Alternative Splicing of the Insulin Receptor Gene

描述(由申请人提供)： 摘要和摘要RNA剪接是在产生最终的RNA之前从初级RNA转录本中去除内含子序列的过程。与低等真核生物不同，哺乳动物的绝大多数基因都是剪接的。大多数基因由于不同的启动子、终止序列或替代外显子的使用而产生多个mRNAs。尽管经常被描述为连续的步骤，但转录和剪接现在被认为是同时发生的，然而支持证据很少。更重要的是，在共转录剪接的背景下，选择性剪接位点是如何识别的尚不清楚。除了燃料代谢外，胰岛素对生长和发育也是必不可少的。胰岛素受体(IR)有两种不同的变体，不同的是激素结合区域中存在12个氨基酸。外显子11缺失的IR广泛表达并与胰岛素和IGF-II结合；含有外显子11的IR主要在胰岛素敏感组织肝脏、肌肉、脂肪细胞和肾脏表达，仅与胰岛素结合。更重要的是，一些疾病状态，如II型糖尿病、衰老、强直性肌营养不良和癌症，减少了外显子11的包含。这使得INSR基因成为研究RNA剪接的一个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，以测试两种可供选择的共转录INSR基因剪接模型。这些研究将解决与共转录选择性剪接的基本生物学过程有关的关键问题，并将把细胞和分子生物学实验与在肝脏中缺乏特定剪接因子的小鼠的生理研究结合起来。具体目标#1：测试共转录剪接和选择性外显子识别的动力学竞争模型。我们将尝试使用新的chrip方法来捕捉仍然与染色质相关的剪接RNA，并将确定外显子11附近是否存在转录暂停。为了测试其充分性，将在外显子11下游设计一个人工暂停位置，并从遗传和药物角度调节转录延长率。特定目的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与染色质相关，外显子11附近是否存在依赖SRp20或SF2的转录暂停，以及SRp20和SF2是否共定位于暂停位点。我们还将测试HEK293细胞中hnRNP-A1水平的升高是否通过干扰SF2结合来阻止共转录剪接。具体目的#3：确定INSR基因的共转录剪接是否需要SRp20的磷酸化。我们将测试PPP1R10是否针对PP1型磷酸酶至外显子11，使SRp20去磷酸化，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和胰岛素的调节，以及PPP1R10是否与RNA结合或通过CUG-BP1。具体目标4：创造肝脏特异的SRp20和SF2基因敲除。将SRp20FLOX/FLOX和SF2FLOX/FLOX小鼠与白蛋白-cre小鼠杂交，以删除肝细胞中的两种剪接因子，将产生小鼠。这些小鼠应该优先表达IR-A亚型。我们将使用一组代谢测试来确定这些小鼠是否存在胰岛素抵抗，并将使用基因组方法评估肝脏中SRp20和SF2的其他潜在靶点。 公共卫生相关性： 糖尿病是一种流行病，预计在未来20年内将翻一番。据估计，15%的退伍军人管理局患者患有糖尿病，其中95%患有2型糖尿病。胰岛素抵抗更常见，估计有42%-43%的60-80岁的人受到影响，是许多代谢综合征的特征，包括肥胖、衰老、高血压、动脉粥样硬化、糖尿病、强直性肌营养不良、糖皮质激素过剩和多囊卵巢综合征。在这些状态中观察到胰岛素受体基因选择性剪接的变化，胰岛素受体表达从对胰岛素敏感的B亚型转变为代谢活性较低的A亚型，可能导致胰岛素抵抗和2型糖尿病。老年VA患者群体也面临神经退行性疾病和癌症的风险，这些疾病也与RNA剪接缺陷有关。因此，详细了解调控选择性剪接的机制可能对困扰退伍军人管理局人群的许多疾病具有广泛的适用性。

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.