Alternative Splicing of the Insulin Receptor Gene

胰岛素受体基因的选择性剪接

基本信息

批准号：
8394584
负责人：
NICHOLAS J WEBSTER
金额：
--
依托单位：
VA SAN DIEGO HEALTHCARE SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8394584
关键词：
Address Adipocytes Aedes Affect Age Aging Albumins Alternative Splicing Am 80 Amino Acids Animals Appearance Atherosclerosis Binding Biological Biological Models Biological Process Caenorhabditis elegans Cells Chromatin Chromosomes, Human, Pair 19 Coupled Cyclic AMP-Dependent Protein Kinases Data Defect Deposition Development Diabetes Mellitus Disease Drosophila genus Embryo Engineering Epidemic Eukaryota Evolution Excision Exhibits Exons Genes Genetic Genetic Transcription Genomics Glucocorticoids Goals Growth Growth and Development function Hepatocyte Homologous Gene Hormones Human Hypertension INSR gene Insulin Insulin Receptor Insulin Resistance Insulin-Like Growth Factor II Kidney Kinetics Liver Longevity Lower Organism Lymnaea Malignant Neoplasms Mammals Messenger RNA Metabolic Metabolic syndrome Metabolism Methods Modeling Molecular Monkeys Mus Muscle Myotonic Dystrophy Neonatal Neurodegenerative Disorders Non-Insulin-Dependent Diabetes Mellitus Obesity Organism Patients Phosphoric Monoester Hydrolases Phosphorylation Physiological Plague Polycystic Ovary Syndrome Population Process Protein Isoforms RNA RNA Primary Transcript RNA Splicing Receptor Gene Regulation Research Risk Rodent Role Series Site Spliced Genes Suggestion Takifugu Testing Tissues Variant abstracting base blood glucose regulation hnRNP A1 interest patient population prevent promoter public health relevance receptor receptor expression receptor function research study

项目摘要

DESCRIPTION (provided by applicant): Summary and Abstract RNA splicing is the process of removal of intronic sequences from the primary RNA transcript before the final mRNA is generated. Unlike lower eukaryotes, the vast majority of mammalian genes are spliced. Most genes give rise to multiple mRNAs resulting from differential promoters, termination sequences, or the use of alternative exons. Although often depicted as sequential steps, transcription and splicing are now thought to occur simultaneously, however supporting evidence is scarce. More importantly, how alternative splice sites are recognized in the context of co-transcriptional splicing is unknown. Insulin is essential for growth and development in addition to fuel metabolism. There are two variants of the insulin receptor (IR), which differ in the presence of 12-amino acids in the hormone-binding domain. The two variants arise from alternative splicing of exon 11. The IR lacking exon 11 is widely expressed and binds both insulin and IGF-II; the IR containing exon 11 is expressed predominantly in the insulin-sensitive tissues liver, muscle, adipocytes and kidney, and only binds insulin. More importantly, a number of disease states, such as type II diabetes, aging, myotonic dystrophy and cancer, have decreased inclusion of exon 11. This makes the INSR gene a particularly interesting model system for the study of RNA splicing. Based on our extensive preliminary data we are proposing a comprehensive but realistic series of experiments to test two alternative models of co-transcriptional INSR gene splicing. These studies will address key questions concerning the fundamental biological process of co-transcriptional alternative splicing and will integrate cell and molecular biological experiments with physiological studies in mice lacking specific splicing factors in liver. Specific Aim #1: To test for co-transcriptional splicing and the kinetic competition model for alternative exon recognition. We will attempt to catch the spliced RNA still associated with chromatin using the new ChRIP method and will determine whether there is a transcriptional pause near exon 11. To test sufficiency, an artificial pause site will be engineered downstream of exon 11 and transcriptional elongation rates will be modulated genetically and pharmacologically. Specific Aim #2: To determine whether SRp20 or SF2 is required for transcriptional pausing and co- transcriptional splicing of the INSR gene. We will test whether exon 11 requires SRp20 or SF2 for association with chromatin, whether there is either a SRp20 or SF2-dependent transcriptional pause near exon 11, and whether SRp20 and SF2 co-localize at the pause site. We will also test whether elevated levels of hnRNP-A1 in HEK293 cells prevents co-transcriptional splicing via interfering with SF2 binding. Specific Aim #3: To determine whether phosphorylation of SRp20 is required for co-transcriptional splicing of the INSR gene. We will test whether PPP1R10 targets PP1-type phosphatases to exon 11 to dephosphorylate SRp20, preventing its release from chromatin and reducing exon inclusion. We will also test whether PP1 activity is regulated by PKA and insulin and whether PPP1R10 binds to RNA or via CUG-BP1. Specific Aim #4: To create genetic liver-specific knock-outs of SRp20 and SF2. Mice will be created by crossing SRp20flox/flox and SF2flox/flox mice with albumin-cre mice to delete the two splicing factors in hepatocytes. These mice should preferentially express the IR-A isoform. We will determine whether these mice are insulin-resistant using a panel of metabolic tests and we will assess other potential targets for SRp20 and SF2 in the liver using genomic approaches.

描述（由申请人提供）：总结和摘要 RNA 剪接是在生成最终 mRNA 之前从初级 RNA 转录物中去除内含子序列的过程。与低等真核生物不同，绝大多数哺乳动物基因都是剪接的。大多数基因会因差异启动子、终止序列或替代外显子的使用而产生多种 mRNA。尽管转录和剪接通常被描述为连续步骤，但现在认为它们是同时发生的，但支持证据很少。更重要的是，如何在共转录剪接的背景下识别选择性剪接位点尚不清楚。除了燃料代谢之外，胰岛素对于生长和发育也至关重要。胰岛素受体 (IR) 有两种变体，其不同之处在于激素结合域中存在 12 个氨基酸。这两种变体源自外显子 11 的选择性剪接。缺乏外显子 11 的 IR 广泛表达并结合胰岛素和 IGF-II；含有外显子11的IR主要在胰岛素敏感组织肝脏、肌肉、脂肪细胞和肾脏中表达，并且仅与胰岛素结合。更重要的是，许多疾病状态，如 II 型糖尿病、衰老、强直性肌营养不良和癌症，都减少了外显子 11 的包含量。这使得 INSR 基因成为研究 RNA 剪接的特别有趣的模型系统。基于我们广泛的初步数据，我们提出了一系列全面但现实的实验来测试共转录 INSR 基因剪接的两种替代模型。这些研究将解决有关共转录选择性剪接的基本生物学过程的关键问题，并将细胞和分子生物学实验与肝脏中缺乏特定剪接因子的小鼠的生理研究相结合。具体目标#1：测试共转录剪接和替代外显子识别的动力学竞争模型。我们将尝试使用新的 ChRIP 方法捕获仍与染色质相关的剪接 RNA，并确定外显子 11 附近是否存在转录暂停。为了测试充分性，将在外显子 11 下游设计一个人工暂停位点，转录延伸率将受到遗传和药理学的调节。具体目标#2：确定 INSR 基因的转录暂停和共转录剪接是否需要 SRp20 或 SF2。我们将测试外显子 11 是否需要 SRp20 或 SF2 来与染色质关联，外显子 11 附近是否存在 SRp20 或 SF2 依赖性转录暂停，以及 SRp20 和 SF2 是否在暂停位点共定位。我们还将测试 HEK293 细胞中 hnRNP-A1 水平升高是否会通过干扰 SF2 结合来阻止共转录剪接。具体目标#3：确定 INSR 基因的共转录剪接是否需要 SRp20 磷酸化。我们将测试 PPP1R10 是否将 PP1 型磷酸酶靶向外显子 11 以使 SRp20 去磷酸化，从而阻止其从染色质中释放并减少外显子包含。我们还将测试 PP1 活性是否受 PKA 和胰岛素调节，以及 PPP1R10 是否与 RNA 结合或通过 CUG-BP1 结合。具体目标#4：创建 SRp20 和 SF2 的肝脏特异性基因敲除。将 SRp20flox/flox 和 SF2flox/flox 小鼠与 albumin-cre 小鼠杂交以删除肝细胞中的两个剪接因子来创建小鼠。这些小鼠应优先表达 IR-A 同工型。我们将使用一组代谢测试确定这些小鼠是否具有胰岛素抵抗，并且我们将使用基因组方法评估肝脏中 SRp20 和 SF2 的其他潜在靶标。