Mechanisms of alternative splicing of pre-mRNA

描述（由申请人提供）： 在这个建议中描述的实验旨在提供洞察的机制和调节的选择性前mRNA剪接（AS）。提出了以下具体目标。1. SRp 38及其相关因素将继续分析在第一步之后阻断体外剪接的新型RNA元件。该序列将被完全定义，并确定其在各种情况下阻止第二步的能力。可能的反式作用的监管机构，确定了RNA亲和力和质谱（MS），将进行表征。将阐明第二步阻滞的机制，包括已知第二步因素的可能参与。这代表了一种新的机制，调节AS的可能性将进行调查。SRp 38需要一个特定的辅激活因子作为序列特异性剪接激活因子，并将继续努力识别和表征它。蛋白质纯化和MS已经鉴定出进化上保守的剪接因子Prp 5作为强有力的候选者。将确定共激活因子的作用机制，并研究其参与体内AS调节的可能性。2.剪接和转录。将继续研究剪接与转录在功能上耦合的机制。与R. Roeder，将组装染色质化的模板，并确定从这些模板产生的转录物剪接的程度和速率。将研究特定组蛋白修饰的可能影响。将继续研究RNA聚合酶II大亚基C末端结构域（CTD）在剪接中的作用的实验。ASF/SF 2-CTD融合蛋白已被用于显示CTD可以在存在共激活剂的情况下增强体外剪接，共激活剂已被纯化并通过MS初步鉴定为剪接因子U2 AF和Prp 19复合物（Prp 19 C）之间的新型复合物。这个复杂的特点是详细的，其功能的意义，特别是在spliceosome组装，确定。将研究偶联转录/剪接期间CTD募集U2 AF-Prp 19 C的作用。3.剪接和疾病。研究丙酮酸激酶M型（PKM）AS在癌症中的调节的实验将继续进行。最近的工作涉及hnRNPA 1/A2和PTB在这个过程中，和其他实验，包括在体内外显子交换，RNA结合和体外剪接测定，将进行，以确定是否有其他蛋白参与PKM剪接控制。这些发现的生理意义将与P. Canoll合作，使用成胶质细胞瘤大鼠模型进行检查。逆转录病毒感染将用于确定hnRNPA 1/A2/PTB shRNA是否可以阻断胶质瘤的发生，以及这些蛋白的过表达是否可以诱导胶质瘤的发生。将分析最近鉴定的TLS靶基因在TLS敲低/过表达细胞中AS的变化，并确定剪接因子向这些基因募集的改变。 公共卫生相关性： 相关性：本提案中描述的实验旨在增加我们对选择性剪接机制和调控及其与转录的联系的理解。许多研究表明，选择性剪接的变化发生在发育和疾病过程中，拟议的实验将为这些变化提供一个机制的理解。

DESCRIPTION (provided by applicant): The experiments described in this proposal are designed to provide insight into the mechanism and regulation of alternative pre-mRNA splicing (AS). The following Specific Aims are proposed. 1. SRp38 and related factors. Analysis of a novel RNA element that blocks in vitro splicing after the first step will be continued. The sequence will be fully defined and its ability to block the second step in a variety of contexts determined. Possible trans-acting regulators, identified by RNA affinity and mass spectrometry (MS), will be characterized. The mechanism of the second-step block, including the possible involvement of known second-step factors, will be elucidated. The possibility that this represents a novel mechanism for regulating AS will be investigated. SRp38 requires a specific coactivator to function as a sequence-specific splicing activator, and efforts to identify and characterize it will be continued. Protein purification and MS have identified the evolutionarily conserved splicing factor Prp5 as a strong candidate. The mechanism by which the coactivator functions will be determined and the possibility that it participates in AS regulation in vivo investigated. 2. Splicing and transcription. Studies to investigate mechanisms by which splicing is functionally coupled to transcription will be continued. In collaboration with R. Roeder, chromatinized templates will be assembled, and the extent and rate of splicing of transcripts produced from these templates determined. Possible effects of specific histone modifications will be investigated. Experiments examining the role of the RNA polymerase II large subunit C- terminal domain (CTD) in splicing will be continued. An ASF/SF2-CTD fusion protein has been used to show that the CTD can enhance splicing in vitro in the presence of a coactivator, which has been purified and tentatively identified by MS as a novel complex between splicing factor U2AF and the Prp19 complex (Prp19C). This complex will be characterized in detail, and its functional significance, especially during spliceosome assembly, determined. The role of U2AF-Prp19C recruitment by the CTD during coupled transcription/splicing will be investigated. 3. Splicing and disease. Experiments to investigate regulation of pyruvate kinase M-form (PKM) AS in cancer will be continued. Recent work has implicated hnRNPA1/A2 and PTB in this process, and additional experiments, including in vivo exon-swapping, RNA binding and in vitro splicing assays, will be performed to determine whether additional proteins participate in PKM splicing control. The physiological significance of these findings will be examined in collaboration with P. Canoll using a rat model for gliablastoma. Retoviral infection will be used to determine whether hnRNPA1/A2/PTB shRNAs can block gliomagenesis, and conversely whether overexpression of these proteins can induce it. Lastly, the function of the Translocated in Liposarcoma (TLS) protein in coupling transcription-splicing will be investigated. Recently identified TLS target genes will be analyzed for changes in AS in TLS knockdown/overexpressing cells, and alterations in recruitment of splicing factors to these genes will be determined. PUBLIC HEALTH RELEVANCE: RELEVANCE: The experiments described in this proposal are designed to increase our understanding of the mechanism and regulation of alternative splicing and its link to transcription. Numerous studies have revealed that changes in alternative splicing occur during development and disease, and the proposed experiments will provide a mechanistic understanding for these changes.