Mechanisms of alternative splicing of pre-mRNA

描述(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前-RNA剪接的机制调控。这项工作主要关注被称为SR蛋白质的保守剪接因子家族的成员，但也将分析参与剪接和/或其调节的其他蛋白质。将继续研究SnRNAs在剪接催化中的作用。提出了以下具体目标。1.ASF/SF2的新功能。最近的发现，ASF/SF2对于通过防止突变的共转录R环来维持基因组稳定性是必要的，这一发现将被详细探讨。将开发体外试验来研究RNP形成的机制和特异性，最终目标是重建R-环形成和随后的DMA双链断裂(DSB)，这些断裂在缺乏适当的RNP形成和/或剪接的情况下发生。并对DMA DSB的形成机理进行了探讨。将继续进行基因筛选，以确定ASF/SF2诱导的细胞死亡的抑制因子。2.SRp38的性质。将继续发现SRp38可以作为序列特异性剪接激活剂发挥作用，并确定SRp38特异性辅激活剂的身份。将对导致SRp38去磷酸化的信号通路进行分析。Hsp27阻止SRp38去磷酸化和诱导剪接耐热性的机制将被继续探讨。对最近创建的SRp38基因敲除小鼠的分析将继续进行。3.其他剪接调节器。我们将分析hnRNP A1导致SMN2外显子7排除的机制。将研究通过突变产生依赖于hnRNP A1的外显子剪接沉默子的可能性，例如在BRCA1和BRCA2转录本中相对频繁地发生。将确定RNAPII CTD特异性剪接共激活子的身份，并分析其作用机制。将确定剪接的第二催化步骤所需的PPP家族磷酸酶的基础。4.小分子RNA与剪接催化。RNA-Y的表征将完成，这是一种由纯化的U2和U6 RNA片段在非常类似于第一步剪接的反应中形成的新产物。将继续开展合作努力，以确定活性U2/U6络合物的晶体结构。将继续进行实验，以确定促进U2/U6介导的催化和/或U2/U6复合体形成的蛋白质辅因子。将对U2/U6中的部分残基进行诱变，以阐明它们在催化中的作用。

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.