Alternative Splicing of the Insulin Receptor Gene

描述（由申请人提供）： 摘要RNA剪接是在mRNA产生之前从初级RNA转录物中去除内含子序列的过程。与低等真核生物不同，哺乳动物的绝大多数基因都是剪接的。大多数基因产生多个mRNA，这是由不同的启动子、终止序列或使用替代外显子引起的。虽然通常被描述为连续的步骤，转录和剪接现在被认为是同时发生的，但支持的证据很少。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被识别的是未知的。 除了燃料代谢外，胰岛素对生长和发育至关重要。胰岛素受体（IR）有两种变体，其不同之处在于胰岛素结合结构域中存在12个氨基酸。这两种变体来自外显子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，并仅结合胰岛素。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，已经减少了外显子11的包含。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，我们提出了一个全面的，但现实的一系列实验，以测试两个替代模型的共转录INSR基因剪接。这些研究将解决关键问题的基本生物过程中的共转录选择性剪接，并将整合细胞和分子生物学实验与生理学研究在小鼠肝脏缺乏特定的剪接因子。 具体目标#1：检测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的下游工程化人工暂停位点，并且将遗传和非遗传地调节转录延伸速率。 具体目标#2：确定SRp 20或SF 2是否是INSR基因的转录暂停和共转录剪接所需的。我们将测试外显子11是否需要SRp 20或SF 2与染色质的关联，是否存在SRp 20或SF 2依赖的转录暂停外显子11附近，以及SRp 20和SF 2是否共定位在暂停位点。我们还将测试HEK 293细胞中hnRNP-A1水平升高是否通过干扰SF 2结合来阻止共转录剪接。 具体目标#3：确定SRp 20的磷酸化是否是INSR基因共转录剪接所必需的。我们将测试PPP 1 R10是否将PP 1型磷酸酶靶向外显子11以使SRp 20去磷酸化，防止其从染色质释放并减少外显子包含。我们还将测试PP 1活性是否受PKA和胰岛素调节，PPP 1 R10是否与RNA结合或通过CUG-BP 1。 具体目标#4：创建SRp 20和SF 2的遗传肝脏特异性敲除。通过将SRp 20 flox/flox和SF 2flox/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来创建小鼠。这些小鼠应优先表达IR-A同种型。我们将使用一组代谢测试来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来评估肝脏中SRp 20和SF 2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。估计15%的VA患者患有糖尿病，其中>95%患有2型糖尿病。胰岛素抵抗甚至更常见，影响估计42-43%的年龄在60-80岁之间的人，并且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、糖皮质激素过量和多囊卵巢综合征。在许多这些状态中观察到胰岛素受体基因的选择性剪接的改变，并且胰岛素受体表达从胰岛素敏感性B同种型转变为代谢活性较低的A同种型可能导致胰岛素抵抗和2型DM。老年VA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剪接缺陷相关。因此，详细了解参与调节选择性剪接的机制可能对困扰VA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.