Molecular Regulation of Alternative Splicing by Nuclear Receptors and Coregulators

核受体和共调节器对选择性剪接的分子调节

• 批准号: RGPIN-2014-03666
• 负责人: Cummins, Carolyn
• 金额: $ 3.42万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=648407
• 关键词: Molecular; Regulation; Alternative; Splicing; Nuclear

The nuclear hormone receptors comprise a superfamily of ligand-activated transcription factors that regulate diverse physiological processes by coordinating patterns of gene expression. Transcriptional activation by a nuclear hormone receptor (NR) depends strongly on its interactions with coactivators and corepressors that either directly or indirectly modify the surrounding chromatin configuration to allow recruitment of the basal transcriptional machinery. GR is expressed in virtually every tissue in the body, however transcriptional regulation of GR is highly promoter and cell type dependent. While transcriptional activation or repression changes absolute levels of mRNA transcripts, an additional layer of complexity is created in the cell by the alternative splicing of the pre-messenger RNA. Much progress has been made in the field of alternative splicing through the study of RNA binding proteins which are important in reading the "splicing code", relatively little is known about how ligand-dependent transcriptional activation and alternative splicing are controlled in cells. **Using a high-throughput mammalian cell co-transfection assay measuring GR-mediated luciferase output we screened a human brain cDNA library and uncovered a highly evolutionarily conserved protein named arginine and glutamate rich 1 (ARGLU1) that strongly potentiated GR transcriptional activation. ARGLU1 had no similarities to already uncovered NR coregulators but was just recently implicated as a co-activator of the estrogen receptor. Using a proteomics approach with a tandem affinity purified ARGLU1 complex we determined that ARGLU1 interacting proteins were primarily ribosomal proteins and RNA splicing factors. Intriguingly, we also found that ARGLU1 associated with JMJD6 (Jumanji domain containing 6), a histone arginine demethylase implicated in chromatin remodeling and alternative gene splicing. The proteomics data suggested that this may be a protein that could function in coupling transcriptional activation to alternative splicing. A number of earlier literature reports have shown that heterologous "mini-genes" driven by NR response elements can be modulated by NR activation. However, the in vivo significance of this was not possible to determine prior to the technological advances in exon sequencing that occurred over the past several years. With the currently available technology we would now be able to identify the endogenous role of these NR binding proteins in alternative splicing. **This research program will examine the molecular mechanisms by which members of the NR superfamily help promote transcriptional diversity by coordinating ligand-dependent transcriptional activation with alternative splicing. Because over 95% of human genes are estimated to undergo alternative splicing, understanding how nuclear receptors contribute to the transcriptional diversity at the molecular level is essential for understanding how NRs function in cell and tissue-specific contexts.

核激素受体包括配体激活的转录因子的超家族，其通过协调基因表达模式来调节多种生理过程。核激素受体（NR）的转录激活强烈依赖于其与辅激活子和辅抑制子的相互作用，辅激活子和辅抑制子直接或间接地修饰周围的染色质构型以允许基础转录机制的募集。 GR几乎在身体的每个组织中表达，然而GR的转录调节是高度依赖于启动子和细胞类型的。虽然转录激活或抑制改变了mRNA转录物的绝对水平，但通过前信使RNA的选择性剪接在细胞中产生了额外的复杂性。通过对阅读“剪接密码”的重要RNA结合蛋白的研究，选择性剪接领域取得了很大进展，但对细胞中配体依赖的转录激活和选择性剪接是如何控制的知之甚少。** 使用测量GR介导的荧光素酶输出的高通量哺乳动物细胞共转染测定，我们筛选了人脑cDNA文库，并发现了一种高度进化保守的蛋白质，名为精氨酸和谷氨酸丰富1（ARGLU1），其强烈增强GR转录激活。ARGLU1与已经发现的NR辅助调节因子没有相似之处，但最近被认为是雌激素受体的辅助激活因子。使用串联亲和纯化的ARGLU 1复合物的蛋白质组学方法，我们确定ARGLU 1相互作用蛋白主要是核糖体蛋白和RNA剪接因子。有趣的是，我们还发现ARGLU1与JMJD6（Jumanji domain containing 6）相关，JMJD6是一种与染色质重塑和选择性基因剪接有关的组蛋白精氨酸脱甲基酶。蛋白质组学数据表明，这可能是一个蛋白质，可以在耦合转录激活选择性剪接。许多早期的文献报道已经表明，由NR应答元件驱动的异源“小基因”可以通过NR活化来调节。 然而，在过去几年外显子测序技术进步之前，无法确定其体内意义。利用现有的技术，我们现在能够确定这些NR结合蛋白在选择性剪接中的内源性作用。** 本研究计划将研究NR超家族成员通过协调配体依赖性转录激活与选择性剪接来帮助促进转录多样性的分子机制。由于超过95%的人类基因估计经历选择性剪接，了解核受体如何在分子水平上促进转录多样性对于了解核受体在细胞和组织特异性环境中的功能至关重要。