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器的募集。我的实验室正在研究的分子机制，有助于在选择性剪接的变化，以响应NR激素信号。我们最近发现，糖皮质激素受体（GR）配体地塞米松（Dex）可以在给药后4小时内改变神经元N2 a细胞中选择性剪接（除了基因表达）的全基因组模式。有趣的是，其mRNA表达水平被Dex改变的基因在很大程度上不同于其选择性剪接（AS）受到影响的基因。因此，AS代表了一个新的基因调控层，以前没有认识到核受体。最近，我们发现了一个新的NR coregulator称为精氨酸和谷氨酸丰富的1（ARGLU 1），发挥了作用，共激活转录以及调制AS，无论是基础和响应糖皮质激素信号。使用蛋白质组学方法，ARGLU 1被证明主要与RNA剪接因子相互作用。有趣的是，我们还发现ARGLU 1与RBM 39（先前显示在类固醇激素介导的转录和AS中发挥作用的U2 AF 65家族的成员）和JMJD 6（含有6的Jumanji结构域）相关，JMJD 6是一种与染色质重塑和AS有关的赖氨酰羟化酶和精氨酸脱甲基酶。先前的研究发现，JMJD 6可以在剪接后修饰剪接因子并影响其功能。我们将使用多种功能获得和丧失模型，结合使用神经细胞和体内诱导神经元特异性ARGLU 1敲除小鼠，通过RNAseq分析响应GC治疗的基因表达/AS变化，并将这些与行为措施相关联。为了直接评估与ARGLU 1结合的全基因组RNA集，我们将进行HITS-CLIP，并将结合RNA的同一性与那些显示为从RNAseq选择性剪接和转录改变的RNA进行比较。我们将使用生物化学，分子和蛋白质组学方法来评估ARGLU 1和GR与染色质修饰剂JMJD 6相互作用的重要性，以介导其不同的功能，并更好地了解GR如何促进AS。对RNA结合蛋白的研究在我们对“剪接密码”的理解方面取得了重大进展，然而，对细胞或组织中依赖剪接的AS是如何控制的知之甚少。该研究计划将研究核受体超家族成员（包括GR）通过协调配体依赖性AS帮助促进转录多样性的分子机制。

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 basal transcriptional machinery. My laboratory is studying the molecular mechanisms contributing to changes in alternative splicing in response to NR hormone signaling. We recently discovered that the glucocorticoid receptor (GR) ligand Dexamethasone (Dex) can alter genome-wide patterns of alternative splicing (in addition to gene expression) in neuronal N2a cells within 4 hours of administration. Intriguingly, genes whose levels of mRNA expression were altered by Dex were largely distinct from those whose alternative splicing (AS) was affected. Thus, AS represents a new layer of gene regulation not previously appreciated for nuclear receptors. Recently, we discovered a new NR coregulator called arginine and glutamate rich 1 (ARGLU1) that plays a role in coactivating transcription as well as modulating AS, both basally and in response to glucocorticoid signaling. Using a proteomics approach, ARGLU1 was shown to interact primarily with RNA splicing factors. Interestingly, we also found that ARGLU1 associated with RBM39 (a member of the U2AF65 family that was previously shown to play a role in steroid-hormone mediated transcription and AS) and JMJD6 (Jumanji domain containing 6), a lysyl hydroxylase and arginine demethylase implicated in chromatin remodeling and AS. Prior studies found that JMJD6 can post-translationally modify splicing factors and impact their function. We will use a variety of gain and loss of function models incorporating the use of neural cells and in vivo inducible neuron-specific ARGLU1 knockout mice to analyze changes in gene expression/AS in response to GC treatment by RNAseq and correlate these to behavioural measures. To directly assess the genome-wide set of RNAs bound to ARGLU1 we will perform HITS-CLIP and compare the identity of the bound RNAs with those shown to be alternatively spliced and transcriptionally altered from RNAseq. We will use biochemical, molecular and proteomic methods to assess the importance of the interaction of ARGLU1 and GR with the chromatin modifier JMJD6 for mediating each of its distinct functions and gain a better understanding of how GR is contributing to AS. The study of RNA binding proteins has yielded significant progress in our understanding of the “splicing code”, however, relatively little is known about how hormone-dependent AS is controlled in cells or tissues. This research program will examine the molecular mechanisms by which members of the nuclear receptor superfamily, including GR, help promote transcriptional diversity by coordinating ligand-dependent AS.