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

 描述（由申请人提供）：大多数真核信使RNA前体（pre-mRNAs）必须在细胞核中经历广泛的共转录加工，然后才能输出到细胞质并作为mRNAs发挥功能。加工事件包括5 ′端加帽、剪接和3 ′端切割和多聚腺苷酸化。大多数前体mRNA的3 '末端加工需要大量蛋白质因子用于其执行，包括切割和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）。酵母中的3 '端加工机制与哺乳动物中的相似，尽管也存在显著差异。复制依赖性组蛋白前mRNA在其3 ′端附近含有保守的茎环，并采用独特的加工机制，尽管它与典型的前mRNA 3 ′端加工机制共享一些蛋白质因子。mRNA的5 ′端加帽发生在RNA聚合酶II转录的早期，一般认为加帽总是进行到完成。我们最近发现Rai 1/DXO蛋白家族是mRNA加帽质量监控机制的一部分。它们可以具有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，但我们对其分子作用机制的认识仍存在重大差距。我们将对蛋白质因子及其复合物进行结构研究，并通过仔细的生化和功能实验评估结构观察结果。该项目将极大地增强我们对mRNA生命周期中这些重要事件的理解。

 DESCRIPTION (provided by applicant): 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 5ʹ′-end capping, splicing, and 3ʹ′- 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 3ʹ′- end and employ a distinct machinery for its processing, although it shares some protein factors with the canonical pre-mRNA 3ʹ′-end processing machinery. mRNA 5ʹ′-end capping occurs early during transcription by RNA polymerase II, and it was generally believed that capping always proceeds to completion. We have recently discovered that the Rai1/DXO family of proteins are part of a mRNA capping quality surveillance mechanism. They can possess RNA 5ʹ′-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 of their molecular mechanisms of action. We will carry out structural studies on the protein factors and their complexes, and assess the structural observations by careful biochemical and functional experiments. The proposed project will greatly enhance our understanding of these important events in mRNA lifecycle.