Alternative splicing regulation of caspase-2 gene

描述(申请人提供)：前-信使核糖核酸剪接是大多数哺乳动物基因表达的关键步骤。选择性剪接是促成遗传多样性的基本机制。我们的长期目标是了解哺乳动物前体mRNA剪接调控的分子基础及其在癌症发病和治疗中的作用。这项应用主要研究剪接位点识别/选择的机制以及caspase-2(CASP-2)的选择性剪接调控的作用，CASP-2是程序性细胞死亡(PCD)中的一个重要角色。许多重要的PCD基因，包括Casp-2，利用选择性剪接产生功能拮抗的产物。最近的研究表明，Casp-2在抗癌药物诱导的细胞死亡中起关键作用。我们的初步结果表明，CASP-2的选择性剪接模式随着化疗药物的反应而改变。为了研究caspase基因选择性剪接调控的机制，我们建立了一个利用Casp-2基因的模型系统。已经构建了一个含有复制Casp-2选择性剪接所必需和足够的顺式元件的Casp-2微型基因。CASP-2选择性剪接已在体外重组。利用体外生化分析和培养细胞的转染，我们已经开始剖析对Casp-2选择性剪接至关重要的顺式元件和调控因子。我们建议通过解剖相关的顺式元件和反式作用因子来研究调控Casp-2选择性剪接的分子机制。我们计划使用分子和生物化学相结合的方法来研究不同的顺式和反式因子如何在调节Casp-2选择性剪接中发挥作用，该选择性剪接控制着抗凋亡和促凋亡产物的微妙平衡。我们将使用靶向敲除的方法来研究in100在调节小鼠Casp-2选择性剪接中的作用。我们将检测PCD基因选择性剪接和剪接调节因子在细胞死亡过程中的表达/活性的变化。这项研究可能会为哺乳动物剪接调控机制提供新的见解，并促进我们对PCD调控的理解。

DESCRIPTION (provided by applicant): Pre-mRNA splicing is a critical step in the expression of most mammalian genes. Alternative splicing is a fundamental mechanism that contributes to genetic diversity. Our long-term goal is to understand the molecular basis underlying the regulation of mammalian pre-mRNA splicing and its role in the pathogenesis and treatment of cancer. This application focuses on investigating mechanisms underlying splice site recognition/selection and the role of alternative splicing regulation of caspase-2 (casp-2), an important player in programmed cell death (PCD). A number of important PCD genes, including casp-2, utilize alternative splicing to generate functionally antagonistic products. Recent studies suggest that casp-2 is critical for the initiation of cell death induced by anti-cancer drugs. Our preliminary results show that alternative splicing pattern of casp-2 changes in response to chemotherapeutic drugs. To investigate mechanisms underlying the alternative splicing regulation of caspase genes, we have established a model system using casp-2 gene. A casp-2 minigene has been constructed containing cis-elements essential and sufficient for reproducing casp-2 alternative splicing. Casp-2 alternative splicing has been reconstituted in vitro. Using both in vitro biochemical assays and transfection in the cultured cells, we have begun to dissect cis-elements and transacting factors critical for casp-2 alternative splicing. We propose to study molecular mechanisms regulating casp-2 alternative splicing by dissecting cis- elements and trans-acting factors involved. We plan to use combined molecular and biochemical approaches to investigate how different cis- and trans-factors function in regulating casp-2 alternative splicing that controls the delicate balance of anti- and pro- apoptotic products. We will use targeted-knock-out approach to study the role of In100 in regulating casp-2 alternative splicing in mice. We will examine changes in PCD gene alternative splicing and in the expression/activities of splicing regulators during cell death. This study is likely to provide new insights into mechanisms of mammalian splicing regulation and to advance our understanding of regulation of PCD.