Characterization of Ars2 function in RNA processing

RNA 加工中 Ars2 功能的表征

• 批准号: RGPIN-2015-06811
• 负责人: Howard, Perry
• 金额: $ 2.19万
• 依托单位: University of Victoria
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683381
• 关键词: Characterization; Ars2; function; RNA; processing

The long term goal of my research program is to address how different families of RNA transcripts are recognized and correctly processed to mature RNAs with distinct destinations and functions and to understand how the different types of RNA contribute to stem and progenitor cell behavior. The generation of mature RNA in the nucleus is a highly coordinated multistep process that requires distinct complexes for the biogenesis of different RNA families. RNAPII transcripts include at least 4 different types of RNA, each with its own processing requirements. How different families of RNAPII transcripts are recognized and processed to generate mature RNA is not understood. My lab discovered a gene called Ars2 that is a critical regulator of RNAPII transcript maturation and surveillance (appendix 2). We were the first to produce a mouse knockout for Ars2 and discovered that it is required for maintenance of stem and progenitor cells. We showed that Ars2 is the mammalian orthologue of the plant SERRATE gene and we predicted that ARS2(protein) would be important for RNA biogenesis. Our publication led to the discovery that ARS2 is an integral part of the nuclear cap complex and is necessary for the biogenesis of several different types of RNAPII transcripts. Therefore understanding ARS2 function is a critical step towards our long term goal. ***The story emerging from our work is that ARS2 has multiple putative elements for binding to both RNA and processing-protein components. Thus ARS2 is poised to act as a key regulator of RNA maturation with the ability to regulate the processing of different RNA molecules. However we do not have a complete understanding of ARS2 protein or RNA interactions. For example, we identified an RRM domain in ARS2. We have shown that the RRM domain is required specifically for replication dependent histone mRNA processing and cell cycle progression, yet mutation of the canonical RNA binding interface does not affect the ability of ARS2 to interact with histone mRNA or histone processing machinery. Similarly there are conserved arginine, glutamate, and proline rich regions highly likely to be forming interactions for which we do not know the function. ***We hypothesize ARS2 is critical nexus of RNA processing that couples the CBC to the appropriate processing machinery. As a first step to address this hypothesis we need to know what ARS2 interacts with and how it interacts with these proteins and RNAs. Our short term aims are 1) to determine the discreet protein interaction surfaces of ARS2 2) to determine the RNA interaction surfaces of ARS2 and 3) to determine which ARS2 interactions underpin ARS2 loss-of-function phenotypes. Our aims are the requisite logical steps towards our long term goal of understanding how ARS2 contributes to stem and progenitor maintenance and differentiation and provide a mechanistic model for the rationale design of in vivo experiments. **

我的研究计划的长期目标是解决如何识别不同的RNA转录物家族并正确加工成具有不同目的地和功能的成熟RNA，并了解不同类型的RNA如何有助于干细胞和祖细胞行为。 成熟RNA在细胞核中的产生是一个高度协调的多步骤过程，需要不同的复合物来进行不同RNA家族的生物发生。RNAPII转录物包括至少4种不同类型的RNA，每种都有自己的加工要求。RNAPII转录物的不同家族如何被识别和加工以产生成熟RNA尚不清楚。我的实验室发现了一个名为Ars2的基因，它是RNAPII转录成熟和监视的关键调节因子（附录2）。我们是第一个产生Ars2敲除小鼠的人，并发现它是维持干细胞和祖细胞所必需的。我们表明，Ars2是哺乳动物的植物SERRATE基因的直系同源物，我们预测，ARS2（蛋白质）将是重要的RNA生物合成。我们的出版物导致发现ARS2是核帽复合物的组成部分，并且对于几种不同类型的RNAPII转录物的生物发生是必需的。 因此，了解ARS2功能是实现我们长期目标的关键一步。* 从我们的工作中出现的故事是，ARS2有多个假定的元件与RNA和加工蛋白组分结合。 因此，ARS2有望作为RNA成熟的关键调节因子，具有调节不同RNA分子加工的能力。 然而，我们对ARS2蛋白或RNA相互作用没有完全的了解。例如，我们在ARS2中鉴定了RRM结构域。我们已经表明，RRM结构域是复制依赖性组蛋白mRNA加工和细胞周期进程所必需的，但典型RNA结合界面的突变不影响ARS 2与组蛋白mRNA或组蛋白加工机制相互作用的能力。类似地，有保守的精氨酸，谷氨酸和脯氨酸丰富的区域很可能形成相互作用，我们不知道其功能。* 我们假设ARS 2是RNA加工的关键联系，将CBC与适当的加工机制偶联。作为解决这一假设的第一步，我们需要知道ARS2与什么相互作用以及它如何与这些蛋白质和RNA相互作用。我们的短期目标是1）确定ARS 2的离散蛋白质相互作用表面，2）确定ARS 2的RNA相互作用表面，3）确定哪些ARS 2相互作用支持ARS 2功能丧失表型。 我们的目标是理解ARS2如何有助于干细胞和祖细胞的维持和分化的长期目标的必要逻辑步骤，并为体内实验的合理设计提供一个机制模型。**