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为替代前mRNA剪接(AS)的机制和调控提供洞察力。提出了以下具体目标。1.SRp38及其相关因素。在第一步之后，将继续分析一种阻止体外剪接的新型RNA元件。该序列将被完全定义，并确定其在各种环境中阻止第二步的能力。通过RNA亲和力和质谱学(MS)确定的可能的反式作用调节因子将被表征。第二步阻断的机制，包括已知的第二步因素的可能参与，将被阐明。这代表着一种监管AS的新机制的可能性将被调查。SRp38需要一种特定的辅活化子来发挥序列特异性剪接激活剂的作用，鉴定和鉴定它的努力将继续下去。蛋白质纯化和MS已证实进化上保守的剪接因子Prp5是一个强有力的候选者。共激活子的作用机制将被确定，以及它参与体内调节的可能性被研究。2.剪接和转录。将继续研究剪接与转录在功能上偶联的机制。与R.Roeder合作，将组装色谱化模板，并确定从这些模板产生的转录的剪接程度和速率。特异组蛋白修饰的可能影响将被调查。检测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将继续进行。一个ASF/SF2-CTD融合蛋白被用来证明CTD在共激活剂存在的情况下可以促进体外剪接，该融合蛋白已经被MS纯化并初步鉴定为剪接因子U2AF与Prp19复合体(Prp19C)之间的一个新的复合体。将对该复合体进行详细的表征，并确定其功能意义，特别是在剪接体组装过程中。将研究U2AF-Prp19C在耦合转录/剪接过程中由CTD招募的作用。3.剪接和疾病。研究丙酮酸激酶M型(PKM)在癌症中的调节作用的实验将继续进行。最近的工作表明hnRNPA1/A2和PTB参与了这一过程，还将进行更多的实验，包括体内外显子交换、RNA结合和体外剪接试验，以确定是否有更多的蛋白质参与PKM剪接控制。这些发现的生理学意义将与P.Canoll合作，使用胶质母细胞瘤的大鼠模型进行检验。逆转录病毒感染将被用来确定hnRNPA1/A2/PTB shRNAs是否可以阻止胶质瘤的发生，反之，这些蛋白的过度表达是否可以诱导胶质瘤的发生。最后，将研究脂肪肉瘤转位蛋白(TLS)在转录-剪接偶联中的作用。最近确定的TLS靶基因将被分析TLS基因敲除/过表达细胞中AS的变化，并将确定这些基因的剪接因子募集的变化。 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及其与转录的联系的机制和调控的理解。大量研究表明，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种机制上的理解。

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.