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

 描述(申请人提供)：大多数真核信使RNA前体(前mRNAs)必须在细胞核内经过广泛的共转录处理后才能输出到细胞质并作为mRNAs发挥作用。加工事件包括5个ʹ‘端封端、剪接和3个ʹ’端切割和聚腺苷酸化。大多数前-mRNAs的3‘端加工需要大量的蛋白质因子来执行，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(A)聚合酶(PAP)。酵母的3‘端加工机制与哺乳动物的相似，但也有显著的差异。依赖复制的组蛋白前ʹ‘端在其3ʹ’端附近含有一个保守的茎环，并使用一种不同的机制进行加工，尽管它与典型的前mRNA3 RNA‘端处理机制有一些共同的蛋白质因子。MRNA5RNA‘端的封顶发生在ʹ聚合酶II转录的早期，人们普遍认为封顶总是进行到完成。我们最近发现，Rai1/DXO蛋白家族是mRNA封顶质量监控机制的一部分。它们可以具有RNA5ʹ‘端焦磷水解酶和解帽活性，并帮助从细胞中去除未完全封顶的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，但我们对它们的分子作用机制的了解仍然存在重大差距。我们将对蛋白质因子及其复合体进行结构研究，并通过仔细的生化和功能实验对结构观察进行评估。该项目将极大地提高我们对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.