Molecular Regulation of Alternative Splicing by Nuclear Receptors and Coregulators

核激素受体包括一个配体激活的转录因子超家族，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）的转录激活在很大程度上取决于其与辅激活因子和辅抑制因子的相互作用，这些辅激活因子和辅抑制因子直接或间接地改变周围染色质的结构，以允许基础转录机制的募集。GR几乎在身体的每一个组织中表达，然而GR的转录调控是高度依赖于启动子和细胞类型的。当转录激活或抑制改变mRNA转录物的绝对水平时，通过前信使RNA的选择性剪接在细胞中创建了额外的复杂性层。通过对RNA结合蛋白的研究，在选择性剪接领域取得了很大的进展，而RNA结合蛋白在读取“剪接密码”中起着重要的作用，但对于细胞中依赖配体的转录激活和选择性剪接是如何被控制的，人们知之甚少。**利用高通量哺乳动物细胞共转染法测量GR介导的荧光素酶输出，我们筛选了一个人脑cDNA文库，发现了一个高度进化保守的蛋白，名为arginine and glutamate rich 1 (ARGLU1)，它强烈增强了GR的转录激活。ARGLU1与已经发现的NR共调节因子没有相似之处，但最近才被认为是雌激素受体的共激活剂。利用串联亲和纯化ARGLU1复合物的蛋白质组学方法，我们确定ARGLU1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是一种组蛋白精氨酸去甲基化酶，与染色质重塑和选择性基因剪接有关。蛋白质组学数据表明，这可能是一种可以耦合转录激活和选择性剪接的蛋白质。许多早期的文献报道表明，由NR应答元件驱动的异源“迷你基因”可以通过NR激活来调节。然而，在过去几年外显子测序技术取得进步之前，这一结果的体内意义是不可能确定的。利用目前可用的技术，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。**本研究项目将研究NR超家族成员通过协调配体依赖性转录激活和选择性剪接来促进转录多样性的分子机制。据估计，超过95%的人类基因经历了选择性剪接，因此了解核受体如何在分子水平上促进转录多样性对于理解rna在细胞和组织特异性背景下的功能至关重要。

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.