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PubMed2006

SPA: a probabilistic algorithm for spliced alignment.

Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5' and 3' ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non-canonical splice site that we also find in the mouse dataset. The SPA software package is available at http://www.biozentrum.unibas.ch/personal/nimwegen/cgi-bin/spa.cgi.

Algorithms↗

PubMed2005

Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes.

Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.

Alternative Splicing↗

PubMed2002

Two alternative exons can result from activation of the cryptic splice acceptor site deep within intron 2 of the dystrophin gene in a patient with as yet asymptomatic dystrophinopathy.

Intron 2 of the dystrophin gene is unusually large, extending 157 kb on the X-chromosome, and is known to contain one cryptic exon 2a. Here, we report that a single nucleotide change in the middle of this huge intron is a source of two novel extra exons. A novel point mutation changing T to A nucleotide was identified at 5591 bp downstream from the 3' end of exon 2 (T310+5591A) in genomic DNA of an asymptomatic dystrophinopathy case. The mutation identification was initiated by detection of two novel dystrophin mRNAs containing a 132-nucleotide or 46-nucleotide insertion between exons 2 and 3 in lymphocytes but one with a 132-nucleotide insertion in skeletal muscle. It was concluded that T310+5591A created a novel consensus sequence for a splice acceptor site leading to the formation of two novel exon structures by using two cryptic splice donor sites at 132 bp or 46 bp downstream. The former maintained the dystrophin reading frame and was expected to insert 44 amino acids in the N-terminal domain of dystrophin, whereas the latter created a premature stop codon. An immunohistochemical study of the skeletal muscle of the patient disclosed that the N-terminal domain of dystrophin was not stained, but the rod- and C-terminal domains were stained in a patchy and discontinuous manner, indicating that the in-frame mRNA was functional. Creation of a splice acceptor site by a single nucleotide change leading to extra exon structures is a novel molecular mechanism in human disease.

Alternative Splicing↗

PubMed2004

Exon junction sequences as cryptic splice sites: implications for intron origin.

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns. To do this, we experimentally identified nine cryptic splice sites within the coding sequence of actin genes from humans, Arabidopsis, and Physarum by inactivating their normal intron splice sites. We found that seven of these cryptic splice sites correspond exactly to the positions of exon junctions in actin genes from other species. Because actin genes are highly conserved, we could conclude that at least seven actin introns are flanked by cryptic splice sites, and from the phylogenetic evidence, we could also conclude that actin introns were inserted into these cryptic splice sites during evolution. Furthermore, our results indicate that these insertion events were dependent upon the splicing machinery. Because most introns are flanked by similar sequences, our results are likely to be of general relevance.

Actins↗

PubMed2003

Sequence information for the splicing of human pre-mRNA identified by support vector machine classification.

Vertebrate pre-mRNA transcripts contain many sequences that resemble splice sites on the basis of agreement to the consensus,yet these more numerous false splice sites are usually completely ignored by the cellular splicing machinery. Even at the level of exon definition,pseudo exons defined by such false splices sites outnumber real exons by an order of magnitude. We used a support vector machine to discover sequence information that could be used to distinguish real exons from pseudo exons. This machine learning tool led to the definition of potential branch points,an extended polypyrimidine tract,and C-rich and TG-rich motifs in a region limited to 50 nt upstream of constitutively spliced exons. C-rich sequences were also found in a region extending to 80 nt downstream of exons,along with G-triplet motifs. In addition,it was shown that combinations of three bases within the splice donor consensus sequence were more effective than consensus values in distinguishing real from pseudo splice sites; two-way base combinations were optimal for distinguishing 3' splice sites. These data also suggest that interactions between two or more of these elements may contribute to exon recognition,and provide candidate sequences for assessment as intronic splicing enhancers.

Artificial Intelligence↗

PubMed2004

A probabilistic model of 3' end formation in Caenorhabditis elegans.

The 3' ends of mRNAs terminate with a poly(A) tail. This post-transcriptional modification is directed by sequence features present in the 3'-untranslated region (3'-UTR). We have undertaken a computational analysis of 3' end formation in Caenorhabditis elegans. By aligning cDNAs that diverge from genomic sequence at the poly(A) tract, we accurately identified a large set of true cleavage sites. When there are many transcripts aligned to a particular locus, local variation of the cleavage site over a span of a few bases is frequently observed. We find that in addition to the well-known AAUAAA motif there are several regions with distinct nucleotide compositional biases. We propose a generalized hidden Markov model that describes sequence features in C.elegans 3'-UTRs. We find that a computer program employing this model accurately predicts experimentally observed 3' ends even when there are multiple AAUAAA motifs and multiple cleavage sites. We have made available a complete set of polyadenylation site predictions for the C.elegans genome, including a subset of 6570 supported by aligned transcripts.

3' Untranslated Regions↗

PubMed2005

Functional studies on the ATM intronic splicing processing element.

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5' and 3' splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a approximately 40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5'-3' order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.

Animals↗

PubMed2002

Non-expression of HLA-A*2901102 N is caused by a nucleotide exchange in the mRNA splicing site at the beginning of intron 4.

We describe the identification of the novel human leukocyte antigen (HLA) blank allele A*2901102 N which was detected in an individual of mixed race. The serological HLA class I typing was A1; B7,44 whereas PCR-SSP indicated the presence of an additional A*29 allele. The pedigree analysis demonstrated that the new blank allele segregated with the haplotype A*29null B*07, inherited from the individual's Vietnamese father. A single G-->Tau transversion was detected at position 1 of intron 4, which is a highly conserved nucleotide position in vertebrate splice donor sites. Accordingly, it is very likely that this nucleotide exchange inhibits the splicing of intron 4, resulting in a premature stop codon further downstream. Despite this alteration, transcription into mRNA was demonstrated.

Alleles↗

PubMed2002

Characterization of U2AF(6), a splicing factor related to U2AF(35).

The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. U2AF(35) has multiple functions in pre-mRNA splicing. First, U2AF(35) has been shown to function by directly interacting with the AG at the 3' splice site. Second, U2AF(35) is thought to play a role in the recruitment of U2AF(65) by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF(35) and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF(35), designated U2AF(26). The N-terminal 187 amino acids of U2AF(35) and U2AF(26) are nearly identical. However, the C-terminal domain of U2AF(26) lacks many characteristics of the U2AF(35) RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF(26) can associate with U2AF(65) and can functionally substitute for U2AF(35) in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF(26) functions by enhancing the binding of U2AF(65) to weak 3' splice sites. These studies identify U2AF(26) as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF(35), U2AF(26) may play a role in regulating alternative splicing.

Amino Acid Sequence↗

PubMed2006

Predicting severity of haemophilia A and B splicing mutations by information analysis.

Bleeding symptoms and clotting activity vary among mutations that alter mRNA splicing of either the factor VIII or factor IX genes. We analyzed splicing mutations in both genes for changes in individual information (R(i), in bits) involving both donor or acceptor sites. Mutations with low or negative R(i) values (<2.4 bits) or significant changes in R(i) (DeltaR(i) > or = 7 bits) exhibited either reduced protein activity, increased clotting time and bleeding frequency and were predictive of severe disease. Thus, information analysis of splicing mutations may be useful in predicting phenotypes in hemophilia.

Factor IX↗

PubMed2005

Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer.

Ancient Alu elements have been shown to be included in mature transcripts by point mutations that improve their 5' or 3' splice sites. We have examined requirements for exonization of a younger, disease-associated AluYa5 in intron 16 of the human ACE gene. A single G>C transversion in position -3 of the new Alu exon was insufficient for Alu exonization and a significant inclusion in mRNA was only observed when improving several potential splice donor sites in the presence of 3' CAG. Since complete Alu exonization was not achieved by optimizing traditional splicing signals, including the branch site, we tested whether auxiliary elements in AluYa5 were required for constitutive inclusion. Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 222-228 and point mutations in highly conserved nucleotides of this heptamer decreased Alu inclusion. In addition, we show that Alu exonization was facilitated by a subset of serine/arginine-rich (SR) proteins through activation of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE minigenes generated similar splicing patterns in both ACE-expressing and non-expressing cells, suggesting that previously reported allelic association with plasma ACE activity and cardiovascular disease is not attributable to differential splicing of introns 16 and 17.

Alu Elements↗

PubMed2003

Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disorder characterised by the absence of the protein dystrophin. Antisense oligonucleotides have been used to re-direct dystrophin pre-mRNA processing by blocking sequences crucial to pre-mRNA splicing, thereby inducing skipping of specific exons. We wished to determine which splicing motifs are most amenable as targets for antisense oligonucleotide induction of efficient and specific skipping of selected exons. METHODS: Antisense oligonucleotides were directed at regions of dystrophin exon 19 involved in pre-mRNA splicing, including the donor and acceptor splice sites and the exon splicing enhancer (ESE). Cultured myotubes were transfected with antisense oligonucleotides at various concentrations and studies undertaken to determine both specificity and efficiency of induced exon 19 skipping. RESULTS: Antisense oligonucleotides as small as 12 nucleotides targeting the ESE induced consistent and specific skipping of only exon 19 in both human and normal and mdx mouse myotubes. Antisense oligonucleotides directed at the donor and acceptor splice sites also induced specific exon 19 skipping while mismatched antisense oligonucleotides could only induce skipping when delivered at higher concentrations. No other dystrophin exons were removed from the mature mRNA as a consequence of these antisense oligonucleotides treatments. CONCLUSIONS: Antisense oligonucleotides directed at the ESE tended to be marginally more efficient than those which targeted the donor or acceptor splice sites, based on their ability to induce specific skipping at lower concentrations. The specificity of exon removal does not appear to be a function of target selection, but may reflect the combination of the splicing motifs and position of that exon in the pre-mRNA.

Animals↗

PubMed2003

Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene.

Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2alpha and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.

Animals↗

PubMed2005

A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro.

The splicing pattern of pre-mRNA is unpredictable in genes harboring a single-nucleotide change within the consensus sequence of a splice-donor site. In the dystrophin gene, a transition from G to A at the fifth position of intron-32 (4518+5G > A) has been reported as a polymorphism within the consensus sequence or a mutation identified in Duchenne muscular dystrophy (DMD). Here, we report both in vivo and in vitro evidence that shows inactivation of the splice-donor site caused by this mutation. In one Japanese DMD case, two novel dystrophin mRNAs were identified in the patient's lymphocytes, one with a 98 bp deletion of the 3' end of exon-32 (dys32-98) and the other with a 28 bp intron retained between exons 32 and 33 (dys32 + 28). Genomic sequencing disclosed a single-nucleotide change from G to A at the fifth position of intron-32 (4518+5G > A). To demonstrate in vitro the inactivation of this splice-donor site by this nucleotide change, mini-dystrophin genes comprising three exons harboring either normal or mutant intron-32 sequences were expressed in HeLa cells, and the splicing products were analyzed by reverse-transcription PCR amplification. A normal transcript consisting of three exons was obtained from the normal construct. From the mutant, we obtained one product containing a 98 bp deletion at the 3' end of exon-32, indicating complete inactivation of the native splice-donor site. Thus, both in vivo and in vitro experiments demonstrate that 4518+5G > A causes a splicing error leading to transcript termination; it did not behave like a silent polymorphism. Our results indicate that the in vitro splicing system is a powerful tool for determining the underlying mechanism of a disease-causing mutation in a splicing consensus sequence.

Base Sequence↗

PubMed2006

AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing.

More than 90% of human genes are rich in intronic latent 5' splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5' splice sites regulates alternative 5' splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin-a drug known to block translation by its ability to recognize an RNA fold-but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism.

Animals↗

PubMed2001

Splicing of constitutive upstream introns is essential for the recognition of intra-exonic suboptimal splice sites in the thrombopoietin gene.

The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon-intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.

Alternative Splicing↗

PubMed2000

The splicing determinants of a regulated exon in the axonal MAP tau reside within the exon and in its upstream intron.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression profile is distinct from that of the other tau regulated exons, implying the utilization of distinct regulatory factors. Previous work had established the use of cryptic splice sites within exon 6 and the influence of flanking exons on the ratio of exon 6 variants. The present work shows that, in addition to the previously identified participants, the ratio of exon 6 isoforms is affected by: (1) suppression of the cryptic sites, (2) deletions of the upstream intron, and (3) the splicing regulators PTB and U2AF, both of which act on the branch point/polypyrimidine tract region. These results strongly suggest that factors binding immediately upstream of exon 6 are involved in regulation of this exon. They also lead to the conclusion that splicing of exon 6 is primarily governed by multiple branch points.

Animals↗

PubMed2003

Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene.

BACKGROUND: Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission. RESULTS: Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A) site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression. CONCLUSIONS: Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A) site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.

3' Untranslated Regions↗