Glu Gly 770 775 780 Glu Val Ala Asn Trp Asp Cys Val Leu Arg Phe 785 790 795 53785PRTAspergillus fumigatus 53Met Glu Val Ser Ala Ala Asn Pro Ala Ser Leu Arg Val Ala Phe Glu 1 5 10 15 Gly Cys Gly His Gly Arg Leu Asp Asp Ile Tyr Asp Ser Val Thr Arg 20 25 30 Ser Ala Thr Arg Arg Gly Trp Asp Gly Val Asp Leu Val Val Ile Gly 35 40 45 Gly Asp Phe Gln Ala Val Arg Asn Ser Asn Asp Leu Ala Cys Met Ser 50 55 60 Val Pro Gln Lys Tyr Lys Ala Ile Gly Asp Phe His Glu Tyr Tyr Ser 65 70 75 80 Gly Lys Lys Thr Ala Pro Tyr Leu Thr Ile Phe Ile Gly Gly Asn His 85 90 95 Glu Ala Ser Asn Tyr Leu Phe Glu Leu Tyr Tyr Gly Gly Trp Val Ala 100 105 110 Pro Asn Ile Tyr Tyr Leu Gly Ala Ala Asn Val Ile Arg Cys Gly Pro 115 120 125 Leu Arg Ile Ala Gly Leu Ser Gly Ile Trp Lys Gly Tyr Asp Tyr Arg 130 135 140 Lys Pro His Phe Glu Arg Leu Pro Tyr Asn Asn Asp Asp Val Gln Ser 145 150 155 160 Ile Tyr His Val Arg Glu Leu Asp Val Arg Lys Leu Leu Gln Ile Arg 165 170 175 Thr Gln Val Asp Leu Gly Leu Ser His Asp Trp Pro Asn Arg Val Glu 180 185 190 Leu Cys Gly Asp His Glu Thr Leu Phe Ala Lys Lys His Gly Phe Arg 195 200 205 Glu Asp Ser Asn Asn Gly Arg Leu Gly Ser Ile Ala Ala Arg Phe Val 210 215 220 Leu Asp Arg Leu Arg Pro Ala Phe Trp Phe Ser Ala His Leu His Val 225 230 235 240 Lys Phe Asn Ala Val Val Gln His Gly Asp Asn Leu Gln Pro Asp Ser 245 250 255 Leu Gly Pro Thr Arg His Ile Ala Ser Ser Gln Arg Thr Ser Ser Asn 260 265 270 Ala Ser Thr Leu Thr Thr Ser Phe Gly Met Asp Gly Ala Ala Val Thr 275 280 285 Ser Leu Val Leu Gly Asp Glu Asp Met Pro Thr Glu Gln Ala Gln Val 290 295 300 Pro His Asn Phe Ser Glu Asn Lys Gly His Ala Ala Asn Thr Leu Gly 305 310 315 320 Glu Asp Glu Arg Leu Glu Glu Pro Pro Arg Glu Leu Pro Thr Ala Gln 325 330 335 Ala Thr Gln Gln Ser Asn Leu Val Gly Leu Ala Arg Thr Ser Ser Pro 340 345 350 Leu Lys Arg Val His Asp Asp Asn Gln Ser Arg Ile Ser Ala Trp Asn 355 360 365 Asn Phe His Ala Val Ala Ala Arg Asp Glu Ala Ala Glu Asn Ala Pro 370 375 380 Arg Leu Glu Glu Ser Gln Asp Asn Ser Ala Ser Gln Leu Pro His Ser 385 390 395 400 Leu Thr Trp Arg Lys Ile Ser Val Asp Glu Asp Asp Pro Val Arg Lys 405 410 415 Val Thr Thr Val Glu Lys Pro Ala Asp Glu Asn Glu Ser Glu Thr Lys 420 425 430 Lys Gln Lys Thr Gly His Ala Val Ser Ala Thr Lys Asn Ser Asp Glu 435 440 445 Ile Pro Leu Asp Leu Asp Ser Asp Ser Asp Gln Gly Ile Ser Thr Thr 450 455 460 Ala Glu Thr Gln Gly Ala Thr Gln Lys Gln Asn Ala Val Val Thr Gln 465 470 475 480 Pro Ala Ala Pro Asp Val Thr Gly Glu Ser Lys Leu Ser Gly Thr Thr 485 490 495 Gln Gln Asp Lys Pro Gln Val Arg Ser Leu Asp Ser Gln Asp Val Arg 500 505 510 Asn Leu Leu Pro Thr Ser Phe Ser Gln Pro Glu Ser Phe Val Ser Gln 515 520 525 Asp Val Arg Asn Gln Leu Pro Ala Ser Phe Ser Arg Val Asp Cys Pro 530 535 540 Val Ser Gln Asp Val Arg Asn Gln Leu Pro Ser Ser Phe Ser Arg Pro 545 550 555 560 Gln Ala Thr Pro Lys Leu Asp Pro Ser Val Ser Glu Pro Val Pro Glu 565 570 575 Thr Ile Thr Asn Lys Thr Thr Arg Phe Leu Ala Leu Asp Lys Cys Glu 580 585 590 Pro Lys Arg His Phe Leu Glu Leu Leu Glu Ile Pro Ile Val Ser Glu 595 600 605 Gln Asn Gly Ser Gln Arg Thr Arg Pro Phe Arg Leu Glu Tyr Asp Lys 610 615 620 Glu Trp Leu Ala Ile Thr Arg Val Phe Ala Asp Glu Leu Gln Leu Gly 625 630 635 640 Asp Leu Ala Val Gln Met Gln Pro Asp Arg Gly Gln Ala Phe Tyr Lys 645 650 655 Pro Leu Ile Glu Glu Ala Glu Gln Trp Val Glu Glu Asn Val Val Lys 660 665 670 Ala Gly Lys Met Met Val Pro Glu Asn Phe Thr Pro Thr Ala Pro Phe 675 680 685 Phe Asp Pro Ala Val Pro Ile Thr Thr Asp Glu Leu Pro Pro Glu Phe 690 695 700 Thr Asn Pro Gln Thr Ala Gln Phe Cys Glu Leu Ile Gly Ile Glu Asn 705 710 715 720 Lys Phe His Leu Ser Asp Glu Glu Arg Gln Ala Arg Val Glu Ala Gly 725 730 735 Pro Arg Pro Asn Lys Pro Lys Pro Glu Gly Gly Trp Asn Arg Gly Arg 740 745 750 Arg Arg Asn Tyr Asn Asn Asn Asn Arg Gly Gly Gly Ser Gln Trp Trp 755 760 765 Gly Arg Gly Ala Gly Arg Asp Arg Gly Arg Ser Gly Gly Asn Gln Arg 770 775 780 Trp 785 54606PRTCryptococcus neoformans 54Met Arg Ile Ala Ile Gln Gly Cys Ser His Gly Ser Leu Ala Gln Ile 1 5 10 15 Tyr Asp Val Val Asn Tyr Tyr Ser Ser Gln Thr Lys Asn Pro Ile Asp 20 25 30 Leu Leu Leu Leu Cys Gly Asp Phe Gln Ala Leu Arg Ser Lys His Asp 35 40 45 Tyr Ala Ser Leu Ala Val Pro Ala Lys Phe Lys Gln Leu Gly Ser Phe 50 55 60 His Gln Tyr Tyr Ser Gly Glu Arg Val Ala Pro Val Leu Thr Ile Val 65 70 75 80 Ile Gly Gly Asn His Glu Ala Ser Asn Tyr Met Trp Glu Leu Tyr His 85 90 95 Gly Gly Trp Leu Ala Pro Ser Ile Tyr Tyr Leu Gly Ala Ala Gly Ser 100 105 110 Val Tyr Val Asn Gly Leu Arg Ile Val Gly Ala Ser Gly Ile Tyr Lys 115 120 125 Gly Phe Asp Tyr Arg Lys Gly His Phe Glu Lys Val Pro Tyr Asn Asp 130 135 140 Lys Glu Leu Arg Ser Ile Tyr His Ile Arg Glu Tyr Asp Val Glu Lys 145 150 155 160 Leu Met His Leu Thr Pro Ser Pro Ser Thr Ile Phe Leu Ser His Asp 165 170 175 Trp Pro Thr Thr Ile Ala His His Gly Asn Lys Asn Ala Leu Leu Lys 180 185 190 Arg Lys Pro Phe Phe Arg Asp Glu Ile Glu Lys Asn Thr Leu Gly Ser 195 200 205 Pro Pro Leu Leu Arg Leu Met Asn His Phe Gln Pro Ser Tyr Trp Phe 210 215 220 Ser Ala His Leu His Val Lys Phe Ala Ala Leu Tyr Glu His Gln Ala 225 230 235 240 Pro Ser His Gly Pro Asp Val Asp Gly Gly Ala Pro Leu Pro Leu Pro 245 250 255 Ala Met Ser Thr Ala Ile Ala Gln Thr Gly Asn Asn Pro Asp Glu Ile 260 265 270 Gln Ile Asp Glu Glu Met Asp Glu Gly Asn Pro Asp Glu Ile Ile Val 275 280 285 Glu Asp Glu Gly Glu Glu Ile Ile Val Arg Pro Arg Gln Val Asn Pro 290 295 300 Asp Glu Ile Val Met Asp Asp Glu Glu Phe Asp Asp Pro Pro Pro Ala 305 310 315 320 Val Pro Gln Pro Leu Pro Ile Thr Thr Ser Ser Val Val Asn Pro Glu 325 330 335 Glu Ile Thr Ile Ser Asp Glu Glu Phe Asp Ala Pro Met Ala Val Ser 340 345 350 Gln Ser Pro Gln Pro Leu Pro Pro Thr Arg Ala Asn Ala Ser Asn Pro 355 360 365 Glu Glu Ile Ala Ile Ser Asp Asp Glu Phe Asp Asp Pro Ala Pro Val 370 375 380 Ala Gln Pro Leu Thr Ala Ile Asp Glu Ser Thr Asp Leu Ile Ala Gln 385 390 395 400 Ser Arg Ser Asn Pro Ser His Pro His Val Ala Gly Thr Ile Ala Pro 405 410 415 Pro Ala Ser Asp Ser Thr Ala Pro Arg Val Met Gln Glu Ala Arg Gln 420 425 430 Glu Gln Gln Lys Trp Glu Leu His Gly Gly Lys Gly Met Glu Gly Val 435 440 445 Thr Lys Phe Leu Ala Leu Asp Lys Cys Gly Pro Gly Lys Asp His Met 450 455 460 Gln Phe Leu Glu Ile Pro Asp Pro Ser Pro Pro Ala Ile Pro Gly Pro 465 470 475 480 Pro Arg Leu Thr Tyr Asp Pro Glu Trp Leu Ala Ile Ser Arg Ala Phe 485 490 495 His Pro Tyr Leu Ser Thr Ser Tyr Gln Pro Ile Pro Leu Pro Ser Pro 500 505 510 Asp Val Leu Glu Gln Met Val Lys Asp Glu Val Thr Arg Ile Lys Glu 515 520 525 Glu Gly Leu Leu Val Pro Ala Val Pro Glu Lys Gly Ala Val Glu Gly 530 535 540 Gln Glu Gly Leu Val Trp Glu Lys Gly Lys Val Asp Val Gly Arg Val 545 550 555 560 Gln Arg Phe Trp Trp Thr Ala Pro Pro Glu Gly His Pro Gly Gly Asn 565 570 575 Asp Ala Ala Trp Tyr Thr Asn Pro Gln Thr Glu Ala Phe Cys Gly Met 580 585 590 Leu Gly Val Gln Asn Lys Ile Asn Pro Pro Val Asn Arg Ser 595 600 605 55340PRTRhizopus delemar 55Met Met His Gln Lys Ile Ala Ile Glu Gly Cys Cys His Gly Glu Leu 1 5 10 15 Asp Lys Ile Tyr Asn Ala Val Arg Glu Glu Glu Ala Arg Tyr Gly Gln 20 25 30 Lys Val Asp Leu Val Leu Ile Cys Gly Asp Phe Gln Ala Leu Arg Asn 35 40 45 Glu Ser Asp Leu Ala Cys Met Ala Val Pro Asp Lys Phe Lys Thr Met 50 55 60 Gly Thr Phe Trp Lys Tyr Tyr Ser Gly Gln Ala Arg Ala Pro Tyr Pro 65 70 75 80 Thr Ile Phe Ile Gly Gly Asn His Glu Ala Ser Asn Tyr Leu Trp Glu 85 90 95 Leu Tyr His Gly Gly Trp Val Cys Asp Asn Ile Tyr Tyr Leu Gly Cys 100 105 110 Ala Gly Val Ile Asn Phe Gly Gly Leu Arg Ile Gly Gly Leu Ser Gly 115 120 125 Ile Tyr Lys Gln Asn Asp Tyr His Ile Gly His His Glu Thr Val Pro 130 135 140 Tyr Asn Ser Ser Glu Met Arg Ser Ile Tyr His Val Arg Glu Tyr Asp 145 150 155 160 Val Arg Lys Leu Leu Gln Val Gln Glu Pro Ile Asp Ile Phe Leu Ser 165 170 175 His Asp Trp Pro Arg Gly Ile Glu Arg Tyr Gly Asp Val Leu Ser Asn 180 185 190 Ser Leu Gly Ser Ser Pro Asn Glu Val Leu Leu Tyr Asn Leu Lys Pro 195 200 205 Ala Arg Trp Phe Ala Ala His Leu His Val Arg Tyr Glu Ala Glu Ile 210 215 220 Asn His Glu Lys Lys Asp Glu Tyr Ser Val Ser Ala Arg Glu Leu Leu 225 230 235 240 Gly Arg Lys Gly Ala Asn Lys Ile Arg Asn Ser Asp Glu Ile Gln Ile 245 250 255 Asp Asp Asp Ser Glu Asp Ile Asn Ala Val Ser Ser Ser Ser Pro Thr 260 265 270 Asn Asp Val Asp Asn Ser Lys Val Val Ser Lys Thr Thr Lys Phe Leu 275 280 285 Ser Leu Asp Lys Cys Leu Pro Arg Arg Gln Phe Leu Glu Glu Gln Arg 290 295 300 Lys Cys Gly Ile Leu Asp Leu Lys Ile Pro His Asn Phe Glu Pro Thr 305 310 315 320 Ala Pro Val Tyr Ile Pro Lys Lys Gln Lys Gly Arg His Ile Phe Lys 325 330 335 Gln Pro Arg Met 340 56706PRTCoccidioides immitis 56Met Ala Ser Asp Leu Pro Thr Gln Lys Gly Phe Arg Leu Ala Ile Glu 1 5 10 15 Gly Cys Gly His Gly Lys Leu His Glu Ile Tyr Asp Ser Val Lys Lys 20 25 30 Ser Ala Glu Ala Lys Gly Trp Asp Gly Val Asp Leu Val Ile Ile Gly 35 40 45 Gly Asp Phe Gln Ala Val Arg Asn Ser Asn Asp Met Ala Cys Met Ala 50 55 60 Val Pro Ala Lys Tyr Lys Lys Ile Gly Asp Phe His Glu Tyr Tyr Ser 65 70 75 80 Gly Ala Arg Val Ala Pro Tyr Leu Thr Ile Phe Val Gly Gly Asn His 85 90 95 Glu Ala Ser Asn His Leu Phe Glu Leu Tyr Tyr Gly Gly Trp Val Ala 100 105 110 Pro Asn Ile Tyr Tyr Leu Gly Ala Ala Asn Val Ile Arg Cys Gly Pro 115 120 125 Leu Arg Ile Ala Gly Met Ser Gly Ile Trp Lys Gly Tyr Asp Tyr Arg 130 135 140 Arg Gln His Phe Glu Arg Leu Pro Tyr Gly Asp Asp Ala Leu Arg Ser 145 150 155 160 Ile Tyr His Val Arg Glu Ile Asp Val Arg Lys Leu Leu Gln Val Arg 165 170 175 Thr Gln Val Asp Ile Gly Ile Ser His Asp Trp Pro Gln Ala Ile Glu 180 185 190 Trp Thr Gly Asp Val Asp Asp Leu Phe Arg Arg Lys Pro His Phe Val 195 200 205 Lys Asp Ala Glu Ser Gly Lys Leu Gly Ser Pro Ala Val Arg Tyr Val 210 215 220 Leu Asp Arg Leu Arg Pro Ala His Trp Phe Ser Ala His Leu His Val 225 230 235 240 Lys Tyr Thr Ser Thr Leu Glu His Lys Ala Tyr Ser Pro Pro Arg Ala 245 250 255 Val Asn Ala His Asn Ile Asp Thr Lys Ser Gln Gln Ser Arg Met Lys 260 265 270 Asp Pro Ala Lys Asp Pro Glu Pro Glu Glu Val Met Ala Lys Pro Met 275 280 285 Gln Ala Cys Val Arg Arg Pro Gln Met Met Thr Pro Gly Ala Ala Ala 290 295 300 Tyr Ser Asp Arg Arg Pro Val Thr Tyr Asn Thr Gln Leu Gln Ser Ser 305 310 315 320 Glu Gln Asp Arg Ile Asn Ala Trp Arg Gly Phe Tyr Glu Val Ala Ser 325 330 335 Lys Arg Glu Ala Glu Glu Asn Ala Glu Tyr Leu Lys Ala Ala Asp Glu 340 345 350 Phe Arg Arg Arg Val Asp Ala Gly Glu Ile Glu Lys Pro Lys Ser Asn 355 360 365 Ile Asp Tyr Gln Val Thr Trp Lys Lys Val Val Thr Asp Asp Gly Leu 370 375 380 Ser Arg Glu Val Ser Asp Val Val Arg Thr Lys Ala Glu Asp Glu Ile 385 390 395 400 Asn Gln Val Gln Lys Glu Thr Ala Pro Ser Pro Val Lys Asn Ala Asp 405 410 415 Glu Ile Asp Leu Glu Met Glu Ser Ala Ser Glu Thr Ala Glu Thr Pro 420 425 430 Asn Glu Ala Leu Asp Ala Ser Ile Thr Lys Gln Ser Phe Ser Thr Gln 435 440 445 Leu Glu Thr Thr Ala Thr Met Pro Met Pro Pro Ala Gln Phe Asp Gly 450 455 460 Val Ser Asp Glu Leu Arg Glu Gln Leu Pro Ala Ser Phe Gln Lys Arg 465 470 475 480 Asp Lys Thr Gln Asp Lys Ala Ile Ala Glu Glu Glu Leu Pro Gly Gly 485 490

495 Ile Thr Asn Lys Ala Thr Gln Phe Leu Ala Leu Asp Lys Cys Glu Pro 500 505 510 His Arg Lys Phe Leu Glu Leu Leu Glu Ile Phe Pro Val Ser Glu Ser 515 520 525 Asp His Thr Asp Glu Gln Arg Pro Tyr Gln Leu Lys Tyr Asp Lys Glu 530 535 540 Trp Leu Ala Ile Thr Arg Val Phe Ala Glu Gly Phe Val Val Gly Lys 545 550 555 560 Lys Ser Gln Val Leu Ile Asp Lys Gly Ser Ala Phe Tyr Lys Pro Lys 565 570 575 Ile Ile Asp Ala Glu Ala Trp Val Glu Glu Asn Ile Val Lys Glu Gly 580 585 590 Lys Met Val Val Pro His Asn Phe Thr Ile Thr Ala Pro Val Tyr Glu 595 600 605 Pro Ser Val Pro Val Thr Thr Pro Glu Gln Pro Phe Glu Tyr Leu Asn 610 615 620 Pro Gln Thr Thr Arg Phe Cys Glu Met Leu Gly Ile Ala Asn Pro Phe 625 630 635 640 Glu Gln Ser Glu Glu Glu Arg Ala Glu Gln Glu Ala Ala Ile Arg Gln 645 650 655 Ala Asn Asp Arg Arg Lys Ser Glu Pro Ala His Ser Gly Arg Arg Gly 660 665 670 Gly Phe Arg Gly Gly Arg Gly Gly Gly Trp Gly Arg Gly Asn Gly Gly 675 680 685 Tyr Gly Gly Arg Gly Gly Trp Gln Gly Gly Arg Gly Arg Gly Arg Gly 690 695 700 Gly Pro 705 57569PRTUstilago maydis 57Met Lys Leu Ala Ile Gln Gly Cys Ser His Gly Glu Leu Asp Ala Ile 1 5 10 15 Tyr Ala Ser Leu Leu Arg Thr Glu Arg Glu Gln Ser Leu His Ile Asp 20 25 30 Ala Leu Leu Leu Cys Gly Asp Phe Gln Ala Ile Arg Asn His Ser Asp 35 40 45 Leu His Ala Leu Ala Val Pro Gln Lys Tyr Arg Gln Leu Gly Asp Phe 50 55 60 His Ser Tyr Tyr Ser Gly Glu Lys Ile Ala Pro Ile Leu Thr Leu Val 65 70 75 80 Ile Gly Gly Asn His Glu Ala Ser Asn Tyr Met His Glu Leu Tyr His 85 90 95 Gly Gly Trp Leu Ala Pro Asn Ile Tyr Phe Leu Gly Ala Ala Gly Val 100 105 110 Ile Glu Leu Asn Gly Ile Val Val Ala Gly Ile Ser Gly Ile Tyr Lys 115 120 125 Glu Lys Asp Tyr Arg Lys Gly Arg Phe Glu Lys Leu Pro Tyr Asp Ala 130 135 140 Gly Ser Ile Arg Ser Cys Tyr His Thr Arg Glu Phe Asp Val Val Arg 145 150 155 160 Leu Lys Ala Leu Lys Asp Gly Gln Val Glu Ile Val Met Ser His Asp 165 170 175 Trp Pro Asn Thr Ile Glu Gln Trp Gly Asn Thr Gln Ala Leu Ile Arg 180 185 190 Lys Lys Pro Phe Phe Lys Glu Glu Ile Glu Ser Arg Thr Leu Gly Ser 195 200 205 Pro Pro Leu Met Glu Leu Leu Gln Cys Leu Lys Pro Ala Phe Trp Phe 210 215 220 Ser Ala His Leu His Val Lys Phe Ala Ala Leu Phe Arg His Gly Gln 225 230 235 240 Met Asp Pro Ala Ile Glu Pro Ser Ser Thr Thr Ala Ala Asn Thr Asn 245 250 255 Pro Glu Ala Leu Asp Ile Ser Leu Asp Ser Asp Asp Asp Leu Pro Glu 260 265 270 Ser Pro Lys Pro Ala Pro Ser Ala Asp Ile Ala Val Asp Gly Thr Val 275 280 285 Thr Lys Ser Ala Thr Ala Thr Arg Phe Leu Ala Leu His Lys Cys Leu 290 295 300 Pro Gln Thr Gln Phe Leu Gln Ile Ile Asn Leu Pro Ser Pro Gln Asp 305 310 315 320 Ala Glu Leu Glu Ser Arg Lys Ala Ser Leu Gly Tyr Thr Gln Arg Ile 325 330 335 Pro Pro Ser Leu Arg Tyr Asn Gln Arg Trp Leu Ala Ile Thr Arg Ala 340 345 350 Phe His Ser His Phe Ser Leu Gln Tyr Arg Gln Pro Asp Leu Pro Asp 355 360 365 Pro Phe Ser Ala Ser Leu Leu Ala Arg Ile Glu Glu Glu Glu Arg Trp 370 375 380 Ile Glu Gln Asn Leu Val Ser Pro Phe Thr Thr Arg Asn Ser Gly Lys 385 390 395 400 Arg Lys Gln Asp Glu Arg Glu Gly Ser Arg Ala Cys Thr Pro Gln Asp 405 410 415 Glu Arg Glu Thr Glu Gly Val Gly Leu Asp Val His Arg Val Gln Gln 420 425 430 Phe Val Arg Thr Ala Pro Ala Pro Phe Glu Pro Gly Gly Leu Ser Gln 435 440 445 Ala Pro Pro Ala Trp Tyr Thr Asn Pro Gln Thr Glu Ala Phe Cys Arg 450 455 460 Phe Leu Gly Ile Glu Asn Lys Ile Asn Pro Arg Pro Asp Ala Phe Gly 465 470 475 480 Gly Ala Ser Cys Tyr Pro His Pro Gln Glu His Leu Ala Ala Ser Phe 485 490 495 His Pro Ala Thr Pro Thr Gly Asp Arg Asp Pro Ser Gln Val His Ser 500 505 510 Glu Ala Val Ser Asp Pro Asn Ala Leu Ala Ile Asp Met Asp Asp Leu 515 520 525 Asp Ser Asp Cys Ser Asp Ala His Ala Asn Gly Asp Gly Phe Lys His 530 535 540 Asp Gly Ser Arg Arg Asp Val Leu Thr Leu Ser Asp Asp Asp Glu Leu 545 550 555 560 His Ala Arg Trp Lys Glu Gly Thr Gly 565 58649PRTMagnaporthe oryzae 58Met Gly Asp Ala Gln Thr Gln Thr Phe Thr Ser Pro Asp Gly Leu Arg 1 5 10 15 Val Ala Val His Gly Cys Gly His Gly Val Leu Asn Ala Ile Tyr Ala 20 25 30 Ala Val Ala Ile Ser Cys Lys Glu Arg Gly Trp Asp Thr Val Asp Leu 35 40 45 Leu Ile Ile Gly Gly Asp Phe Gln Ala Val Arg Asn Ala Ala Asp Leu 50 55 60 Ser Val Met Ser Cys Pro Val Lys Tyr Arg Thr Ile Gly Asp Phe His 65 70 75 80 Glu Tyr Tyr Ser Gly Ser Arg Thr Ala Pro Tyr Leu Thr Ile Phe Ala 85 90 95 Gly Gly Asn His Glu Ala Ala Ser His Ser Trp Glu Leu Phe Tyr Gly 100 105 110 Gly Trp Val Ala Pro Asn Ile Tyr Tyr Leu Gly Pro Ala Asn Val Val 115 120 125 Arg Leu Gly Pro Leu Arg Ile Ala Ala Leu Gly Gly Ile Trp Ala Gly 130 135 140 Tyr Asp Tyr Arg Lys Pro His His Glu Arg Leu Pro Phe Ser Glu Ser 145 150 155 160 Asn Ile Lys Ser Phe Tyr His Val Arg Glu Met Asp Val Arg Lys Leu 165 170 175 Leu Gln Ile Arg Thr Gln Val Asp Ile Gly Leu Ser His Asp Trp Pro 180 185 190 Arg Ala Val Glu Arg His Gly Asp Glu Gly Ala Leu Phe Arg Lys Lys 195 200 205 Pro Phe Leu Arg Asp Glu Ser Lys Ala Gly Thr Leu Gly Asn Pro Ala 210 215 220 Ala Thr Tyr Val Met Asp Arg Leu Arg Pro Ala Tyr Trp Phe Ala Ser 225 230 235 240 His Met His Cys Lys Phe Ala Ala Leu Lys Val Tyr Thr Asp Glu Pro 245 250 255 Pro Thr Glu Asp Asp Gly Val Glu Ala His Lys Ile Asp His Gly Pro 260 265 270 Val Ala Gln Ala Lys Asp Leu Thr Ala Glu Ala Ser Ala Pro Thr Ile 275 280 285 Glu Asn Pro Asp Glu Ile Asp Leu Asp Met Asp Asp Asn Asp Asp Ala 290 295 300 Ala Gly Ala Gly Ala Ala Ala Ala Ala Ser Thr Ser Thr Asn Gly Glu 305 310 315 320 Thr Ala Ala Ala Lys Asp Val Val Ser Glu Asn Thr Ser Asn Gly Lys 325 330 335 Val Val Asn Pro Asp Ala Ile Asp Leu Asp Leu Asp Asp Asp Glu Ala 340 345 350 Gln Asp Thr Ala Pro Gly Ala Pro Gly Gly Gln Pro Glu Glu Asp Gly 355 360 365 Glu Gly Lys Ala Lys Pro Leu Ser Thr Glu Lys Ala Thr Asn Glu Asn 370 375 380 Asn Thr Thr Thr Thr Ala Ala Ser Ser Phe Ile Ser Gln Asp Ile Arg 385 390 395 400 Asn Gln Leu Pro Ala Ser Phe Ala Pro Pro Pro Gln Gln Ala Pro Thr 405 410 415 Glu Ser Arg Ala Lys Arg Thr Pro Gly Gln Pro Val Pro Glu Gly Ile 420 425 430 Thr Asn Lys Glu Val Arg Phe Leu Ala Leu Ser Lys Cys Leu Pro Gly 435 440 445 His Asp Phe Leu Gln Leu Cys Asp Ile Ser Pro Leu Asp Arg Ser Ser 450 455 460 Thr Gly Ser Ser Asn Asp Thr Pro Pro Lys Tyr Arg Leu Glu Tyr Asp 465 470 475 480 Pro Glu Trp Leu Ala Ile Thr Arg Val Phe Ala Ser Glu Leu Ile Ile 485 490 495 Gly Asp Ser Asn Ala Thr Ala Thr Thr Asp Leu Gly Glu Glu His Tyr 500 505 510 Lys Pro Leu Ile Gln Ala Glu Arg Thr Trp Val Glu Glu Asn Ile Val 515 520 525 Ala Lys Asp Lys Leu Ala Ile Pro Glu Asn Phe Val Ile Thr Ala Pro 530 535 540 Pro His Ile Pro Gly Gln Pro Glu Gly Val Pro Glu Gln Pro Asp Glu 545 550 555 560 Tyr Thr Asn Pro Gln Thr Ser Ala Phe Cys Glu Leu Leu Gly Val Lys 565 570 575 Asn Leu Trp Asn Ala Thr Asp Glu Glu Arg Leu Glu Arg Lys Asn Gln 580 585 590 Gly Pro Pro Pro Asp Gln Gly Gly Phe Arg Gly Gly Arg Gly Gly Gly 595 600 605 Ser Gly Gly Gly Arg Gly Gly Arg Gly Gly Phe Gly Gly Arg Gly Arg 610 615 620 Gly Gly Arg Gly Gly Gln Gly Gly Gly Gly Arg Gly Arg Gly Phe Arg 625 630 635 640 Gly Gly His Gly Gly Arg Gly Arg Tyr 645 59546PRTFusarium graminearum 59Met Thr Thr Asn Ala Phe Glu Ala Gln Gly Val Arg Val Ala Ile Glu 1 5 10 15 Gly Cys Thr Gln Gly His Gly Thr Leu Asp Ala Ile Tyr Ala Ser Val 20 25 30 Glu Glu Ser Cys Lys Gln Arg Gly Trp Asp Gly Val Asp Ile Leu Ile 35 40 45 Ile Gly Gly Asp Phe Gln Ser Val Arg Asn Ala Glu Asp Leu Ser Ile 50 55 60 Met Ser Cys Pro Val Lys Tyr Arg His Leu Gly Asp Phe Pro Lys Tyr 65 70 75 80 Tyr Ser Gly Glu Arg Lys Ala Pro Tyr Leu Thr Ile Phe Ile Ala Gly 85 90 95 Asn His Glu Ala Ser Ser His Leu Trp Glu Leu Tyr Tyr Gly Gly Trp 100 105 110 Val Ala Pro Asn Ile Tyr Tyr Met Gly Ala Ala Asn Ile Leu Arg Phe 115 120 125 Gly Pro Leu Arg Ile Ala Gly Leu Ser Gly Ile Trp Lys Gly Phe Asp 130 135 140 Tyr Arg Lys Pro His His Glu Arg Leu Pro Phe Ser Gly Gly Asp Val 145 150 155 160 Lys Ser Trp Tyr His Val Arg Glu Ile Asp Val Arg Lys Leu Leu Gln 165 170 175 Val Gln Thr Gln Val Asp Val Gly Leu Ser His Asp Trp Pro Arg Ala 180 185 190 Val Glu Leu His Gly Asp His Glu Trp Leu Phe Arg Lys Lys Pro Asp 195 200 205 Phe Arg Asn Glu Ser Arg Asp Gly Thr Leu Gly Ser Val Ala Ala Glu 210 215 220 Tyr Val Met Asp Arg Leu Arg Pro Pro His Trp Phe Ser Ala His Met 225 230 235 240 His Val Lys Phe Ala Ala Ile Lys Thr Tyr Ser Glu Ala Gln Pro Glu 245 250 255 Val Glu Glu Thr Lys Gln Glu Leu Ala Pro Ala Ala Ala Pro Val Pro 260 265 270 Ala Thr Glu Asn Asn Pro Asp Glu Ile Asp Leu Asp Met Asp Asp Glu 275 280 285 Asp Glu Asp Thr Lys Pro Asn Pro Glu Pro Glu Ala Lys Lys Ser Glu 290 295 300 Pro Glu Val Glu Glu Ala Lys Glu Ala Ser Asn Glu Val Ser Asp Glu 305 310 315 320 Leu Arg Ala Gln Leu Pro Ala Ser Phe Ala Arg Pro Gln Pro Lys Lys 325 330 335 Thr Pro Gly Gln Pro Val Pro Pro Gly Ile Thr Asn Lys Glu Val Arg 340 345 350 Phe Leu Ala Leu Asp Lys Cys Leu Pro Gly Arg His Phe Leu Gln Leu 355 360 365 Cys Asp Leu Gln Pro Phe Asn Pro Glu Thr Ser Ser Glu Tyr Pro Pro 370 375 380 Ala Gln Glu Ser Pro Arg Trp Arg Leu Gln Tyr Asp Pro Glu Trp Leu 385 390 395 400 Ala Ile Thr Arg Val Phe His Asp Ser Leu Val Ile Gly Asp Ser Asn 405 410 415 Ala Gln Ser Pro Pro Asp Leu Gly Glu Glu His Tyr Gln Pro Leu Ile 420 425 430 Lys Lys Glu Arg Glu Trp Val Glu Asp Asn Ile Val Lys Ala Gly Lys 435 440 445 Leu Asp Val Pro Tyr Asn Phe Glu Ile Thr Ala Pro Pro His Val Pro 450 455 460 Gly Gly Pro Glu Ile Ala Ser Glu Gln Pro Ser Glu Tyr Thr Asn Pro 465 470 475 480 Gln Thr Ser Lys Phe Cys Glu Ile Met Glu Leu Ser Asn Ile Trp Asp 485 490 495 Ala Thr Asp Glu Glu Arg Arg Gln Arg Lys Ala Gln Gly Pro Pro Lys 500 505 510 Thr Asp Gln Arg Phe Thr Gly Gly Gly Arg Gly Gly Gly Arg Gly Arg 515 520 525 Gly Gly Arg Gly Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly 530 535 540 Arg Trp 545 60566PRTPlasmodium vivax 60Met Ile Ile Ala Val Val Gly Cys Thr His Gly Glu Leu Asn Phe Ile 1 5 10 15 Tyr Ala Thr Ile Glu Lys Leu Glu Gln Asp Asn Asn Phe Lys Val Asp 20 25 30 Leu Leu Ile Cys Cys Gly Asp Phe Glu Cys Val Arg Tyr Gly Val Asp 35 40 45 Asn Asp Cys Leu Asn Val Pro Asn Lys Tyr Lys Lys Glu Glu Asn Asp 50 55 60 Phe Arg Asp Tyr Phe Thr Gly Lys Lys Lys Ala Lys Val Leu Thr Ile 65 70 75 80 Phe Ile Gly Gly Asn His Glu Ala Val Asn Val Leu Lys Gln Leu Tyr 85 90 95 Tyr Gly Gly Trp Val Ala Pro Asn Ile Tyr Phe Leu Gly Tyr Ser Asn 100 105 110 Val His Asn Ile Asn Asp Phe Arg Ile Cys Ser Leu Ser Gly Ile Tyr 115 120 125 Lys Lys Tyr Asn Phe Tyr Lys Lys Tyr Asn Glu His Tyr Pro Tyr Asp 130 135 140 Glu Ile Ser Lys Val Ser Ala Tyr His Ile Arg Lys Phe Glu Ile Glu 145 150 155 160 Lys Leu Lys Leu Leu Lys Glu Lys Ile Asp Ile Val Val Thr His Asp 165 170 175 Trp Pro Asn Asn Ile Glu Lys His Gly Asp Val Asn Asp Leu Val Arg 180 185 190 Arg Lys Phe His Phe Gln Ser Asp Ile Tyr Asn Asn Thr Leu Gly Asn 195 200 205 Pro His Thr Glu Phe Leu Leu Asn Lys Leu Lys Pro Tyr Phe Trp Phe 210 215 220 Ser Ser His Leu His Val Lys Tyr Ser Ala Ile Phe Leu His Ser Asp 225 230 235 240 Lys Arg Asn Tyr Thr Arg Phe Leu Ser Leu Asp Lys Ala Glu Pro Arg 245 250 255 Lys His Phe Ile Gln Ile Leu Asn Ile Glu Lys Arg Asn Asn Ile Pro 260 265 270 Tyr Leu Ser Phe Asp His Leu Pro Arg Pro Ser Ala Asn Asp Pro Asp 275 280 285 Gly Lys Ser His Phe Phe Asn Glu Asp Tyr Glu Glu Leu Leu Gln His 290 295 300 Val Glu Asp Val Gln Arg Arg Asp Ala Glu Gly Gly Gly Lys Gly His 305 310 315

320 Ser Gly Gly Ala Ala Gln Ala Lys Glu Asn Ala Pro Val Glu Ala Ala 325 330 335 Thr Arg Glu Ala Ala Thr Arg Glu Ala Val Lys Gln Glu Asn Ala Ala 340 345 350 Val Glu Thr Ser Pro Gly Glu Ala Ala Thr Lys Glu Asp Ala Pro Gly 355 360 365 Glu Ala Asp Pro Gln Glu Pro Pro Pro Gln Glu Asn Ala Ala Pro Glu 370 375 380 Arg Lys Lys Leu Phe Ile Cys Tyr Asp Glu Glu Trp Leu Ala Ile Leu 385 390 395 400 Lys Ala Asn Gln His Leu Val Ser Glu Gly Cys Asp Lys Asp Tyr Asn 405 410 415 Leu Glu Lys Leu Lys Cys Pro Ser Lys Glu Asp Phe Glu Tyr Ile Arg 420 425 430 Asp Lys Leu Lys Glu Leu Glu Lys Thr Ser Val Lys Gly Lys Asp Tyr 435 440 445 Tyr Leu Val His Gly Tyr Asn Thr Pro Ser Tyr Lys His Leu Trp Glu 450 455 460 Gln Arg Gln Leu Phe Leu Ser Arg Phe Asp Phe Glu Glu Leu Arg Met 465 470 475 480 Tyr Asp Asp Phe Glu Arg Leu Phe Phe Ala Glu Glu Val Arg Lys Met 485 490 495 Asp Ala Gly Leu Pro Leu Asp Pro Pro Lys Val Glu Glu Asp Glu Glu 500 505 510 Glu Asp Gly Glu Glu Asp Glu Pro Glu Glu Val Gly Gln Asn Asn Gln 515 520 525 Ala Asp Glu His Gly Gly Gly Asn Pro Asn Gly Asp Asn Ala Pro His 530 535 540 Ser Gly Asn Ala Pro Asn Val Glu Gly Ala Ser Glu Thr Asn Glu Ile 545 550 555 560 Ser Leu Ser Ile Asp Cys 565 61565PRTPlasmodium falciparum 61Met Phe Ile Ala Val Val Gly Cys Thr His Gly Glu Leu Asp Leu Ile 1 5 10 15 Tyr Ser Thr Leu Glu Lys Ile Glu Glu Glu Asn Lys Ile Lys Val Asp 20 25 30 Leu Leu Ile Cys Cys Gly Asp Phe Gln Ser Val Arg Tyr Asn Val Asp 35 40 45 Asn Glu Cys Leu Asn Val Pro Ala Lys Tyr Lys Lys Glu Gln Asn Asp 50 55 60 Phe Val Asp Tyr Phe Thr Gly Lys Lys Lys Ala Lys Ile Leu Thr Ile 65 70 75 80 Phe Val Gly Gly Asn His Glu Ala Met Asn Val Leu Lys Gln Leu Tyr 85 90 95 Tyr Gly Gly Trp Val Ala Pro Asn Ile Tyr Tyr Leu Gly Tyr Ser Ser 100 105 110 Val His Asn Ile Asn Asn Phe Arg Ile Cys Ser Leu Ser Gly Ile Tyr 115 120 125 Lys Lys Tyr Ser Phe Phe Lys Lys Tyr Tyr Glu Ser Tyr Pro Tyr Thr 130 135 140 Asp Ile Thr Lys Val Ser Ala Tyr His Ile Arg Lys Tyr Glu Ile Glu 145 150 155 160 Lys Leu Lys Leu Leu Lys Asn Asn Val Asp Ile Val Val Thr His Asp 165 170 175 Trp Pro Asn Asn Ile Glu Lys His Gly Asp Val His Asp Leu Leu Arg 180 185 190 Arg Lys Tyr His Phe Gln Ser Asp Val Tyr Asn Asn Thr Leu Gly Asn 195 200 205 Pro His Thr Glu Ile Leu Leu Asn Lys Leu Lys Pro Tyr Phe Trp Phe 210 215 220 Ala Ser His Leu His Val Lys Tyr Ser Ala Leu Tyr Ile His Asn Asp 225 230 235 240 Gln Lys Gln Tyr Thr Arg Phe Leu Ser Leu Asp Lys Ala Gln Glu Tyr 245 250 255 Lys His Phe Ile Gln Ile Leu Asn Ile Val Lys Lys Lys Asp Ser Ser 260 265 270 Ile His Leu Asn Phe Asp His Val Pro Lys Val Leu Leu Pro Glu Pro 275 280 285 Gly Ser Lys Met Asp Ile Gln Asn Asp Ala Gln Pro Asn His Asp Leu 290 295 300 Glu Asn Cys Pro Asn Thr Lys Thr Asn Thr Cys Asn Asn Asn Asp His 305 310 315 320 His Asn Asp Asp Ser Ile Asn Leu Asp Tyr Asp His Glu Lys Ala Leu 325 330 335 Tyr Glu Leu Asp Arg Asn Met Gln Leu Asp Gln Glu Lys Asn Asp Glu 340 345 350 Lys Asn Val Asp Lys Ser Ala Asp Lys Asn Val Cys Asn Lys Asp Ile 355 360 365 Ser Leu Glu Asp Lys Asn Gln His Asn Asn Asn Asn Asn Asn Asn Asp 370 375 380 Asp Asp Asp Asp Gly Val Asp Ile Gln Ala Asp Thr Ser Thr Asn Val 385 390 395 400 Ala Asp Gln Asn Asn Asn Ser Val Pro Thr Asn Leu Lys Glu Asn Glu 405 410 415 Glu Glu Ser Leu Asn Asp Gln Asn Glu Asn Lys Asp Glu Glu Thr Ser 420 425 430 Gln Asp Glu Asn Ile Thr Asp Glu Lys Lys Lys Lys Lys Phe Tyr Leu 435 440 445 Cys Tyr Asp Ile Glu Trp Leu Ala Ile Val Lys Ala Asn His His Leu 450 455 460 Ile Ser Ala Ser Cys Asp Pro Thr Lys Glu Asp Phe Asp Phe Val Glu 465 470 475 480 Asn Lys Leu Lys Glu Leu Asp Asn Lys Ile Thr Ile Lys Gly Lys Asp 485 490 495 Tyr Tyr Cys Val Asn Gly Tyr Asn Thr Pro Asn Tyr Lys Asn Leu Gln 500 505 510 Glu Gln Arg Gln Leu Phe Leu Lys Arg Phe Glu Leu Glu Glu Leu Ser 515 520 525 Ile Tyr Thr Glu Ser Glu Leu Asn Phe Phe Ala Glu Glu Met Lys Thr 530 535 540 Leu Glu Lys Met Asn Thr Asp Ile His Asn Glu Glu Asp Lys Asn Glu 545 550 555 560 Cys Thr Ile Glu Ala 565 62496PRTToxoplasma gondii 62Met Lys Ile Ala Ile Glu Gly Cys Cys His Gly Glu Leu Asp Ala Ile 1 5 10 15 Tyr Ser Ser Leu Ala Arg Leu Glu Glu Met His Lys Met Lys Val Asp 20 25 30 Leu Leu Ile Cys Cys Gly Asp Phe Gln Cys Val Arg Asp Ser Asn Asp 35 40 45 Leu Gln Phe Leu Ala Cys Pro Pro Lys Tyr Arg Asp Leu Arg Asp Phe 50 55 60 Pro Ala Tyr Phe Arg Gly Glu Lys Glu Ala Pro Cys Leu Thr Val Phe 65 70 75 80 Val Gly Gly Asn His Glu Ala Pro Thr Val Leu Arg Glu Leu Tyr Tyr 85 90 95 Gly Gly Trp Val Ala Pro Lys Ile Phe Tyr Leu Gly His Ala Gly Val 100 105 110 Val Asn Val Gly Gly Val Arg Ile Ala Gly Leu Ser Gly Ile Phe Lys 115 120 125 Ser Gln Asp Tyr Arg Lys Gly Tyr Phe Glu Arg Pro Pro Tyr Thr Glu 130 135 140 Asp Thr Met Arg Ser Ala Tyr His Val Arg Glu Phe Glu Ile Ala Lys 145 150 155 160 Leu Ser Glu Leu Thr Gly Arg Val Asp Ile Val Val Thr His Asp Trp 165 170 175 Pro Glu Gly Ile Tyr Asp Phe Gly Asp Lys Thr Glu Leu Ile Arg Gln 180 185 190 Lys Pro Phe Leu Glu Lys Asp Ile Gln Ala His Glu Leu Gly Asn Pro 195 200 205 His Ser Met Glu Leu Leu Lys Lys Leu Lys Pro Ala Phe Trp Phe Ala 210 215 220 Ala His Leu His Thr Arg Phe Ala Ala Val Tyr Val His Pro Gly Pro 225 230 235 240 Glu Gly Lys Ala Thr Arg Phe Leu Ala Leu Asp Lys Val Leu Pro Arg 245 250 255 Arg Glu Phe Leu Gln Ile Leu Glu Val Glu Pro Leu Leu Pro Ala Gly 260 265 270 Tyr Val Gln Gln Leu Ser Pro Gly Ile Ser Arg Arg Ser Pro Thr Leu 275 280 285 Cys Tyr Asp Glu Glu Trp Leu Ala Ile Leu Arg Ala Asn Gln Gln Val 290 295 300 Leu Pro Val Ser Arg Phe Pro Gln Lys Ser Cys Leu Val Thr Lys Ala 305 310 315 320 Thr Ala Asp Asp Leu Ala Thr Val Lys Lys Asn Leu Ala Ser Leu Gly 325 330 335 Leu Arg Asn Tyr Arg Glu Thr Ser Ser Pro Lys Arg Leu Ser Leu Asn 340 345 350 Ser Val Gly Ala Ala Ala Ala Ala Glu Asp Ala Arg Arg Glu Ser Asp 355 360 365 Gly Asp Arg Arg Ser Ala Arg Glu Glu Lys Glu Gly Cys Glu Glu Ala 370 375 380 Ala Ala Gly Val Ser Ala Gly Ala Ser Val Gln Arg Thr Asp Val Ala 385 390 395 400 Ala Glu Thr Pro Pro Gln Pro Gln Gly Gly Gln Glu Glu Ser Thr Val 405 410 415 Phe Glu Trp Ile Asn Trp Ala Asp Pro Arg Ala Pro Tyr Thr Glu Leu 420 425 430 Lys Glu Gln Arg Leu Phe Leu Leu Arg Asn Ile Leu Gly Phe Asp Glu 435 440 445 Ala Asp Asp Lys Phe Gly Glu Ala Arg Gln Arg Glu Ala Ala Asp Val 450 455 460 Asp Val Pro Val Asp Trp Thr Ser Gly His Val Asp Pro Gln Arg Thr 465 470 475 480 Thr Glu Glu Val Asp Ile Cys Leu Asp Leu Ser Asp Glu Glu Thr Ala 485 490 495 63521PRTTrypanosoma brucei 63Met Ser Ser Leu Val His His Phe Phe Asn Val Lys Gly Gly Val Thr 1 5 10 15 Glu Arg Thr Ala Pro Ser Ser Ser Gly Gly Ala Thr Glu Thr Phe Ala 20 25 30 Asn Leu His Val Ala Val Val Gly Cys Cys His Gly Glu Leu Asp Lys 35 40 45 Ile Tyr Leu Ala Cys Ser Asp His Glu Val Ser Ser Gly Lys Lys Ile 50 55 60 Asp Phe Val Ile Cys Ala Gly Asp Phe Gln Ala Leu Arg Arg Glu Glu 65 70 75 80 Asp Leu Lys Cys Met Ala Val Pro Glu Lys Tyr Arg Ser Leu Gly Asp 85 90 95 Phe Val Lys Tyr Tyr Gln Gly Glu Lys Arg Ala Pro Tyr Leu Thr Leu 100 105 110 Phe Val Gly Gly Asn His Glu Cys Ser Asp Trp Leu Ala Glu Glu Ser 115 120 125 Tyr Gly Gly Phe Leu Ala Pro Asn Ile Tyr Tyr Leu Gly His Ser Gly 130 135 140 Val Val Val Val Asp Gly Cys Ile Thr Val Ala Gly Ile Ser Gly Ile 145 150 155 160 Phe Lys Ala His Asp Tyr Val Arg Pro Tyr Pro Asn Arg Pro Phe His 165 170 175 Val Ser Glu Ala Ser Lys Arg Ser Ala Tyr His Val Arg Arg Ile Glu 180 185 190 Val Glu Lys Leu Arg Ala Phe Val Arg Ala Leu Arg His Met Gln Gln 195 200 205 Trp Gly Arg Lys Trp Gly Ala Gln Ser Val Ser Pro Leu Ala Thr Ala 210 215 220 Ala Asn Ile Ala Asn Pro Ala Gln Lys Val Ser Gln Asp Gly Gly Asn 225 230 235 240 Asp Thr Thr Asn Ser His Ile Thr Leu Pro Pro Val Asp Ile Phe Val 245 250 255 Ser His Asp Trp Pro Thr Gly Val Thr Lys Tyr Gly Asp Glu Glu Gln 260 265 270 Leu Leu Arg Tyr Lys Pro Tyr Phe Arg Glu Asp Ile Arg His Gly Val 275 280 285 Leu Gly Asn Pro His Thr Val Lys Leu Leu Gln Asp Ile Lys Pro Arg 290 295 300 Tyr Trp Ile Ala Ala His Leu His Cys Arg Phe Glu Ala Thr Val Pro 305 310 315 320 His Glu Asn Thr Ser Gly Lys Cys Thr Thr Ala Gly Thr Thr Ser Pro 325 330 335 Val Ala Thr Gln Gln Lys Thr Lys Phe Leu Ala Leu Asp Lys Pro Ala 340 345 350 Lys Gly Lys Gly Phe Ile Asp Phe Ile Asp Val Pro Gly Glu Arg Gly 355 360 365 Ala Val Gly Arg Lys Ser Asp Val Asp Arg Val Val His His Pro Leu 370 375 380 Trp Leu Arg Val Leu Arg Glu Ser His Asn Tyr Leu Ser Ala Asn Asp 385 390 395 400 Asp Ser Trp Ser Ser Glu Thr Cys Asn Phe Leu Gln Ser Ser Glu Glu 405 410 415 Glu Pro Ile Ser Thr Glu Val Ser Ile Pro Ala His Ser Thr Lys Gln 420 425 430 Leu Leu Gln Ser Leu Gly Leu Pro Pro Ser Pro Ile Gln Gln Ala Gln 435 440 445 Pro Gln Ser Thr Ile Ala Val Val Ala Gly Gly Gly Ser Gly His His 450 455 460 Arg Pro Val Thr Gly Ser Gly His Ala Lys Leu Asp Asp Lys Ala Gly 465 470 475 480 Ala Pro Asp Ala Asn Cys Ser Ser Val Ala Thr Arg Pro Ala Asp Trp 485 490 495 Asn Gly Ala Arg Thr Glu Asp Gly Val Asp Ala Gly Asn Asp Leu Pro 500 505 510 Trp Val Glu Asp Ala Val Gly Asp Val 515 520 64503PRTTrypanosoma cruzi 64Met Cys Phe Val Val Val Val Phe Ala Val Phe Leu Leu Leu Leu Pro 1 5 10 15 Trp Val Pro Met Cys Gly Val Val Cys Pro His Tyr Ser Ser Phe Phe 20 25 30 Phe Val Arg Phe Val Phe Tyr Tyr Arg Leu Ser Gly Gly Lys Gly Cys 35 40 45 Arg Phe Val Leu Tyr Lys Met Ser Leu Val His His Phe Phe His Val 50 55 60 Lys Gly Gly Val Thr Thr Asn Thr Ala Lys Asn Asn Thr Gly Ser Ser 65 70 75 80 Asp Ser Gly Thr Ala Ala Glu Thr Ile His Val Ala Val Gln Gly Cys 85 90 95 Cys His Gly Glu Leu Asp Arg Ile Tyr Ala Ala Cys Ala Ala His Glu 100 105 110 Lys Ala Thr Gly Arg Arg Ile Glu Phe Leu Leu Cys Cys Gly Asp Phe 115 120 125 Gln Ala Val Arg Asp Glu Val Asp Leu Arg Ser Met Ala Val Pro Gln 130 135 140 Lys Tyr Cys Val Leu Gly Asp Phe Leu Ala Tyr His Arg Arg Glu Lys 145 150 155 160 His Ala Pro Tyr Leu Thr Leu Phe Val Gly Gly Asn His Glu Gly Ser 165 170 175 Asp Trp Leu Ala Thr Glu Cys Tyr Gly Gly Phe Leu Ala Pro Asn Ile 180 185 190 Tyr Tyr Ile Gly His Ser Gly Ala Val Ile Val Asp Asp Cys Val Thr 195 200 205 Val Ala Gly Leu Ser Gly Ile Phe Lys Gly His Asp Tyr Ala Arg Pro 210 215 220 Tyr Pro Gly Arg Pro Phe His Ala Ser Glu Ala Ala Lys Arg Ser Ala 225 230 235 240 Tyr His Val Arg Arg Ile Glu Val Glu Lys Leu Arg Ala Phe Ser Gln 245 250 255 Ala Leu Glu Arg Met Arg Gln Pro Ala Ser Ser Pro Met Thr Ala Ser 260 265 270 Met Ala Gly Pro Gly Ala Ser Pro Ser Arg Cys Ala Gly Glu Phe Pro 275 280 285 His Ile Asp Leu Phe Leu Ser His Asp Trp Pro Ala Gly Ile Thr Lys 290 295 300 Tyr Gly Asp Glu Thr Gln Leu Leu Arg Tyr Lys Pro Phe Phe Glu Glu 305 310 315 320 Asp Ile Arg His Gly Ala Leu Gly Asn Pro His Thr Met Thr Leu Leu 325 330 335 Arg Ala Val Lys Pro Arg Tyr Trp Leu Ala Ala His Leu His Cys Gln 340 345 350 Phe Glu Ala Thr Ile Pro His His Asp Val Glu Asn Asp Ala Ala Ala 355 360 365 Ala Gly Val Pro Arg Ala Thr Lys Phe Leu Ala Leu Asp Lys Cys Ser 370 375 380 Lys Gly Lys Gly Phe Ile Asp Phe Ile Asp Val Arg Val Ser Arg Gly 385 390 395 400 Pro His Leu Thr Lys Glu Lys Asn Arg Glu Arg Thr Ala Arg Glu Gln 405 410 415 Glu Arg Val Val His His Pro Leu Trp Leu Glu Val Leu Arg Glu Thr 420 425 430 His Gly Phe Leu Thr Ser Asn Asn Asn Glu Trp Ser Ala Gly Ser Cys 435 440 445 Ala Leu Leu Arg Leu Thr Pro Asp Glu Leu Arg Gln Arg Gly Val Trp 450 455 460

Leu Leu Ala Arg Ser Thr Ala Ser Val Leu Glu Ala Leu Val Leu Pro 465 470 475 480 Pro Ala Pro Leu Gln Arg Pro Ser Ala Glu Gly Glu Trp Arg Arg Arg 485 490 495 Arg Thr His Ala Ser Ala Leu 500 65620PRTLeishmania donovani 65Met Ser Leu Ala His Lys Ile Phe Ala Thr Leu Lys Ala Ser Gly Asp 1 5 10 15 Gly Thr Phe Pro Ser Pro Ala Ala Pro Thr Ser Gly Gln Ser Glu Thr 20 25 30 Ala Thr Thr Thr Ser Ala Ser Pro Ser Gly Ser Trp Asn Glu Arg Phe 35 40 45 Tyr His Ile Ala Val Gln Gly Cys Cys His Gly Glu Leu Asp Arg Ile 50 55 60 Tyr Asp Ser Cys Ser Glu His Glu Arg Gln Thr Gly Lys Arg Ile Asp 65 70 75 80 Val Leu Leu Cys Cys Gly Asp Phe Gln Ala Val Arg Thr Ala Arg Asp 85 90 95 Met Asp Ser Met Ala Val Pro Asp Lys Tyr Lys Val Leu Gly Asp Phe 100 105 110 His Lys Tyr Tyr Ala Asp Val Ser Gly Ala Phe Thr Gly His Lys Ala 115 120 125 Gln Thr Leu Ala Pro Tyr Leu Thr Ile Phe Val Gly Gly Asn His Glu 130 135 140 Asn Ser Asp Leu Leu Ala Gln Glu Ser Tyr Gly Gly Phe Val Ala Pro 145 150 155 160 Asn Val Phe Tyr Leu Gly His Ser Ser Val Val Thr Val Asp Asp Cys 165 170 175 Leu Thr Ile Ala Gly Leu Ser Gly Ile Phe Lys Asp Pro Asp Tyr Asp 180 185 190 Arg Pro Tyr Pro Pro Arg Pro Tyr Ala Val Asn Pro Val Ala Lys Lys 195 200 205 Ser Ala Tyr His Val Arg Arg Ile Glu Val Ala Lys Leu His Ala Tyr 210 215 220 Leu Arg Ala Thr Gln Lys Ile Arg Ser Asn Ser Thr Ile Glu Ala Ala 225 230 235 240 Lys Thr Thr Ser Ala Thr Ser Pro Ala Ala Ser Pro Pro Met Val Asp 245 250 255 Leu Phe Leu Ser His Asp Trp Pro Val Gly Ile Thr Gly Tyr Gly Asp 260 265 270 Glu Ala Gln Leu Leu Arg Phe Lys Pro Tyr Phe Lys Asp Asp Ile Arg 275 280 285 Arg His Ala Leu Gly Asn Pro Tyr Thr Met Arg Leu Leu Gln Glu Ala 290 295 300 Lys Ala Pro Tyr Trp Phe Ala Ala His Leu His Cys Tyr Phe Glu Ala 305 310 315 320 Thr Val Glu His Pro Ser Ala Gly Ala Thr Glu Thr Met Ala Ala Thr 325 330 335 Ala Ala Ala Ser Thr Lys Phe Val Ala Leu Asp Lys Cys Ala Lys Gly 340 345 350 His Gly Phe Leu Thr Phe Ile Asp Leu Pro Arg Val Arg Arg Gly Gly 355 360 365 Val Arg Ala Ala Pro Pro Ser Glu Ser His Pro His Gly Thr Ala Thr 370 375 380 Val Leu Gly Thr Ser Arg Ile Arg Arg Asp Pro Val Trp Leu Glu Val 385 390 395 400 Leu Arg Val Ser His Gln Phe Val Ala Ala Asn Arg Thr Val Glu Ala 405 410 415 Gly Leu Gly Gly Gly Gly Phe Asp Val Asp Glu Ala Val Lys Glu Val 420 425 430 Val Ala Ser Tyr Arg Ser Ala Thr Arg Pro Ser Ala Ala Ala Leu Leu 435 440 445 Ala Pro Thr Thr Glu Thr Leu Leu Ala Ala Leu Gln Leu Ser Pro Ala 450 455 460 Leu Pro Leu Gln Gln Met Ala Pro Ala Ala Ala Ser Pro Glu Ser Pro 465 470 475 480 Thr Lys Gly Ala Asp Gly Arg Ala Ser Pro Ser Ala Thr Arg Arg Asp 485 490 495 Glu Thr Val Trp Gln Asn Arg Asn Ser Thr Arg Cys Ile Gly Gly Ser 500 505 510 Leu Gln Pro His His Pro Arg Ala Arg Thr Glu Ala Thr Arg Ala Ser 515 520 525 Ser Val Ser Thr Ala Ala Pro Lys Ser Ser Thr Pro Leu Trp Tyr Thr 530 535 540 Ala Gly Thr Gln Pro Leu Gln Gln Pro Pro Thr Ser Ala Leu Arg Ile 545 550 555 560 Phe Glu Asp Val Gly Pro Thr Gly Cys Ser Ser Ala Pro Ser Ser Thr 565 570 575 Ser Gly Met Val Ala Gly His Val Ser Ser Ser Phe Ala Cys Thr Asp 580 585 590 Gly Asp Gly Gly Ala Pro Pro Arg Glu Pro Ala Ala Thr Thr Leu Ser 595 600 605 Trp Phe Glu Asp Thr Thr Gln Gln Gln Gln Gln Ser 610 615 620 66369PRTCryptosporidium parvum 66Val Ala Val Ile Gly Cys Cys His Gly Glu Leu Asn Arg Leu Tyr Met 1 5 10 15 Glu Val Glu Lys Tyr Glu Asn Glu Lys Asn Glu Lys Val Asp Leu Ile 20 25 30 Leu Cys Cys Gly Asp Met Gln Thr Ile Arg Asp Glu Asn Asp Leu Gln 35 40 45 Asp Met Ala Val Lys Ser His Arg Ser Lys Lys Gly Asp Phe Trp Glu 50 55 60 Tyr Tyr Glu Gly Leu Lys Lys Ala Pro Lys Leu Thr Ile Phe Ile Gly 65 70 75 80 Gly Asn His Glu Thr Pro Asn Val Leu Ile Pro Leu Tyr Tyr Gly Gly 85 90 95 Trp Val Ala Pro Asn Ile Phe Tyr Leu Gly Ser Ser Gly Val Ile Arg 100 105 110 Val Gly Asp Val Arg Val Ala Gly Ile Ser Gly Ile Tyr Lys Asn Tyr 115 120 125 Asp His Phe Arg Gly Tyr Tyr Glu Ser Lys Pro Phe Thr Glu Glu Ser 130 135 140 Lys Arg Ser Trp Tyr His Ile Arg Trp Leu Glu Ile Gln Lys Leu Leu 145 150 155 160 Leu Ile Glu Asn Ile Lys Ser Asn Phe Leu Gly Ser Thr Glu Ser Arg 165 170 175 Lys Val Asp Val Met Ile Ser His Asp Trp Pro Asn Gly Ile Glu Arg 180 185 190 Phe Gly Asn Leu Asn Tyr Leu Ile Arg Arg Lys Pro Tyr Leu Lys Glu 195 200 205 Asp Ile Glu Leu Gly Arg Leu Gly Ile Pro Gly Cys Ile Glu Leu Ile 210 215 220 Glu His Leu Arg Pro Thr Phe Trp Phe Ser Gly His His His Cys Phe 225 230 235 240 Phe Asp Ala Ser Ile Glu Phe Glu Asn Gln Leu Tyr Ser Ser Glu Phe 245 250 255 Arg Ala Ile Asp Lys Phe Lys Asn Ser Asn Ser Pro Val Arg Tyr Phe 260 265 270 Asp Ile Asn Ser Asn Lys Asn Asp Val Arg Ile Tyr Leu Asp Phe Glu 275 280 285 Trp Leu Thr Ile Leu Arg Ser Val Lys Ala Asn Ile Pro Lys Gly Asn 290 295 300 Phe Thr Ile Asp Lys Asn Ser Ile Pro Lys Leu Ser Gly Pro Thr Lys 305 310 315 320 Ser Asp Ile Asp Ala Ile Tyr Lys Asn Leu Lys Glu Val Ile Gly Asp 325 330 335 Phe Asp Glu Asn His Tyr Glu Trp Pro Leu Trp Gly Gln Ala Asn Gly 340 345 350 Asn Phe Lys Asn Leu Gln Asp Gln Tyr Asn Phe Ile Asn Arg Ile Ile 355 360 365 Glu

Claims

1. An isolated debranching enzyme comprising an amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66.

2. The isolated debranching enzyme of claim 1, wherein the sequence identity is selected from the group consisting of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more.

3. The isolated debranching enzyme of claim 1, wherein the amino acid sequence has at least 75% sequence identity to the metallophosphatase domain of a sequence selected from the group consisting of SEQ ID NO: 46-66.

4. The isolated debranching enzyme of claim 3, wherein the sequence identity is selected from the group consisting of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more.

5. A method of enriching an RNA population for lariat RNA comprising: a. providing an RNA population; and, b. contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population.

6. The method of claim 5 further comprising contacting the RNA population with a debranching enzyme.

7. The method of claim 6, wherein the debranching enzyme comprises an amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66.

8. The method of claim 5, wherein the linear RNA degrading enzyme is selected from the group consisting of 3' exonuclease, 5' exonuclease, 5'/3' exonuclease, or combinations thereof.

9. A method of analyzing lariat RNA in an RNA sample comprising the steps of: a. providing an RNA population; b. contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population; and, c. creating a cDNA library from the lariat RNA population.

10. The method of claim 9, wherein the linear RNA degrading enzyme is selected from the group consisting of 3' exonuclease, 5' exonuclease, 5'/3' exonuclease, or combinations thereof.

11. The method of claim 9 further comprising contacting the lariat RNA enriched population with a debranching enzyme.

12. The method of claim 10, wherein the debranching enzyme comprises an amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66.

13. The method of claim 9 further comprising sequencing the cDNA library.

14. A kit comprising: a. a linear RNA degrading enzyme; b. buffer; and, c. instructions.

15. The kit of claim 14 further comprising a debranching enzyme.

16. The kit of claim 14 further comprising a debranching enzyme buffer.

17. The kit of claim 15, wherein the debranching enzyme comprises a amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66.

18. The kit of claim 17, wherein the sequence identity is selected from the group consisting of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more.

19. The kit of claim 14, wherein the linear RNA degrading enzyme is selected from the group consisting of 3' exonuclease, 5' exonuclease, 5'/3' exonuclease, or combinations thereof.

20. The kit of claim 14 further comprising a 5' decapping enzyme.

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