Alternative splicing regulation of caspase-2 gene

描述（由申请人提供）：前体mRNA剪接是大多数哺乳动物基因表达的关键步骤。选择性剪接是促进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机制和治疗中的作用。该应用程序的重点是调查剪接位点识别/选择的机制和选择性剪接调控的caspase-2（casp-2），程序性细胞死亡（PCD）的重要球员的作用。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在抗癌药物诱导的细胞死亡中起着关键作用。我们的初步研究结果显示casp-2的选择性剪接模式会因化疗药物而改变。为了研究caspase基因选择性剪接调控的机制，我们建立了一个以casp-2基因为模型的系统。一个casp-2小基因已被构建含有casp-2可变剪接复制所必需的顺式元件。Casp-2可变剪接已在体外重建。利用体外生化分析和转染培养细胞，我们已经开始解剖casp-2选择性剪接关键的顺式元件和反式作用因子。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casp-2选择性剪接的分子调控机制。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在调节casp-2选择性剪接中的作用，casp-2选择性剪接控制着抗凋亡和促凋亡产物的微妙平衡。我们将采用靶向敲除的方法研究In 100在小鼠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的表达/活动的变化。本研究为哺乳动物剪接调控机制的研究提供了新的思路，并将进一步加深我们对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.