Chan R C, Black D L
Molecular Biology Institute, University of California, Los Angeles, USA.
Mol Cell Biol. 1997 Aug;17(8):4667-76. doi: 10.1128/MCB.17.8.4667.
The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.
c-src前体mRNA的神经细胞特异性N1外显子在非神经细胞中受到负调控，而在神经元中受到正调控。我们之前鉴定出了位于N1两侧的保守内含子元件，它们在非神经的HeLa细胞提取物中指导N1剪接的抑制。上游抑制元件位于N1外显子3'剪接位点的多嘧啶序列内。包含该3'剪接位点序列的短RNA可在HeLa提取物中隔离反式作用因子，从而允许N1的剪接。我们现在表明，这些上游抑制元件与多嘧啶序列结合蛋白（PTB）特异性相互作用。多嘧啶序列中的突变会降低PTB的结合以及竞争RNA解除剪接抑制的能力。此外，纯化的PTB蛋白可在被竞争RNA解除抑制的提取物中恢复N1剪接的抑制。在这个系统中，PTB蛋白作用于N1外显子，以调节N1与下游外显子4的剪接。这种机制与PTB进行剪接调控的其他情况不同，在其他情况中，该蛋白会抑制其结合的剪接位点。
