Functions of CDK12 and CDK13 in pre-mRNA Processing

CDK12 和 CDK13 在前 mRNA 加工中的功能

• 批准号: RGPIN-2022-04740
• 负责人: Morin, Gregg
• 金额: $ 2.33万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760762
• 关键词: Functions; CDK12; CDK13; pre; mRNA

This application will define the roles of the cyclin dependent kinases (CDKs) CDK12 and CDK13 in the coordinate regulation of transcription and RNA processing. While there is a deep understanding of the key proteins and biochemical mechanisms required for RNA splicing by the spliceosome, little is known about the processes that govern coordinate transcription elongation, polyadenylation, and spliceosome activity. CDKs typically respond to signal transduction cascades and regulate central cellular processes (e.g. cell cycle, transcription) through phosphorylation of target proteins. The paralogous proteins CDK12 and CDK13 phosphorylate RNA Polymerase 2 to maintain processive transcription elongation. These kinases also contain domains typically found in splicing factors and signaling proteins, and we have shown that they interact with a broad range of splicing and RNA processing factors. Recently, we demonstrated that CDK12 also regulates a specific form of alternative 3' end mRNA processing. This regulation is highly cell type- and gene-specific, suggesting additional layers of regulation that have yet to be defined. The basic biological functions of both CDK12 and CDK13 remain poorly understood. Thus, understanding their functional mechanisms is essential for defining key or novel aspects of the coordinated regulation of transcription and splicing. We will study CDK12 and CDK13 using these approaches: Aim 1. Determine mechanisms of CDK12 and CDK13 complexes in regulating RNA processing. Proximity labeling will be used to identify proteins and RNA sequences associated with CDK12 and CDK13. This will determine the temporal association of CDK12 and CDK13 with RNA, RNA binding and splicing proteins to determine how these kinases regulate RNA processing. Aim 2. Map phospho-regulation by CDK12 and CDK13 complexes in RNA processing. Using global phospho-proteomics and a potent CDK12/CDK13 covalent inhibitor we will independently identify the dynamics of signaling pathways downstream of CDK12 and CDK13. Aim 3. Define cellular roles of CDK12 and CDK13 by functional genomics. Using RNA-seq and proteomics, we will ascertain the genes and pathways regulated by CDK12 and CDK13. We will employ transient transcriptome (TT)-seq to examine temporal changes in RNA regulated by CDK12 and CDK13. These experiments will define the cellular roles of CDK12 and CDK13. While CDK12 and CDK13 have specific features to suggest that they perform the coordinated regulation of transcription and RNA processing, the molecular mechanism of these processes have not been thoroughly investigated. Our program will define the specific functions of CDK12 and CDK13 complexes in RNA processing, transcription, polyadenylation, and spliceosome regulation. The general significance of our program will be an increased understanding of the critical cellular regulation processes that control coordinated transcription and RNA processing affect the global gene expression state.

本申请将定义细胞周期蛋白依赖性激酶（CDK）CDK 12和CDK 13在转录和RNA加工的协调调节中的作用。虽然有一个深刻的理解所需的RNA剪接的剪接体的关键蛋白质和生化机制，很少有人知道有关的过程，管理协调转录延伸，多聚腺苷酸化，剪接体活性。CDK通常响应于信号转导级联并通过靶蛋白的磷酸化调节中心细胞过程（例如细胞周期、转录）。旁系同源蛋白CDK 12和CDK 13磷酸化RNA聚合酶2以维持进行性转录延伸。这些激酶还含有通常在剪接因子和信号蛋白中发现的结构域，我们已经证明它们与广泛的剪接和RNA加工因子相互作用。最近，我们证明了CDK 12还调节一种特定形式的替代3'端mRNA加工。这种调节是高度细胞类型和基因特异性的，这表明还有待确定的其他调节层。CDK 12和CDK 13的基本生物学功能仍然知之甚少。因此，理解它们的功能机制对于定义转录和剪接的协调调节的关键或新方面是必不可少的。我们将使用以下方法研究CDK 12和CDK 13：目的1。确定CDK 12和CDK 13复合物调节RNA加工的机制。邻近标记将用于鉴定与CDK 12和CDK 13相关的蛋白质和RNA序列。这将确定CDK 12和CDK 13与RNA、RNA结合和剪接蛋白的时间关联，以确定这些激酶如何调节RNA加工。目标2. RNA加工中CDK 12和CDK 13复合物的磷酸化调控图谱。使用全局磷酸化蛋白质组学和有效的CDK 12/CDK 13共价抑制剂，我们将独立鉴定CDK 12和CDK 13下游信号通路的动力学。目标3.通过功能基因组学来确定CDK 12和CDK 13的细胞作用。使用RNA-seq和蛋白质组学，我们将确定由CDK 12和CDK 13调控的基因和途径。我们将采用瞬时转录组（TT）-seq来检查由CDK 12和CDK 13调节的RNA的时间变化。这些实验将确定CDK 12和CDK 13的细胞作用。虽然CDK 12和CDK 13具有特定的特征，表明它们执行转录和RNA加工的协调调节，但这些过程的分子机制尚未得到彻底研究。我们的计划将定义CDK 12和CDK 13复合物在RNA加工，转录，多聚腺苷酸化和剪接体调节中的特定功能。我们计划的一般意义将是增加对控制协调转录和RNA加工影响全球基因表达状态的关键细胞调控过程的理解。