Functions of CDK12 and CDK13 in pre-mRNA Processing

这项应用将确定细胞周期蛋白依赖性蛋白激酶(CDKs)CDK12和CDK13在转录和RNA加工的协调调控中的作用。虽然人们对剪接体剪接RNA所需的关键蛋白质和生化机制有了深入的了解，但对控制协调转录延伸、多聚腺苷酸化和剪接体活性的过程知之甚少。CDK通常通过靶蛋白的磷酸化来响应信号转导通路，并调节中枢细胞过程(如细胞周期、转录)。同源蛋白CDK12和CDK13使RNA聚合酶2磷酸化，以维持转录延伸。这些激酶还包含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处理因子相互作用。最近，我们证明了CDK12还调节一种特定形式的选择性3‘端mRNA的加工。这一调控是高度针对细胞类型和基因的，这表明还有其他层面的调控尚未确定。CDK12和CDK13的基本生物学功能仍然知之甚少。因此，了解它们的功能机制对于确定转录和剪接的协调调控的关键或新方面是至关重要的。我们将用这些方法研究CDK12和CDK13：目的1.确定CDK12和CDK13复合体调节RNA加工的机制。邻近标记将用于识别与CDK12和CDK13相关的蛋白质和RNA序列。这将确定CDK12和CDK13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的加工。目的2.CDK12和CDK13复合体在RNA加工中的磷酸化调控。利用全球磷酸化蛋白质组学和一种有效的CDK12/CDK13共价抑制剂，我们将独立地确定CDK12和CDK13下游信号通路的动态。目的3.从功能基因组学角度明确CDK12和CDK13在细胞中的作用。利用RNA-seq和蛋白质组学，我们将确定CDK12和CDK13调控的基因和途径。我们将使用瞬时转录组(TT)-SEQ来检测由CDK12和CDK13调控的RNA的时间变化。这些实验将确定CDK12和CDK13的细胞角色。虽然CDK12和CDK13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被彻底研究。我们的计划将确定CDK12和CDK13复合体在RNA加工、转录、多聚腺苷化和剪接体调节中的特定功能。我们计划的总体意义将是增加对控制协调转录和RNA处理影响全球基因表达状态的关键细胞调控过程的理解。

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.