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

项目摘要(来自资助的Mira申请) 大多数真核信使RNA前体(Pre-mRNAs)必须经历广泛的协同作用 在核中进行转录加工，然后才能输出到细胞质和功能 作为mRNAs。加工事件包括5？端封顶、剪接和3？端切割和 多聚腺苷酸化。大多数前-mRNAs的3‘端加工需要大量蛋白质 影响其执行的因素包括切割和多聚腺苷酸化特异性因子(CPSF)、切割 刺激因子(CstF)、切割因子I和II以及多聚(A)聚合酶(PAP)。3‘端 酵母中的加工机制与哺乳动物中的相似，尽管也有显著的 不同之处。复制依赖的组蛋白前mRNAs在其3？ 结束并使用不同的加工机制，尽管它有一些关键的蛋白质因素 使用标准的Pre-mRNA3‘端处理设备。 在RNA聚合酶II转录过程中，mRNA5‘端的封顶发生在早期，通常是 相信封顶总是会完成的。我们早些时候发现DXO/RAI1 蛋白质家族是信使核糖核酸质量监控机制的一部分。他们可以拥有 RNA5‘端焦磷水解酶(PPH)和解帽活性，并有助于清除不完全 从细胞中提取的mRNAs。 尽管对这些信使核糖核酸的加工和质量控制因素进行了广泛的研究，但显著的差距 我们对它们的分子作用机制仍然知之甚少。在之前的资助期间 期间，我们通过冷冻-电子显微镜确定了活性的、完全重组的人组蛋白前... MRNA3末端加工机械，是第一台结构活跃的加工机械.我们有 还表明，DXO/Rai1和Nudex家族的酶可以去除核苷酸代谢物的帽子 在RNA上，如NAD(DeNADding)、FAD(DeFADding)和DePho-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关于前信使核糖核酸的新的结构信息。 和SnRNA3的末端加工机制，并了解分子基础的多样性 DXO/Rai1和Nudex酶的脱帽活性。我们会进行结构研究。 蛋白质因子及其复合体的冷冻-电子显微镜和结晶学，并评估其结构 通过仔细的生化和机能实验进行观察。拟议中的项目将极大地 增强我们对信使核糖核酸生命周期这些重要事件的理解。 1

Project Summary (from the funded MIRA application) 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. 1