Alternative splicing regulation of caspase-2 gene

描述（由申请人提供）：Pre-mRNA剪接是大多数哺乳动物基因表达的关键步骤。选择性剪接是遗传多样性的一种基本机制。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。本应用程序的重点是研究剪接位点识别/选择的机制以及caspase-2 （casp-2）的选择性剪接调节的作用，casp-2是程序性细胞死亡（PCD）的重要参与者。许多重要的PCD基因，包括casp-2，利用选择性剪接产生功能性拮抗产物。最近的研究表明，casp-2在抗癌药物诱导细胞死亡的启动过程中起着关键作用。我们的初步结果表明，casp-2的选择性剪接模式在化疗药物的反应中发生了变化。为了研究caspase基因的选择性剪接调控机制，我们建立了casp-2基因的模型系统。已经构建了一个casp-2迷你基因，它包含了复制casp-2选择性剪接所必需的和足够的顺式元件。体外重建了Casp-2选择性剪接。利用体外生化分析和转染培养细胞，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交易因子。我们拟通过分析casp-2选择性剪接的顺式元件和反式作用因子来研究其调控机制。我们计划使用分子和生化相结合的方法来研究不同的顺式和反式因子如何调节casp-2选择性剪接，从而控制抗和促凋亡产物的微妙平衡。我们将使用靶向敲除方法来研究In100在调节小鼠casp-2选择性剪接中的作用。我们将研究细胞死亡过程中PCD基因选择性剪接和剪接调节因子表达/活性的变化。这项研究可能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.