Structural and functional studies of mRNA processing, stability and quality control

项目摘要 大多数真核信使RNA前体(Pre-mRNAs)必须经历广泛的协同作用 在它们被输出到细胞质之前，在核中进行转录处理 并作为mRNAs发挥作用。加工事件包括5美分末端封口、拼接和3美分- 末端切割和多聚腺苷酸化。大多数前mRNAs的3‘端处理需要 它的执行需要大量的蛋白质因子，包括卵裂和 多聚腺苷酸特异性因子(CPSF)、切割刺激因子(CstF)、切割 因子I和因子II，以及多聚(A)聚合酶(PAP)。中国的3‘端加工机械 酵母与哺乳动物有相似之处，但也有显著的不同。 复制依赖的组蛋白前mRNAs在其3？ 结束并使用不同的机器进行处理，尽管它共享一些关键 蛋白质因子与规范的Pre-mRNA3？端加工机制。 在RNA聚合酶II转录的早期，mRNA5‘端封顶，并且它 人们普遍认为，封顶总是会进行到完成。我们发现 早些时候，DXO/Rai1家族的蛋白质是mRNA封顶质量的一部分 监督机制。它们可以拥有RNA5‘端焦磷水解酶(PPH) 以及去包裹活动，并帮助从细胞中移除未完全封顶的mRNA。 尽管对这些mRNA的加工和质量控制因素进行了广泛的研究， 我们对它们的分子作用机制的认识仍然存在很大差距。 在之前的资助期间，我们通过冷冻EM确定了一个活性物质的结构， 首台全重组人组蛋白前信使核糖核酸末端处理机 一种主动加工机械的结构。我们还展示了DXO/RAI1 和Nudex家族的酶可以去除RNA上的核苷酸代谢物帽子，例如 NAD(DeNADding)、FAD(DeFADding)和去磷化CoA(DeCoAping)。这些 成功为拟议的项目提供了良好的基础。 我们当前资助期的主要目标是产生新的结构性信息。 关于前-信使核糖核酸和信使核糖核酸3‘端的加工机制，并理解 DXO/Rai1和Nudex不同脱壳活性的分子基础 酵素。开展蛋白质因子及其复合体的结构研究 通过冷冻-EM和结晶学，并仔细评估结构观察 生物化学和机能实验。拟议中的项目将大大增强我们的 了解信使核糖核酸生命周期中的这些重要事件。

Project Summary Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo extensive co- transcriptional processing in the nucleus before they can be exported to the cytoplasm and function as mRNAs. The processing events include 5¢-end capping, splicing, and 3¢- end cleavage and polyadenylation. The 3’-end processing of most pre-mRNAs requires a large number of protein factors for its execution, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II, and poly(A) polymerase (PAP). The 3’-end processing machinery in yeast has similarity to that in mammals, although there are also significant differences. Replication-dependent histone pre-mRNAs contain a conserved stem-loop near their 3¢- end and employ a distinct machinery for its processing, although it shares some key protein factors with the canonical pre-mRNA 3¢-end processing machinery. mRNA 5¢-end capping occurs early during transcription by RNA polymerase II, and it was generally believed that capping always proceeds to completion. We discovered earlier that the DXO/Rai1 family of proteins are part of a mRNA capping quality surveillance mechanism. They can possess RNA 5¢-end pyrophosphohydrolase (PPH) and decapping activities, and help remove incompletely capped mRNAs from cells. Despite the extensive studies on these mRNA processing and quality control factors, significant gaps remain in our knowledge on their molecular mechanisms of action. During the previous funding period, we determined the structure by cryo-EM of an active, fully-reconstituted human histone pre-mRNA 3¢-end processing machinery, the first structure of an active processing machinery. We have also shown that the DXO/Rai1 and Nudix family of enzymes can remove nucleotide metabolite caps on RNAs, such as NAD (deNADding), FAD (deFADding) and dephospho-CoA (deCoAping). These successes provide an excellent foundation for the proposed project. Our main goals for the current funding period are to produce new structural information on pre-mRNA and snRNA 3¢-end processing machineries, and to understand the molecular basis for the diverse decapping activities of the DXO/Rai1 and Nudix enzymes. We will carry out structural studies on the protein factors and their complexes by both cryo-EM and crystallography, and assess the structural observations by careful biochemical and functional experiments. The proposed project will greatly enhance our understanding of these important events in the mRNA lifecycle.