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. ? ?
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