Characterization of Ars2 function in RNA processing

我的研究计划的长期目标是解决如何识别不同家族的RNA转录本，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如何促进干细胞和祖细胞的行为。成熟RNA在细胞核中的产生是一个高度协调的多步骤过程，不同RNA家族的生物发生需要不同的复合物。RNAPII转录本包括至少4种不同类型的RNA，每种RNA都有自己的加工要求。RNAPII转录本的不同家族如何被识别和加工以产生成熟的RNA尚不清楚。我的实验室发现了一个叫做Ars2的基因，它是RNAPII转录物成熟和监视的关键调节因子（附录2）。我们是第一个产生小鼠敲除Ars2，并发现它是维持干细胞和祖细胞所必需的。我们发现Ars2是植物SERRATE基因的哺乳动物同源基因，我们预测Ars2（蛋白）将在RNA生物发生中发挥重要作用。我们的研究发现，ARS2是核帽复合体的一个组成部分，是几种不同类型RNAPII转录本的生物发生所必需的。因此，了解ARS2的功能是实现我们长期目标的关键一步。***从我们的工作中得出的结论是，ARS2具有多个假定的与RNA和加工蛋白组分结合的元件。因此，ARS2具有调节不同RNA分子加工的能力，可以作为RNA成熟的关键调节因子。然而，我们对ARS2蛋白或RNA的相互作用还没有完全的了解。例如，我们在ARS2中识别了一个RRM域。我们已经证明，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，然而规范RNA结合界面的突变并不影响ARS2与组蛋白mRNA或组蛋白加工机制相互作用的能力。同样，有一些保守的精氨酸、谷氨酸和脯氨酸丰富的区域很可能形成相互作用，但我们不知道其功能。我们假设ARS2是RNA加工的关键纽带，将CBC偶联到适当的加工机制。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ARS2的离散蛋白相互作用表面2)确定ARS2的RNA相互作用表面3)确定哪些ARS2相互作用支持ARS2功能丧失表型。我们的目标是实现我们长期目标的必要逻辑步骤，即了解ARS2如何促进干细胞和祖细胞的维持和分化，并为体内实验的基本原理设计提供机制模型。**

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. **