Gromak Natalia, Rideau Alexis, Southby Justine, Scadden A D J, Gooding Clare, Hüttelmaier Stefan, Singer Robert H, Smith Christopher W J
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
EMBO J. 2003 Dec 1;22(23):6356-64. doi: 10.1093/emboj/cdg609.
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.
α-原肌球蛋白（TM）互斥外显子2和3的调控性剪接涉及平滑肌细胞中外显子3的抑制。多嘧啶序列结合蛋白（PTB）对于TM剪接的调控是必要的，但并不充分。Raver1在双杂交筛选中通过与细胞骨架蛋白辅肌动蛋白和纽蛋白的相互作用被鉴定出来，并且还发现它与PTB相互作用。与这些相互作用一致，Raver1可以定位于细胞核或细胞质中。在这里我们表明，Raver1能够通过增强PTB介导的外显子3抑制来促进TM的平滑肌特异性可变剪接。Raver1的这种活性依赖于特定的PTB结合调控元件以及Raver1与PTB相互作用所必需的区域。Raver1或其C末端的异源募集诱导了非常高水平的外显子3跳跃，绕过了外显子3下游通常对PTB结合位点的需求。这提示了一种由PTB介导的剪接抑制的新机制，涉及募集Raver1作为一种有效的剪接共抑制因子。
