The smooth muscle (SM) and nonmuscle (NM) isoforms of ␣-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat ␣-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3 splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5 splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacercontaining transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3 end of the actinin intron or regulatory sequences from ␣-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated ␣-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of ␣-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, ␣-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3 splice site and by PTB.Many eukaryotic genes employ alternative splicing as a means of generating protein diversity. This differential incorporation of exons into the mature RNA is often under developmental and/or tissue-specific control and enables the cell to tailor the protein to suit its own particular requirements (61, 67). The basic splicing mechanism involves a two-step process which takes place in a ribonucleoprotein complex called a spliceosome and results in adjacent exons being joined together with the intron between released in the form of a lariat (reviewed in references 1 and 57). There is a further level of complexity in alternative splicing in that different combinations of 5Ј and 3Ј splice sites are ligated. The mechanisms that determine which splice sites are utilized and how this is regulated in different cell types or developmental stages have still not been precisely defined. Much progress has been made in identifying the cis-acting elements involved in alternative splicing, and the roles of some general factors have been demonstrated (1, 67). cis-Acting determinants that influence competing splicing pathways include the relative strengths of the competing 5Ј splice sites (e.g., 9, 78), branch point sequences (e.g., 53, 79), and polypyrimidine tracts...

No abstract

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.

PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.