Identification of alternative 5'/3' splice sites based on the mechanism of splice site competition

Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5'/3' splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified approximately 70% of the splice sites into alternative and constitutive, as well as approximately 80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology.     

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

Background  

Despite some degeneracy of sequence signals that govern splicing of eukaryotic pre-mRNAs, it is an accepted rule that U2-dependent introns exhibit the 3' terminal dinucleotide AG. Intrigued by anecdotal evidence for functional non-AG 3' splice sites, we carried out a human genome-wide screen.     

FUBP1 is a general splicing factor facilitating 3′ splice site recognition and splicing of long introns

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Activation of cryptic splice sites is a frequent splicing defect mechanism caused by mutations in exon and intron sequences of the OPA1 gene

Mutations in OPA1 are the most frequent cause underlying autosomal dominant optic atrophy (adOA). Until now only few putative splicing mutations in the OPA1 gene have been investigated at the mRNA level and all these result in exon skipping. Here, we report the identification and cDNA analysis of four intronic and three exonic OPA1 gene mutations that cause a variety of splicing defects including activation of cryptic splice sites in either flanking exon or intron sequences, and a leaky splicing mutation. Our results show that cDNA analysis is of prime importance for the full evaluation of the effect of putative splicing mutations in the OPA1 gene.     

Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).     

Control of hnRNP A1 alternative splicing: an intron element represses use of the common 3' splice site

Alternative splicing of exon 7B in the hnRNP A1 pre-mRNA produces mRNAs encoding two proteins: hnRNP A1 and the less abundant A1B. We have reported the identification of several intron elements that contribute to exon 7B skipping. In this study, we report the activity of a novel element, conserved element 9 (CE9), located in the intron downstream of exon 7B. We show that multiple copies of CE9 inhibit exon 7B-exon 8 splicing in vitro. When CE9 is inserted between two competing 3' splice sites, a single copy of CE9 decreases splicing to the distal 3' splice site. Our in vivo results also support the conclusion that CE9 is a splicing modulator. First, inserting multiple copies of CE9 into an A1 minigene compromises the production of fully spliced products. Second, one copy of CE9 stimulates the inclusion of a short internal exon in a derivative of the human beta-globin gene. In this case, in vitro splicing assays suggest that CE9 decreases splicing of intron 1, an event that improves splicing of intron 2 and decreases skipping of the short internal exon. The ability of CE9 to act on heterologous substrates, combined with the results of a competition assay, suggest that the activity of CE9 is mediated by a trans-acting factor. Our results indicate that CE9 represses the use of the common 3' splice site in the hnRNP A1 alternative splicing unit.     

TIA-1 and TIAR activate splicing of alternative exons with weak 5' splice sites followed by a U-rich stretch on their own pre-mRNAs

TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.     

Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing

Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing.