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-2020-07212
• 负责人: Cummins, Carolyn
• 金额: $ 2.62万
• 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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741037
• 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 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.

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。我的实验室正在研究响应NR激素信号的选择性剪接变化的分子机制。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4小时内改变神经元N2a细胞的选择性剪接（除了基因表达）的全基因组模式。有趣的是，那些mRNA表达水平被Dex改变的基因与那些选择性剪接（AS）受到影响的基因在很大程度上不同。因此，AS代表了一个新的基因调控层，以前没有发现的核受体。最近，我们发现了一种新的NR共调节因子，称为arginine and glutamate rich 1 (ARGLU1)，它在协同激活转录和调节as中发挥作用，无论是基础的还是对糖皮质激素信号的响应。利用蛋白质组学方法，ARGLU1被证明主要与RNA剪接因子相互作用。有趣的是，我们还发现ARGLU1与RBM39 （U2AF65家族成员，先前被证明在类固醇激素介导的转录和AS中发挥作用）和JMJD6 （Jumanji结构域6）相关，JMJD6是一种与染色质重塑和AS有关的lysyl羟化酶和精氨酸去甲基化酶。已有研究发现，JMJD6可以在翻译后修饰剪接因子并影响其功能。我们将利用神经细胞和体内可诱导的神经元特异性ARGLU1敲除小鼠的多种功能增益和丧失模型来分析基因表达/AS在RNAseq处理GC后的变化，并将这些变化与行为测量相关联。为了直接评估与ARGLU1结合的全基因组rna集，我们将执行HITS-CLIP，并将结合的rna与RNAseq显示的选择性剪接和转录改变的rna的身份进行比较。我们将使用生化、分子和蛋白质组学方法来评估ARGLU1和GR与染色质修饰子JMJD6相互作用的重要性，以介导其每种不同的功能，并更好地了解GR如何促进AS。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，对于细胞或组织中激素依赖性AS是如何控制的，我们所知相对较少。本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来促进转录多样性的分子机制。