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

项目摘要 大多数真核生物的信使RNA前体（pre-mRNAs）必须经过广泛的共- 在它们被输出到细胞质之前， 并作为mRNA发挥作用。加工事件包括5 ′-末端加帽、剪接和3 ′-末端加帽。 末端裂解和多聚腺苷酸化。大多数前体mRNA的3 '末端加工需要 大量的蛋白质因子用于其执行，包括切割， 多聚腺苷酸化特异性因子（CPSF），裂解刺激因子（CstF），裂解 因子I和II，以及聚（A）聚合酶（PAP）。3 '端加工机械 酵母菌与哺乳动物有相似之处，但也有显著差异。 复制依赖的组蛋白前体mRNA在其3 ′端附近含有一个保守的茎环， 结束和雇用一个独特的机器处理，虽然它共享一些关键 蛋白质因子与典型的前体mRNA 3 β末端加工机制。 mRNA 5 ′-末端加帽发生在RNA聚合酶II转录的早期， 人们普遍认为，封顶总是进行到完成。我们发现 早些时候，DXO/Rai 1蛋白家族是mRNA加帽质量的一部分， 监督机制。它们具有RNA 5 ′-末端焦磷酸水解酶（PPH） 和去帽活性，并帮助从细胞中去除不完全加帽的mRNA。 尽管对这些mRNA加工和质量控制因子进行了广泛的研究， 我们对其分子作用机制的了解仍存在重大差距。 在上一个资助期间，我们通过冷冻电镜确定了一种活性， 完全重组的人组蛋白前体mRNA 3 ′-末端加工机制， 主动加工机械的结构。我们还表明，DXO/Rai 1 和Nutrition家族的酶可以去除RNA上的核苷酸代谢物帽，例如 NAD（去NADding）、FAD（去FADding）和去磷酸辅酶A（去CoAping）。这些 成功的经验为拟议的项目提供了良好的基础。 我们当前融资期的主要目标是提供新的结构性信息 前体mRNA和snRNA 3 ′-末端加工机制，并了解 DXO/Rai 1和Nutrient不同去帽活性的分子基础 内切酶我们将对蛋白质因子及其复合物进行结构研究 通过cryo-EM和晶体学，并通过仔细的结构观察来评估 生化和功能实验。建议的项目将大大提高我们的 了解mRNA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.