Sphingolipid Regulation of Caspase 9 Alternative Splicing

Caspase 9 选择性剪接的鞘脂调节

• 批准号: 7681614
• 负责人: CHARLES E. CHALFANT
• 金额: $ 22.35万
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7681614
• 关键词: A549; Affect; Agonist; Alternative Splicing; Antisense RNA; Apoptosis; Apoptotic; Be++ element; Beryllium; Biological Assay; CASP9 gene; Caspase; Cell Death; Cells; Ceramides; Coupled; Daunorubicin; Down-Regulation; Elements; Exclusion; Exons; Generations; Glutamic Acid; Glycine; Indium; Laboratories; Laboratory Finding; Link; Lipids; Lung; Mediating; Mutagenesis; Mutate; Mutation; Oligonucleotides; Oncogenes; Oncogenic; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Predisposition; Process; Protein Kinase; Protein phosphatase; Proteins; Purines; RNA; RNA Interference; RNA Splicing; Regulation; Resistance; Role; Second Messenger Systems; Signal Transduction Pathway; Site; Specificity; Sphingolipids; Stimulus; System; Technology; Testing; Transcript; Variant; Whole Organism; base; c-myc Genes; cancer therapy; caspase-9; cell transformation; ceramide 3; chemotherapy; mRNA Precursor; mutant; novel; protein phosphatase inhibitor-1; purine; response; second messenger; sphingosine 1-phosphate

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）：该项目的长期目标集中于确定凋亡途径失调如何赋予对化学疗法的抵抗力和对致癌转化的敏感性。新研究表明，caspase 9的RNA剪接变体的表达称为caspase 9b，赋予了对许多凋亡刺激的抗性。在PI的新研究中，脂质第二信使，神经酰胺和蛋白质磷酸酶-1（PP1）的激活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纳入成熟的caspase 9转录本。 PI实验室的初步结果揭示了caspase 9的替代剪接与SR蛋白SRP30A（ASF/SF2）本质上相关。我们的实验室发现，使用RNA干扰技术对SRP30A的下调极大地抑制了3、4、5、6外显子盒在成熟的caspase 9转录本中的纳入。此外，在caspase 9基因的3、4、5和6个外显子盒中，在每个外显子的内外鉴定了SRP30A的六个可能的相互作用位点。在PI实验室的其他机械研究中，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，有丝分裂生物活性脂质鞘氨醇1-磷酸盐会诱导SR蛋白的磷酸化升高。最后，对caspase 9的替代剪接的直接调节调节了细胞对化学疗法和致癌转化的敏感性。基于上述发现，我们假设SRPSOA是通过与特定RNA C/S元素相互作用来响应神经酰胺的CASPASE 9预先mRNA处理的重要调节剂，并且SRP30A通过其磷酸stusus phospho stuspus stuspus caspase caspase 9的纳入3、4、4、4、4、4、5和6的外部盒子。我们还假设Provival激动剂（例如S-1-P）通过激活Clk/sty诱导SRP30A的磷酸化，这又增加了caspase 9b的表达。最后，我们假设caspase 9的替代剪接可以调节细胞对化学疗法和致癌转化的敏感性。为了验证我们的假设，我们提出了以下具体目的：1）确定caspase 9替代剪接在细胞对化疗的敏感性和C-MYC/RASV12的致癌转化的敏感性中的作用； 2）确定caspase 9的3、4、5和6外显子盒响应于神经酰胺的3、4、5和6外显子盒的神经酰胺反应C/S元素； 3）确定srp30a在调节caspase 9的3、4、5和6外显子盒中的作用中的作用； 4）确定srp30a的磷酸态在caspase 9的3、4、5和6外显子盒响应于神经酰胺中的作用； 5）确定clk/sty在响应有丝分裂激动剂的caspase 9的外显子3、4、5和6盒的调节中的作用。这些研究将在很大程度上定义了调节caspase 9的信号转导途径，以响应凋亡激动剂。这不能低估，因为这些信号转导途径的定义不是针对抗癌疗法创造的，而是许多新目标。