科研之友
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摘要
The human nonmuscle myosin heavy chain B gene contains a 30-nucleotide alternative exon, N30, that is included in the mRNA from neural cells but is skipped in all other cells. We have previously identified an intronic distal downstream enhancer (IDDE) region that is required for neural cell-specific inclusion of N30, In this study, me investigated the mechanism by which the IDDE promotes N30 exon usage. In vitro splicing analysis using neural cell nuclear extracts and two-exon pre-mRNA substrates, which consist of the N30 exon and either the upstream (E5) or downstream (E6) exon, demonstrates that the IDDE activates upstream E5-N30 splicing by facilitating early prespliceosome complex formation. The IDDE has no effect on N30-E6 splicing where the IDDE resides. Inspection of splice site consensus sequences shows that a polypyrimidine (Py) tract preceding N30 is suboptimal for U2AF binding. Optimizing the Py tract completely relieves the requirement for the IDDE in E5-N30 splicing in vitro. In transfected cells, the wild-type minigene transcripts, which consist of three exons, E5, N30, and E6, undergo neural cell-specific and IDDE-dependent alternative splicing of N30, Optimizing the Py tract in minigenes also completely relieves the requirement for the IDDE in N30 inclusion. Furthermore, overexpression of the truncated U2AF65, which contains the arginine and serine dipeptide-rich domain and linker domain, but lacks the RNA binding domain, selectively inhibits the IDDE-mediated N30 inclusion in mRNA from the wild-type minigene in a dominant negative fashion. These results support the hypothesis that the IDDE facilitates the recognition of the 3' splice site preceding N30 by a network of protein-protein interactions implicated in the recruitment of U2AF to a suboptimal Py tract.
- 出版日期2000-10-27
