Mechanisms of alternative splicing of pre-mRNA

描述（由申请人提供）：本提案中描述的实验旨在深入了解脊椎动物细胞中pre-RNA剪接的调节机制。这项工作的重点是被称为SR蛋白的保守剪接因子家族的成员，但其他参与剪接和/或其调控的蛋白质也将被分析。研究snrna在剪接催化中的作用将继续进行。提出以下具体目标。1. ASF/SF2的新功能。最近发现ASF/SF2是通过防止诱变共转录R环来维持基因组稳定性所必需的，我们将详细探讨这一发现。研究RNP形成的机制和特异性的体外实验将被开发出来，最终目标是在没有适当的RNP形成和/或剪接的情况下重建r环形成和随后的DMA双链断裂（dsb）。并对DMA DSB的形成机理进行了研究。将继续进行基因筛选，以鉴定ASF/ sf2诱导的细胞死亡的抑制因子。2. SRp38的性质。SRp38可以作为序列特异性剪接激活因子的发现将继续进行，并确定SRp38特异性共激活因子的身份。我们将对导致srp38去磷酸化的信号通路进行分析。Hsp27阻止SRp38去磷酸化并诱导剪接耐热性的机制将继续研究。将继续对最近创建的SRp38基因敲除小鼠进行分析。3. 其他剪接调节因子。hnRNP A1参与SMN2外显子7排除的机制将被分析。通过突变产生hnRNP a1依赖的外显子剪接沉默子的可能性，例如在BRCA1和BRCA2转录本中，相对频繁发生的可能性将被研究。建立RNAPII ctd特异性剪接共激活子的身份并分析其作用机制。将确定PPP家族磷酸酶用于剪接第二催化步骤的基础。4. snrna和剪接催化。RNA-Y是一种由U2和U6 rna的纯化片段在与剪接的第一步非常相似的反应中形成的新产物，将被完成表征。确定活性U2/U6配合物晶体结构的合作工作将继续进行。将进行实验以鉴定促进U2/U6介导的催化和/或U2/U6复合物形成的蛋白质辅因子。选择U2/U6中的残基进行诱变，以阐明它们如何在催化中起作用。

DESCRIPTION (provided by applicant): The experiments described in this proposal are designed to gain insights into the mechanism regulation of pre-RNA splicing in vertebrate cells. The work focuses on members of a family of conserved splicing factors known as SR proteins, but other proteins that participate in splicing and/or its regulation will also be analyzed. Studies examining the roles of snRNAs in splicing catalysis will be continued. The following specific aims are proposed. 1. Novel functions of ASF/SF2. The recent discovery that ASF/SF2 is necessary for the maintenance of genome stability by preventing mutagenic co-transcriptional R loops will be pursued in detail. In vitro assays to investigate the mechanism and specificity of RNP formation will be developed, with the ultimate goal of recreating R-loop formation and the subsequent DMA double strand breaks (DSBs) that occur in the absence of proper RNP formation and/or splicing. The mechanism of DMA DSB formation will also be investigated. A genetic screen to identify suppressors of ASF/SF2-induced cell death will be continued. 2. Properties of SRp38. The discovery that SRp38 can function as a sequence-specific activator of splicing will be pursued and the identity of an SRp38-specific co-activator determined. Analysis of the signaling pathway that leads to SRp38-dephosphorylation will be investigated. The mechanism by which Hsp27 functions to prevent SRp38 dephosphorylation and induce splicing thermo tolerance will be pursued. Analysis of a recently created SRp38 knockout mouse will be continued. 3. Other splicing regulators. The mechanism by which hnRNP A1 contributes to SMN2 exon 7 exclusion will be analyzed. The possibility that creation of hnRNP A1-dependent exonic splicing silencers by mutation, for example in BRCA1 and BRCA2 transcripts, occurs relatively frequently will be investigated. The identity of an RNAPII CTD-specific splicing co-activator will be established and its mechanism of action analyzed. The basis for the requirement of a PPP family phosphatase for the second catalytic step of splicing will be determined. 4. snRNAs and splicing catalysis. Characterization of RNA-Y, a novel product formed with purified segments of U2 and U6 RNAs in a reaction very similar to the first step of splicing, will be completed. Collaborative efforts to determine the crystal structure of the active U2/U6 complex will be continued. Experiments to identify protein cofactors that facilitate U2/U6- mediated catalysis and/or U2/U6 complex formation will be pursued. Select residues in U2/U6 will be subjected to mutagenesis to elucidate how they function in catalysis.