Molecular Regulation of Alternative Splicing by Nuclear Receptors and Coregulators

核激素受体包括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不同的生理过程。核激素受体(NR)的转录激活在很大程度上依赖于它与辅助激活因子和辅助抑制因子的相互作用，这些辅助激活因子和辅助抑制因子可以直接或间接地改变周围染色质的构型，从而允许基本转录机制的重新启动。GR在体内几乎所有组织中都有表达，但GR的转录调控高度依赖于启动子和细胞类型。虽然转录激活或抑制改变了mRNA转录本的绝对水平，但通过前信使RNA的选择性剪接，细胞中又产生了一层额外的复杂性。通过对RNA结合蛋白的研究，在选择性剪接领域已经取得了很大的进展，RNA结合蛋白是阅读剪接密码的重要因素，但对于细胞中配体依赖的转录激活和选择性剪接是如何控制的，人们知之甚少。**使用高通量哺乳动物细胞共转染实验测量GR介导的荧光素酶产量，我们筛选了一个人脑cDNA文库，并发现了一种在进化上高度保守的蛋白质，称为富含精氨酸和谷氨酸的1(ARGLU1)，它强烈增强了GR的转录激活。ARGLU1与已发现的NR辅助调节因子没有相似之处，但最近才被认为是雌激素受体的辅助激活剂。利用串联亲和纯化的ARGLU1复合体的蛋白质组学方法，我们确定ARGLU1相互作用的蛋白质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结构域包含6)相关，JMJD6是一种组蛋白精氨酸去甲基酶，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，这可能是一种蛋白质，可以将转录激活与选择性剪接结合起来。一些早期的文献报道表明，由NR反应元件驱动的异源“微基因”可以被NR激活所调控。然而，在过去几年外显子测序的技术进步之前，无法确定这在体内的意义。利用目前可用的技术，我们现在可以确定这些NR结合蛋白在选择性剪接中的内源性作用。**这项研究计划将研究NR超家族成员通过协调配体依赖的转录激活和选择性剪接来促进转录多样性的分子机制。由于估计超过95%的人类基因经历了选择性剪接，因此在分子水平上了解核受体如何对转录多样性做出贡献，对于理解NRs在细胞和组织特定环境中的功能至关重要。

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.