Characterization of Ars2 function in RNA processing

我的研究计划的长期目标是解决不同家族的RNA转录本如何被识别和正确处理为具有不同目的地和功能的成熟RNA，并了解不同类型的RNA如何对干细胞和祖细胞行为做出贡献。成熟RNA在细胞核中的生成是一个高度协调的多步骤过程，需要不同RNA家族的不同复合体来进行生物发生。RNAPII转录本包括至少4种不同类型的RNA，每种类型都有自己的处理要求。不同家族的RNAPII转录本是如何被识别和处理以产生成熟的RNA的，目前尚不清楚。我的实验室发现了一种名为Ars2的基因，它是RNAPII转录成熟和监控的关键调节因子(附录2)。我们是第一个为Ars2基因敲除小鼠基因的人，并发现它是维持干细胞和祖细胞所必需的。我们证明了ARS2是植物锯齿状基因的哺乳动物同源基因，我们预测ARS2(蛋白质)将在RNA的生物发生中发挥重要作用。我们的研究发现，ARS2是核帽复合体的组成部分，对于几种不同类型的RNAPII转录本的生物发生是必要的。因此，了解ARS2的功能是实现我们长期目标的关键一步。*从我们的工作中出现的故事是，ARS2有多个假定的元件可以与RNA和加工蛋白质成分结合。因此，ARS2有望成为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的相互作用还没有完全的了解。例如，我们在ARS2中确定了一个RRM域。我们已经证明，RRM结构域是复制依赖的组蛋白mRNA加工和细胞周期进展所特需的，然而规范的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，还有一些保守的精氨酸、谷氨酸和脯氨酸富集区，很可能形成我们不知道其功能的相互作用。*我们假设ARS2是RNA处理的关键纽带，它将CBC连接到适当的处理机械。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的离散蛋白质相互作用面；2)确定ARS2的RNA相互作用面；3)确定哪些ARS2相互作用支持ARS2功能丧失表型。我们的目标是朝着我们的长期目标--了解ARS2如何有助于干细胞和祖细胞的维持和分化--的必要的逻辑步骤，并为活体实验的基本设计提供一个机制模型。**

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. **