Sphingolipid Regulation of Caspase 9 Alternative Splicing

Caspase 9 选择性剪接的鞘脂调节

• 批准号: 7284893
• 负责人: CHARLES E. CHALFANT
• 金额: $ 22.32万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-07 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7284893
• 关键词: A549; Affect; Agar; Agonist; Alternative Splicing; Amino Acid Sequence; Antisense RNA; Apoptosis; Apoptotic; Be++ element; Beryllium; Binding; Biological Assay; CASP9 gene; Caspase; Catalytic Domain; Cell Death; Cells; Ceramides; Complex; Coupled; Data; Daunorubicin; Down-Regulation; Elements; Exclusion; Exons; Family; Generations; Glutamic Acid; Glycine; Heterogeneous Nuclear RNA; Indium; Laboratories; Laboratory Finding; Length; Link; Lipids; Lung; Lung Adenocarcinoma; Measures; Mediating; Mutagenesis; Mutate; Mutation; Oligonucleotides; Oncogenes; Oncogenic; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Predisposition; Process; Protein Kinase; Protein phosphatase; Proteins; Purines; RNA; RNA Interference; RNA Splicing; Radiation; Regulation; Reporting; Resistance; Role; Scheme; Second Messenger Systems; Signal Transduction Pathway; Site; Specificity; Sphingolipids; Stimulus; Stress; System; Technology; Testing; Time; Transcript; Variant; Whole Organism; apoptotic protease-activating factor 1; base; c-myc Genes; cancer therapy; caspase-9; cell transformation; ceramide 3; chemotherapy; human CASP4 protein; mRNA Precursor; mutant; novel; protein phosphatase inhibitor-1; purine; response; second messenger; sphingosine 1-phosphate; tumor

DESCRIPTION (provided by applicant): The long-term objectives of this project focus on determining how dysregulation of apoptotic pathways confers resistance to chemotherapy and sensitivity to oncogenic transformation. New studies have shown that the expression of an RNA splice variant of caspase 9, termed caspase 9b, confers resistance to many apoptotic stimuli. In novel studies by the PI, the generation of the lipid second messenger, ceramide, and the activation of protein phosphatase-1 (PP1) were defined as major components of the signal transduction pathway that induces the inclusion of the four exon cassette into the mature caspase 9 transcript. Preliminary results by the PI's laboratory disclose that the alternative splicing of caspase 9 is intrinsically linked to the SR protein, SRp30a (ASF/SF2). Our laboratory found that downregulation of SRp30a using RNA interference technology dramatically inhibited the inclusion of the 3, 4, 5, 6 exon cassette in the mature caspase 9 transcript. Furthermore, six possible interaction sites for SRp30a were identified within and downstream of each exon in the 3, 4, 5, and 6 exon cassette of the caspase 9 gene. In other mechanistic studies by the PI's laboratory, the protein kinase, Clk/Sty, was found to regulate the phospho-status of SR proteins in A549 cells (23). Furthermore, sphingosine-1-phosphate, a mitogenic bioactive lipid induces an increase in the phosphorylation of SR proteins. Lastly, direct modulation of the alternative splicing of caspase 9 modulated the sensitivity of cells to chemotherapy and oncogenic transformation. Based on the above findings, we hypothesize that SRpSOa is an important regulator of caspase 9 pre- mRNA processing in response to ceramide via interaction with specific RNA c/s-elements, and that SRp30a regulates the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 via its phospho-status. We also hypothesize that prosurvival agonists (e.g. S-1-P) induce the phosphorylation of SRp30a via activation of Clk/Sty, which in turn increases the expression of caspase 9b. Lastly, we hypothesize that the alternative splicing of caspase 9 can modulate the susceptibility of cells to chemotherapy and oncogenic transformation. To validate our hypotheses, we propose the following specific aims: 1) To determine the role of the alternative splicing of caspase 9 in the sensitivity of cells to chemotherapy and oncogenic transformation by c-Myc/RasV12; 2) To determine the ceramide-responsive c/s-elements that regulate the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 in response to ceramide; 3) To determine the role of SRp30a in regulating the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 in response to ceramide; 4) To determine the role of the phospho-status of SRp30a on the inclusion of the 3, 4, 5, and 6 exon cassette of caspase 9 in response to ceramide; and 5) To determine the role of CLK/STY in the regulation of the inclusion of the exon 3, 4, 5, and 6 cassette of caspase 9 in response to mitogenic agonists. These studies will largely define the signal transduction pathway regulating caspase 9 alternative splicing in response to apoptotic agonists. This cannot be understated because the definition of these signal transduction pathways creates, not one, but many new targets, for anti-cancer therapies.

描述（由申请人提供）：该项目的长期目标侧重于确定细胞凋亡途径的失调如何赋予化疗耐药性和对致癌转化的敏感性。新的研究表明，caspase 9 的 RNA 剪接变体（称为 caspase 9b）的表达赋予对许多细胞凋亡刺激的抵抗力。在 PI 的新研究中，脂质第二信使神经酰胺的产生和蛋白磷酸酶 1 (PP1) 的激活被定义为信号转导途径的主要组成部分，该途径诱导四个外显子盒包含在成熟细胞中。半胱天冬酶 9 转录本。 PI 实验室的初步结果表明，caspase 9 的选择性剪接与 SR 蛋白 SRp30a (ASF/SF2) 有内在联系。我们的实验室发现，使用RNA干扰技术下调SRp30a可显着抑制成熟caspase 9转录物中3、4、5、6外显子盒的包含。此外，在 caspase 9 基因的第 3、4、5 和 6 外显子盒中的每个外显子内部和下游鉴定了 SRp30a 的 6 个可能的相互作用位点。在 PI 实验室的其他机制研究中，发现蛋白激酶 Clk/Sty 可以调节 A549 细胞中 SR 蛋白的磷酸化状态 (23)。此外，1-磷酸鞘氨醇（一种促有丝分裂生物活性脂质）可诱导 SR 蛋白磷酸化的增加。最后，直接调节 caspase 9 的选择性剪接可调节细胞对化疗和致癌转化的敏感性。基于上述发现，我们假设 SRpSOa 是 caspase 9 前 mRNA 加工的重要调节因子，通过与特定 RNA c/s 元件相互作用来响应神经酰胺，并且 SRp30a 调节 3、4、5、和 caspase 9 的 6 外显子盒（通过其磷酸化状态）。我们还假设促存活激动剂（例如 S-1-P）通过激活 Clk/Sty 诱导 SRp30a 磷酸化，从而增加 caspase 9b 的表达。最后，我们假设 caspase 9 的选择性剪接可以调节细胞对化疗和致癌转化的敏感性。为了验证我们的假设，我们提出以下具体目标：1）确定caspase 9的选择性剪接在细胞对化疗和c-Myc/RasV12致癌转化的敏感性中的作用； 2) 确定响应神经酰胺调节 caspase 9 的 3、4、5 和 6 外显子盒包含的神经酰胺响应 c/s 元件； 3) 确定SRp30a在响应神经酰胺调节caspase 9的3、4、5和6外显子盒的包含中的作用； 4) 确定 SRp30a 的磷酸化状态对响应神经酰胺而包含 caspase 9 的 3、4、5 和 6 外显子盒的作用； 5) 确定 CLK/STY 在响应促有丝分裂激动剂而调节 caspase 9 外显子 3、4、5 和 6 盒包含的过程中的作用。这些研究将在很大程度上定义调节 caspase 9 选择性剪接以响应细胞凋亡激动剂的信号转导途径。这不能被低估，因为这些信号转导途径的定义为抗癌治疗创造了不是一个，而是许多新靶点。