Alternative Splicing of the Insulin Receptor Gene

胰岛素受体基因的选择性剪接

• 批准号: 8259049
• 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
• 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/8259049
• 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; receptor; receptor expression; receptor function; research study

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. PUBLIC HEALTH RELEVANCE: Diabetes mellitus is epidemic and expected to double in the next 20 years. An estimated 15% of VA patients have diabetes and >95% of these have Type 2 diabetes. Insulin resistance is even more common, affects an estimated 42-43% of people between ages 60-80, and is a feature of many metabolic syndromes including obesity, aging, hypertension, atherosclerosis, diabetes, myotonic dystrophy, glucocorticoid excess, and polycystic ovary syndrome. Alterations in alternative splicing of the insulin receptor gene is observed in a number of these states and a shift in insulin receptor expression from the insulin sensitive B isoform to the less metabolically active A isoform may contribute to insulin resistance and Type 2 DM. The aging VA patient population is also at risk for neurodegenerative diseases and cancer and these disorders are also associated with defects in RNA splicing. Therefore, a detailed understanding of the mechanisms involved in regulating alternative splicing may have wide applicability to many diseases that plague the VA population.

描述（由申请人提供）： 摘要和抽象的RNA剪接是在产生最终mRNA之前从初级RNA转录本中去除内含子序列的过程。与较低的真核生物不同，绝大多数哺乳动物基因是剪接的。大多数基因产生由差异启动子，终止序列或替代外显子的使用引起的多个mRNA。尽管经常被描述为顺序步骤，但现在认为转录和剪接同时发生，但是支持证据很少。更重要的是，在共同剪接的背景下如何识别替代剪接位点是未知的。 除燃料代谢外，胰岛素对于生长和发展至关重要。胰岛素受体（IR）有两种变体，它们在激素结合结构域中的12-氨基酸存在上有所不同。这两个变体来自外显子11的替代剪接。缺乏外显子11的IR被广泛表达并结合胰岛素和IGF-II。含有外显子11的IR主要在胰岛素敏感的组织，肌肉，脂肪细胞和肾脏中表达，仅结合胰岛素。更重要的是，许多疾病状态，例如II型糖尿病，衰老，肌发育症和癌症，已降低了外显子11的纳入。这使INSR基因成为研究RNA剪接研究的特别有趣的模型系统。 根据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，以测试两个共同转录insr基因剪接的替代模型。这些研究将解决有关共转录替代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缺乏肝脏中缺乏特定剪接因子的小鼠的生理研究整合。 特定目的＃1：测试共同剪接和动力学竞争模型的替代外显子识别模型。我们将尝试使用新的Chrip方法来捕获与染色质相关的剪接RNA，并确定是否在外显子11附近有转录暂停。为了测试足够，人造暂停位点将在外显子11下游设计，转录伸长率将在遗传学和药理上进行调节。 特定目标＃2：确定SRP20或SF2是否需要转录暂停和转录剪接的INSR基因。我们将测试外显子11是否需要SRP20或SF2与染色质关联，是否有SRP20或依赖SF2依赖性转录暂停外显子11附近，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水平的升高是否通过干扰SF2结合来防止共转录剪接。 特定目的＃3：确定SRP20的磷酸化是否是INSR基因共转录剪接所必需的。我们将测试PPP1R10是否将PP1型磷酸酶靶向外显子11以去磷酸化SRP20，从而阻止其从染色质中释放并减少外显子包容性。我们还将测试PP1活性是由PKA和胰岛素调节的，以及PPP1R10是否与RNA结合还是通过CUG-BP1结合。 特定目标＃4：创建SRP20和SF2的遗传肝特异性敲除。小鼠将通过将SRP20Flox/Flox和SF2Flox/Flox小鼠与白蛋白-CRE小鼠杂交创建，以删除肝细胞中的两个剪接因子。这些小鼠应优先表达IR-A同工型。我们将使用一系列代谢检测来确定这些小鼠是否具有胰岛素耐药性，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。 公共卫生相关性： 糖尿病是流行病，预计在未来20年内将翻一番。估计有15％的VA患者患有糖尿病，其中> 95％的2型糖尿病。胰岛素抵抗甚至更为普遍，估计有42-43％的60-80岁人群，并且是许多代谢综合征的特征，包括肥胖，衰老，高血压，高血压，动脉粥样硬化，糖尿病，肌动型性疾病，肌动症，糖皮质激素过量和多余的卵巢卵巢综合征。在许多状态中观察到胰岛素受体基因的替代剪接的改变，并且胰岛素受体表达从胰岛素敏感的B同工型转移到代谢较低的活性A同工型可能有助于胰岛素抵抗和2型DM。 VA老化的患者人群也有神经退行性疾病和癌症的风险，这些疾病也与RNA剪接中的缺陷有关。因此，对调节替代剪接所涉及的机制的详细理解可能对困扰VA人群的许多疾病具有广泛的适用性。