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

项目摘要（从资助的MIRA应用程序） 大多数真核生物的信使RNA前体（pre-mRNAs）必须经过广泛的共- 在它们可以被输出到细胞质并发挥功能之前， 作为mRNA。加工事件包括5 ′-末端加帽、剪接和3 ′-末端切割， 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， 其执行因子，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以及聚腺苷酸聚合酶（PAP）。3 '端 酵母中的加工机制与哺乳动物中的加工机制相似，尽管也存在显著的差异。 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， 结束并使用一种独特的机器进行加工，尽管它共享一些关键的蛋白质因子， 与典型的前mRNA 3 ′末端加工机制。 mRNA的5 ′-末端加帽发生在RNA聚合酶II转录的早期， 认为封顶总是会进行到完成。我们之前发现DXO/Rai 1 蛋白质家族是mRNA加帽质量监督机制的一部分。他们可以拥有 RNA 5 ′-末端焦磷酸水解酶（PPH）和脱帽活性，并有助于不完全去除 加帽的mRNA。 尽管对这些mRNA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了解还很有限。在过去的融资中 在此期间，我们通过冷冻电镜确定了一种活性的、完全重建的人类组蛋白的结构， mRNA的3端加工机制，是第一个结构活跃的加工机制。我们有 还表明DXO/Rai 1和Nuxine家族的酶可以去除核苷酸代谢物帽， 在RNA上，例如NAD（deNADding）、FAD（deFADding）和脱磷酸辅酶A（deCoAping）。这些 成功的经验为拟议的项目提供了良好的基础。 我们在当前资助期的主要目标是产生关于前mRNA的新结构信息。 和snRNA 3端加工机制，并了解不同的分子基础， DXO/Rai 1和Nutrient酶的去盖活性。我们会进行结构研究， 通过冷冻电镜和晶体学研究蛋白质因子及其复合物，并评估其结构 通过仔细的生物化学和功能实验进行观察。该项目将大大 增强我们对mRNA生命周期中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