The PTB interacting protein raver1 regulates  -tropomyosin alternative splicing

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.

Raver1 is an integral component of muscle contractile elements

“…Since raver1 harbours three RRMs and affects PTB-mediated RNA splicing in vitro (Gromak et al 2003) it has been speculated that raver1 could be involved in processing and delivery of specific mRNAs to the sarcomere and thus mediate site-specific synthesis of sarcomeric proteins (Jockusch et al 2003). Such sarcomeric localization has been reported for the mRNAs of vimentin, desmin and vinculin (Morris and Fulton 1994).…”

“…Furthermore, raver1-PTB complexes can be immunoprecipitated from vertebrate cells (Huttelmaier et al 2001). While direct RNA binding of raver1 has yet to be demonstrated, the protein may assist in PTB-mediated splicing events in smooth muscle (Gromak et al 2003;Spellman et al 2005). Apart from its association with PTB in the nucleus, raver1 interacts with the microfilament-associated proteins vinculin, its muscle-specific splice variant metavinculin and alpha-actinin (Huttelmaier et al 2001).…”

Structure-function relationships of the polypyrimidine tract binding protein

“…Hence, mechanisms that release PTB are important for tissue-specific regulation of splicing. Another facet of splicing regulation by PTB is the existence of co-repressors like Raver1 [38,39]. Raver1 recruitment is essential for effective repressor function of PTB in certain pre-mRNAs, for example for the splicing repression of a-tropomyosin exon 3, while in other systems PTB alone is sufficient.…”

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

“…In the presence of unr, this structure is altered such that some of the stem-loops are opened, and this allows nPTB to bind at two sites (P1 and P2). The interactions of these proteins thus allow the Apaf-1 IRES RNA to attain the correct structural conformation for ribosome recruitment by creating a single-stranded region, which allows the ribosome-landing site to become accessible metvinculin, and also interacts with PTB to regulate splicing; 57,58 hnRNP-L, a PTB homologue that was found to bind PTB in a yeast two-hybrid screen; 59 PSF (PTB-associated splicing factor) and p54 nrb /NonO are related multifunctional nuclear factors that contain both RNA-binding and DNAbinding functions. 60 Other ITAFs that help to regulate IRES-mediated expression of proteins that function during apoptosis include PCBP2 and hnRNPK.…”