MOTIVATION: A new protocol for sequencing the messenger RNA in a cell, known as RNA-Seq, generates millions of short sequence fragments in a single run. These fragments, or 'reads', can be used to measure levels of gene expression and to identify novel splice variants of genes. However, current software for aligning RNA-Seq data to a genome relies on known splice junctions and cannot identify novel ones. TopHat is an efficient read-mapping algorithm designed to align reads from an RNA-Seq experiment to a reference genome without relying on known splice sites. RESULTS: We mapped the RNA-Seq reads from a recent mammalian RNA-Seq experiment and recovered more than 72% of the splice junctions reported by the annotation-based software from that study, along with nearly 20,000 previously unreported junctions. The TopHat pipeline is much faster than previous systems, mapping nearly 2.2 million reads per CPU hour, which is sufficient to process an entire RNA-Seq experiment in less than a day on a standard desktop computer. We describe several challenges unique to ab initio splice site discovery from RNA-Seq reads that will require further algorithm development. AVAILABILITY: TopHat is free, open-source software available from http://tophat.cbcb.umd.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
We present an improved splice site predictor for the genefinding program Genie. Genie is based on a generalized Hidden Markov Model (GHMM) that describes the grammar of a legal parse of a multi-exon gene in a DNA sequence. In Genie, probabilities are estimated for gene features by using dynamic programming to combine information from multiple content and signal sensors, including sensors that integrate matches to homologous sequences from a database. One of the hardest problems in genefinding is to determine the complete gene structure correctly. The splice site sensors are the key signal sensors that address this problem. We replaced the existing splice site sensors in Genie with two novel neural networks based on dinucleotide frequencies. Using these novel sensors, Genie shows significant improvements in the sensitivity and specificity of gene structure identification. Experimental results in tests using a standard set of annotated genes showed that Genie identified 86% of coding nucleotides correctly with a specificity of 85%, versus 80% and 84% in the older system. In further splice site experiments, we also looked at correlations between splice site scores and intron and exon lengths, as well as at the effect of distance to the nearest splice site on false positive rates.
A systematic analysis of the RNA splice junction sequences of eukaryotic protein coding genes was carried out using the GENBANK databank. Nucleotide frequencies obtained for the highly conserved regions around the splice sites for different categories of organisms closely agree with each other. A striking similarity among the rare splice junctions which do not contain AG at the 3' splice site or GT at the 5' splice site indicates the existence of special mechanisms to recognize them, and that these unique signals may be involved in crucial gene-regulation events and in differentiation. A method was developed to predict potential exons in a bare sequence, using a scoring and ranking scheme based on nucleotide weight tables. This method was used to find a majority of the exons in selected known genes, and also predicted potential new exons which may be used in alternative splicing situations.
EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.
Splice junction sequences from a large number of nuclear and viral genes encoding protein have been collected. The sequence CAAG/GTAGAGT was found to be a consensus of 139 exon-intron boundaries (or donor sequences) and (TC)nNCTAG/G was found to be a consensus of 130 intron-exon boundaries (or acceptor sequences). The possible role of splice junction sequences as signals for processing is discussed.
This paper analyses losses caused by the misalignment of two fibers joined in a splice. We consider the possibility that the two fibers of different dimensions are separated in longitudinal direction and are tilted or offset with respect to each other. Central to our discussion is the observation that the modes of single-mode fibers are very nearly gaussian in shape regardless of the fiber type step-index or graded-index. The splice losses are thus related to the corresponding losses of gaussian beams. We specify the relation between the actual mode field and the gaussian beam that matches this field optimally. The trade-off between slice tolerances with respect to tilt and offset is expressed as an “uncertainty principle. “ Because of the near-gaussian nature of single-mode fiber fields, our results are immediately applicable to the excitation of single-mode fibers by gaussian-shaped laser beams.
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An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope,branched forms were observed in which 160 nucleotides of RNA from the 5' terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.
INTRODUCTIONThe ProblemThe average vertebrate gene consists of multiple small exons (average size, 137 nucleotides) separated by introns that are considerably larger(1Hawkins J.D. Nucleic Acids Res. 1988; 16: 9893-9908Crossref PubMed Scopus (441) Google Scholar). Thus, the vertebrate splicing machinery has the task of finding small desired exons amid much longer introns. The splice site consensus sequences that drive exon recognition are located at the very termini of introns(2Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar, 3Moore M.J. Query C.C. Sharp P.A. Gestland R. Atkins R. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-357Google Scholar). Despite the discriminatory challenge faced during exon recognition in large multiexon premessenger RNAs, vertebrate splice sites are short and poorly conserved. In fact, splice site sequences in mammals are less conserved than their yeast counterparts despite the fact that only a minority of genes in Saccharomyces cerevisiae have introns; and those genes that are split by introns usually have only a single intron(4Guthrie C. Science. 1991; 253: 157-163Crossref PubMed Scopus (314) Google Scholar, 5Ruby S. Abelson J. Trends Genet. 1991; 7: 79-85Abstract Full Text PDF PubMed Scopus (184) Google Scholar). Thus, vertebrate splicing contends with a more complex specificity problem via recognition of less precise consensus sequences. Any mechanism for the orchestration of splicing in multiexon vertebrate genes must provide an explanation for this puzzle.Part of the solution of the puzzle comes from the observation that individual splice sites are not independently recognized consensus sequences. In both yeast and vertebrate splicing, interactions between 5′ and 3′ splice sites and the factors that recognize them have been observed during the earliest steps of spliceosome assembly(4Guthrie C. Science. 1991; 253: 157-163Crossref PubMed Scopus (314) Google Scholar, 5Ruby S. Abelson J. Trends Genet. 1991; 7: 79-85Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 6Jamison S.F. Crow A. Garcia-Blanco M.A. Mol. Cell. Biol. 1992; 12: 4279-4287Crossref PubMed Scopus (80) Google Scholar, 7Lammond A.L. Konarska M.M. Sharp P.A. Genes & Dev. 1987; 1: 532-543Crossref PubMed Scopus (106) Google Scholar, 8Kuo H.-C. Nasim F.H. Grabowski P.J. Science. 1991; 251: 1045-1050Crossref PubMed Scopus (130) Google Scholar, 9Michaud S. Reed R. Genes & Dev. 1993; 7: 1008-1020Crossref PubMed Scopus (140) Google Scholar, 10Robberson B.L. Cote G.J. Berget S.M. Mol. Cell. Biol. 1990; 10: 84-94Crossref PubMed Scopus (547) Google Scholar, 11Rosbash M. Seraphin B. Science. 1991; 16: 187-190Scopus (115) Google Scholar, 12Wu J.Y. Maniatis T. Cell. 1993; 75: 1061-1070Abstract Full Text PDF PubMed Scopus (618) Google Scholar). Usually these interactions are depicted as occurring between the 5′ and 3′ splice sites across an intron. Experimentally, such interactions have been observed with in vitro splicing precursor RNAs having naturally short or artificially shortened introns. It is difficult to extrapolate initial interactions between the factors that recognize the 5′ and 3′ splice sites flanking a small vertebrate intron to introns that can naturally be 100 kilobases in length, especially given the likelihood that such introns will contain sequences that are as good a match to consensus splice sites as the actual utilized sites.Exon DefinitionModels that invoke pairing between the splice sites across an exon, as contrasted with pairing across an intron, are useful perspectives of splice site pairing for the splicing of pre-mRNAs with large introns and small exons. Such an exonic perspective of splice site recognition has been termed “exon definition”(10Robberson B.L. Cote G.J. Berget S.M. Mol. Cell. Biol. 1990; 10: 84-94Crossref PubMed Scopus (547) Google Scholar). This review discusses exon definition and contrasts it with intron-oriented perspectives that are more useful when considering splicing in lower eukaryotes with small introns. The basic exon definition model proposes that in pre-mRNAs with large introns, the splicing machinery searches for a pair of closely spaced splice sites in an exonic polarity (Fig. 1). When such a pair is encountered, the exon is defined by the binding of U1 and U2 snRNPs ( 1The abbreviations used are: snRNPsmall nuclear ribonucleoproteinSRarginine- and serine-rich splicing factorshnRNPheterogeneous nuclear ribonucleoprotein. )and associated splicing factors, including the 3′ splice site recognizing factors U2AF and SC35 and the 5′ splice site-recognizing factor ASF/SF2(2Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar, 13Eperon I.C. Ireland D.C. Smith R.A. Mayeda A. Krainer A.R. EMBO J. 1993; 12: 3607-3617Crossref PubMed Scopus (169) Google Scholar, 14Fu X.-D. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11224-11228Crossref PubMed Scopus (198) Google Scholar, 15Kohtz J.D. Jamison S.F. Will C.L. Zuo P. Luhrmann R. Garcio-Blanco M.A. Manley J.L. Nature. 1994; 368: 119-124Crossref PubMed Scopus (527) Google Scholar, 16Zuo P. Manley J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3363-3367Crossref PubMed Scopus (90) Google Scholar). Following definition of the exon, neighboring exons must be juxtaposed, presumably via interactions between the factors that recognize individual exons. Thus, from this perspective, assembly of the active vertebrate spliceosome consists of the sequential steps of exon definition and exon juxtaposition.Predictions of Exon DefinitionThe exon definition model offers predictions of pre-mRNA behavior. Several of these predictions have been tested in the last several years, and the results lend credence to an exonic perspective of splice site recognition.Exon SkippingExon-oriented and intron-oriented perspectives of splice site pairing predict different phenotypes resulting from mutation of splice sites bordering an internal exon (Fig. 2). Models invoking an initial pairing of splice sites across introns predict that such mutations should inhibit splicing of the intron in which they occur but should have minimal impact on the splicing of neighboring introns. In contrast, exon definition predicts that mutation of a splice site bordering an internal exon should depress recognition of the exon with concomitant inhibition of splicing of the adjoining intron, i.e. mutations in an intron will inhibit the splicing of two introns, the intron containing the mutation and the intron on the other side of the exon bearing the mutation. This hypothesis has been tested in vitro, where it was observed that mutation of a 5′ splice site depressed the removal of the upstream intron 20-fold(17Talerico M. Berget S.M. Mol. Cell. Biol. 1990; 10: 6299-6305Crossref PubMed Scopus (158) Google Scholar). The converse experiments have also been reported. Strengthening a naturally weak 5′ splice site of an internal exon by making it a better fit to the consensus site increased in vitro splicing of the upstream intron(8Kuo H.-C. Nasim F.H. Grabowski P.J. Science. 1991; 251: 1045-1050Crossref PubMed Scopus (130) Google Scholar, 18Grabowski P.J. Nasim F.H. Kuo H.-C. Burch R. Mol. Cell. Biol. 1991; 11: 5919-5928Crossref PubMed Scopus (36) Google Scholar). In vivo, mutant 5′ splice sites were genetically suppressed by second mutations that improved the 3′ splice site across the exon(19Carothers A.M. Urlaub G. Grunberger D. Chasin L. Mol. Cell. Biol. 1993; 13: 5085-5098Crossref PubMed Scopus (67) Google Scholar, 20Tsukahara T. Casciato C. Helfman D.M. Nucleic Acids Res. 1994; 22: 2318-2325Crossref PubMed Scopus (35) Google Scholar).Figure 2Predictions of the phenotype of mutation of the 5′ splice site bordering an internal exon. Exon pairing of splice sites predicts exon skipping or the activation of a proximal cryptic 5′ splice site (left), whereas intronic pairing of splice sites predicts intron inclusion or distal cryptic site activation (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutation of vertebrate splice sites also leads to exon skipping. A survey of mammalian mutations available in the data base in the summer of 1994 indicated that over 100 splice site mutations have been characterized in disease gene DNA(21Nakai K. Sakamoto H. Gene (Amst.). 1994; 141: 171-177Crossref PubMed Scopus (255) Google Scholar). Four phenotypes were observed: exon skipping, activation of a cryptic splice site, creation of a pseudo-exon within an intron, and intron retention, in ratios of 51, 32, 11, and 6%, respectively. The most frequent phenotype was exon skipping. Exon skipping is a predicted phenotype from an exon perspective because mutation of the splice site at one side of an exon should inhibit pairing of splice sites across exons and inhibit recognition of the exon. Rejection of the exon leads directly to exon skipping.The observation of exon skipping strongly indicates that splice sites are recognized as exonic pairs. It is presumably this dependence upon a pair of sites that minimizes recognition of isolated cryptic sites within large vertebrate introns. Occasionally, mutation of human genes has created a strong splice site deep within an intron. Such created sites have been observed to be utilized via the activation of a nearby cryptic splice site of the opposite polarity to create a pseudo-exon from within an intron. Again, the observation is that only pairs of splice sites can be recognized and that cryptic splices in introns can only be activated by creation of a nearby site of the opposite type in an exonic polarity.Occasionally, mutation of an internal splice site results in intron retention. Exon definition would not predict intron retention, except perhaps for very small introns. Of the splice site mutations mentioned above, only 6% caused intron retention. Four of the included introns were very short, and three were terminal introns, suggesting abrogation of exon definition modes of recognition when introns are very small or at the ends of pre-mRNAs (see below). Three examples involved large internal introns and cannot be explained by current exon perspectives.Exon Size MaximumIn addition to exon skipping, the other major phenotype resulting from mutation of a splice site in a human gene is activation of a cryptic site of the same type. The activated cryptic site always lies close to the mutated site, suggesting that splice sites are acceptable only if they reside close to a site of the opposite polarity and that, therefore, internal vertebrate exons may have a size maximum imposed in part by the splicing machinery. Fig. 3 indicates the size distribution of 1600 primate internal exons. Of these exons, only 3.5% are longer than 300 nucleotides and less than 1% are longer than 400 nucleotides, indicating that large internal exons are rare. In vitro, the assembly of ATP-dependent spliceosomes is inhibited if internal exons with strong constitutive splice sites are internally expanded to greater than 300 nucleotides(10Robberson B.L. Cote G.J. Berget S.M. Mol. Cell. Biol. 1990; 10: 84-94Crossref PubMed Scopus (547) Google Scholar). In vivo, expansion of internal exons residing in vertebrate genes with moderate to large introns has two phenotypes: activation of internal cryptic splice sites within the expanded exon to create small exons or skipping of the entire exon (see below). ( 2D. A. Sterner, T. Carlo, and S. M. Berget, unpublished data. )These phenotypes are consistent with splicing-imposed restriction on exon length. Presumably, such a size limitation helps explain why cryptic splice sites located inside of long vertebrate introns are not occasionally misrecognized to create large internal exons when the normal sites are mutated. A few spectacularly long vertebrate internal exons exit; the mechanism whereby such exons bypass restrictions on exon length is unknown.Figure 3Internal exon size distribution. Length distribution of 1600 primate internal exons from a library normalized to represent highly related exons only a single time (top) (library kindly provided by D. Searles, University of Pennsylvania) or 194 alternative vertebrate cassette exons (bottom) compiled by Stamm et al.(46Tian M. Maniatis T. Genes & Dev. 1994; 8: 1703-1712Crossref PubMed Scopus (132) Google Scholar) or by S. Smith and T. A. Cooper (Baylor College of Medicine).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Exon Size MinimumSimultaneous recognition of splice sites bordering an exon also suggests that a minimal separation between the sites might be required to prevent steric hindrance between the factors that recognize individual sites. When a constitutively recognized internal exon was internally deleted below 50 nucleotides it was skipped by the in vivo splicing machinery(23Dominski Z. Kole R. Mol. Cell. Biol. 1991; 11: 6075-6083Crossref PubMed Scopus (178) Google Scholar). Increasing the strength of the splice sites alleviated problems in recognition, suggesting that exon size and splice site strength are additive factors in exon recognition(24Dominski Z. Kole R. Mol. Cell. Biol. 1992; 12: 2108-2114Crossref PubMed Scopus (71) Google Scholar). Some very small natural internal exons exist. Six and seven nucleotide exons are frequently found in muscle protein genes; N-CAM has a three-nucleotide exon. Although few very small exons have been studied, those that have suggest that very small exons require special enhancing sequences in addition to strong splice sites for inclusion(25Black D.L. Genes & Dev. 1991; 5: 389-402Crossref PubMed Scopus (114) Google Scholar, 26Black D.L. Cell. 1992; 69: 795-807Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 27Sterner D.A. Berget S.M. Mol. Cell. Biol. 1993; 13: 2677-2687Crossref PubMed Scopus (68) Google Scholar). Deletion of these elements causes exon skipping when the exon is small but not when it has been internally expanded to a more normal length. The small exon enhancers are located within the neighboring introns outside of the normal splice sites. It seems likely that such enhancers function as binding sites for splicing factors that artificially extend the exon domain during exon recognition.Terminal ExonsExon definition suggests that terminal exons, both first and last exons, will require special mechanisms for their recognition. First exons end with a 5′ splice site but have no processing signal at their beginning. They do, however, bear a modification at their beginning via the 7-methylguanosine cap attached to all polymerase II transcripts. The cap and nuclear proteins that bind the cap are essential for in vitro splicing of simple one-intron pre-mRNAs(28Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (425) Google Scholar). In two-intron pre-mRNAs, changing the guanosine cap to an adenosine cap depressed removal of the first intron in vitro but had only minimal impact on the second intron (29Ohno M. Sakamoto H. Shimura Y. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5187-5191Crossref PubMed Scopus (97) Google Scholar). These results indicate both that pre-mRNAs are recognized segmentally in vitro and that the cap is essential for recognition and removal of the first intron. Or as stated from an exon perspective, first exons can be recognized via interactions between the factors that recognize caps and 5′ splice sites.Last exons begin with a 3′ splice site and terminate with a poly(A) site(30Manley J.L. Proudfoot N.J. Genes & Dev. 1993; 8: 259-264Crossref Scopus (35) Google Scholar). They are often the largest exon in a vertebrate gene, with an average size of approximately 600 nucleotides(1Hawkins J.D. Nucleic Acids Res. 1988; 16: 9893-9908Crossref PubMed Scopus (441) Google Scholar, 31Brunak S. Engelbrecht J. Knudsen S. J. Mol. Biol. 1991; 220: 49-65Crossref PubMed Scopus (623) Google Scholar). Exon recognition predicts that factors recognizing 3′ splice sites interact with factors recognizing poly(A) sites to recognize last exons. Indeed mutation of 3′ splice sites inhibits the in vitro polyadenylation cleavage reaction(32Niwa M. Rose S.R. Berget S.M. Genes & Dev. 1990; 4: 1552-1559Crossref PubMed Scopus (228) Google Scholar). Just as with first exons, mutation of the signal at the distal end of a 3-terminal exon, the poly(A) site, inhibits in vitro removal of proximal but not distal introns(33Niwa M. Berget S.M. Genes & Dev. 1991; 5: 2086-2095Crossref PubMed Scopus (149) Google Scholar). These results suggest that splicing and polyadenylation factors interact across 3′-terminal exons. The mechanism of this interaction is unclear, although recent observations have suggested that U1 snRNPs or the U1 snRNP A protein are involved, either positively or negatively, via recognition of exon internal sequences upstream of the polyadenylation signal AAUAAA(34Boelens W.C. Jansen E.J. Ven Venrooij W.J. Stripeke R. Mattaj I.W. Gunderson S.I. Cell. 1993; 72: 881-892Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 35Lutz C.S. Alwine J.C. Genes & Dev. 1994; 8: 576-586Crossref PubMed Scopus (126) Google Scholar, 36Wasserman K.M. Steitz J.A. Genes & Dev. 1993; 7: 647-659Crossref PubMed Scopus (91) Google Scholar).Exon Enhancer Sequences and Differential SplicingExon definition has proven to be a useful framework for considering differential splicing, especially those differential splicing events involving cassette exons that are differentially included. Generally, differentially recognized exons have either weaker splicing signals or a suboptimal length compared with constitutive exons (3Moore M.J. Query C.C. Sharp P.A. Gestland R. Atkins R. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-357Google Scholar, 37Stamm S. Zhang M.Q. Marr T.G. Helfman D.M. Nucleic Acids Res. 1994; 22: 1515-1526Crossref PubMed Scopus (87) Google Scholar) (Fig. 3), suggesting that the constitutive exon definition process is so strong as to be difficult to regulate unless the involved exon recognition signals are weak. Exon inclusion in these cases appears to be via recognition of special sequences by tissue or development-specific splicing factors(38Cooper T.A. Ordahl C.P. Nucleic Acids Res. 1989; 17: 6999-7011Crossref PubMed Scopus (59) Google Scholar, 39Dirksen W.P. Hampson R.K. Sun Q. Rottman F.M. J. Biol. Chem. 1994; 269: 6431-6436Abstract Full Text PDF PubMed Google Scholar, 40Hedley M.L. Maniatis T. 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Morfin J.P. Merillat N. Rosenfield M.G. Emerson R.B. Mol. Cell. Biol. 1993; 13: 5999-6011Crossref PubMed Scopus (71) Google Scholar). One class of sequences commonly found associated with differential exons, referred to as exon enhancers, resides within the target exon. The existence of exon internal consensus sequences was initially surprising because of the constraints imposed upon such sequences by coding requirements. A family of such sequences, often purine-rich and coding for a wide variety of amino acids, has been observed to be important for recognition of weak exons. These sequences appear to be the binding site for a family of splicing factors known as SR proteins because of the arginine and serine repeats that characterize them(50Zahler A.M. J.A. Genes & Dev. 1992; PubMed Scopus Google Scholar). In addition to binding exon sequences via their RNA binding the SR proteins also via their SR with U2AF the 3′ splice site and U1 snRNPs to the 5′ splice site via in J.Y. Maniatis T. Cell. 1993; 75: 1061-1070Abstract Full Text PDF PubMed Scopus (618) Google Scholar, 15Kohtz J.D. Jamison S.F. Will C.L. Zuo P. Luhrmann R. Garcio-Blanco M.A. Manley J.L. Nature. 1994; 368: 119-124Crossref PubMed Scopus (527) Google Scholar, D. Reed R. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). Such recognition the SR proteins for proteins involved in exon across exons has been in that of U2AF to the 3′ splice site of an isolated exon is by the strength of the 5′ splice site the Grabowski P.J. Genes & Dev. 1992; PubMed Scopus Google SR proteins have not been found in S. those yeast splicing proteins that are in known function to vertebrate proteins containing SR SR in their yeast N. M. Genes & Dev. 1994; 8: PubMed Scopus Google Scholar, J. M. Genes & Dev. 1993; 7: PubMed Scopus Google Scholar). an exon definition this may not be surprising in that with small introns, such as S. may not exon definition and may not or all of the SR In to suggest that pre-mRNAs with small introns the intron, than the exon, as the initial of pairing between splice C. Science. 1991; 253: 157-163Crossref PubMed Scopus (314) Google Scholar, 5Ruby S. Abelson J. Trends Genet. 1991; 7: 79-85Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 11Rosbash M. Seraphin B. Science. 1991; 16: 187-190Scopus (115) Google Scholar). In Saccharomyces pre-mRNAs have multiple small introns of less than 100 M.Q. Marr T.G. Nucleic Acids Res. 1994; 22: PubMed Scopus Google Scholar). In of the introns are less than 100 nucleotides and are often by large J.D. Nucleic Acids Res. 1988; 16: 9893-9908Crossref PubMed Scopus (441) Google Scholar, S.M. C. G. C. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar). small introns in either inhibits splicing of the intron or cryptic sites within the expanded M. S.M. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar, M. Berget S.M. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). ( in genes with small exons, the exon leads to splicing, whereas in genes with small introns, the introns leads to These observations suggests that the pairing utilized is that the between two splice of splice sites in genes with small introns has a different phenotype than the same mutation in genes with large introns. In pre-mRNAs with small introns, mutation of an internal 5′ splice site not to exon skipping. the mutated intron is included in the and the splicing of neighboring introns is M. Berget S.M. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). A in splicing signals between the two of introns has also been S.M. C. G. C. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar, M. S.M. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar). introns often the located between the and the 3′ splice site of vertebrate but not S. cerevisiae introns. small introns appear to have different signals and to be recognized than large pairing of splice sites across an exon may be to initial pairing across an intron. for the SR the vertebrate factors known to be required for splicing are found in yeast and are required as Several of also suggest that either the intron or exon can be the pairing during pre-mRNA recognition. mentioned expansion of an internal exon in a vertebrate gene can exon skipping. the same exons and their flanking splice however, are in a gene in which the introns flanking the expanded exon are the expanded exon is constitutively Chasin Mol. Cell. Biol. 1994; PubMed Scopus Google of the small introns the phenotype to exon skipping. These observations suggest that large exons are only a problem in genes with large introns, and more that the same splice sites can be recognized in either intronic or exonic polarity (Fig. complex in via exon definition that in lower eukaryotes via intron Large Image Figure ViewerDownload Hi-res image Download in also suggests multiple of pairing splice sites within the same Although genes fit two characterized as genes with small introns and large exons or as genes with small exons and large introns, are a of genes that have a suggesting that over part of their length the exon is the of recognition and over part of their length the intron is the of recognition. two such recognition mechanisms can within the same precursor RNA a in exon recognition or exon is one of the for exon exon definition suggests exons and their splice sites are initially recognized by the splicing it not a solution to the second in spliceosome assembly (Fig. 1). of exons across large vertebrate introns is a especially if exon skipping is to be is available as to such A likely interactions between the SR proteins to one exon with the SR proteins to an adjoining exon. In addition to the SR class of nuclear proteins found only in with large introns is the G. M.J. S. Annu. Rev. 1993; PubMed Scopus Google Scholar). one protein 5′ splice site recognition and is likely to have a major in differential splicing M.R. S. Y. Chabot B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, Stamm S. Helfman D.M. Krainer A.R. Science. 1994; PubMed Scopus Google Scholar). the SR the proteins contain both an recognition domain and a recognition the SR the available suggests that the proteins recognize intronic consensus sequences than exonic sequences. their to differentially recognize RNA sequences and their for intronic sequences, the proteins in both differential splicing and exon INTRODUCTIONThe ProblemThe average vertebrate gene consists of multiple small exons (average size, 137 nucleotides) separated by introns that are considerably larger(1Hawkins J.D. Nucleic Acids Res. 1988; 16: 9893-9908Crossref PubMed Scopus (441) Google Scholar). Thus, the vertebrate splicing machinery has the task of finding small desired exons amid much longer introns. The splice site consensus sequences that drive exon recognition are located at the very termini of introns(2Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar, 3Moore M.J. Query C.C. Sharp P.A. Gestland R. Atkins R. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-357Google Scholar). Despite the discriminatory challenge faced during exon recognition in large multiexon premessenger RNAs, vertebrate
Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells.
Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA-protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA-RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.
MOTIVATION: Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. RESULTS: To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. AVAILABILITY AND IMPLEMENTATION: STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from http://code.google.com/p/rna-star/.
Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-beta Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5' and 3' splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project.
BACKGROUND: Sm-like proteins are highly conserved proteins that form the core of the U6 ribonucleoprotein and function in several mRNA metabolism processes, including pre-mRNA splicing. Despite their wide occurrence in all eukaryotes, little is known about the roles of Sm-like proteins in the regulation of splicing. RESULTS: Here, through comprehensive transcriptome analyses, we demonstrate that depletion of the Arabidopsis supersensitive to abscisic acid and drought 1 gene (SAD1), which encodes Sm-like protein 5 (LSm5), promotes an inaccurate selection of splice sites that leads to a genome-wide increase in alternative splicing. In contrast, overexpression of SAD1 strengthens the precision of splice-site recognition and globally inhibits alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates with an increase in splicing accuracy and efficiency for stress-responsive genes. CONCLUSIONS: We conclude that SAD1 dynamically controls splicing efficiency and splice-site recognition in Arabidopsis, and propose that this may contribute to SAD1-mediated stress tolerance through the metabolism of transcripts expressed from stress-responsive genes. Our study not only provides novel insights into the function of Sm-like proteins in splicing, but also uncovers new means to improve splicing efficiency and to enhance stress tolerance in a higher eukaryote.
Rous sarcoma virus was shown to induce in chicken embryo fibroblasts (CEF) a 4.1-kilobase mRNA (designated CEF-147) encoding a 603-amino acid protein. Analysis of the protein sequence showed that it shared 59% amino acid identity with sheep prostaglandin G/H synthase, the enzyme that catalyzes the rate-limiting steps in the production of prostaglandins. Significant differences, at both the protein and mRNA levels, existed between the src oncogene product-inducible prostaglandin synthase and the protein isolated and cloned from sheep seminal vesicle, suggesting that the src-inducible prostaglandin synthase may be a new form of the enzyme. A distinguishing feature of src-inducible prostaglandin synthase mRNA is its low abundance in nonproliferating chicken embryo fibroblasts and its relatively high abundance in src-transformed cells. Additionally, the majority of the src-inducible prostaglandin synthase RNA present in nonproliferating cells was found to be nonfunctional because of the presence of an unspliced intron that separated the signal peptide from the remainder of the protein. Upon mitogenic stimulation, this intron was removed, resulting in the induction of fully-spliced CEF-147 mRNA.
To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine.
Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or "code" for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions.