Biochemical and structural investigation of UPF1 function in eukaryotic mRNA turnover pathways

这项研究计划旨在使用生物化学和结构（X射线晶体学）方法剖析ATP依赖性RNA解旋酶UPF 1在细胞mRNA周转途径中的作用。mRNA的降解是转录后基因调控中的重要步骤，因为它不仅用于调节基因表达的水平，而且用于调节基因表达的质量。因此，mRNA衰变的途径涉及几种炎性和自身免疫性疾病，以及由mRNA转录物畸变引起的遗传疾病。尽管mRNA水平调节（mRNA转换）和mRNA质量控制（mRNA监测）存在不同的途径，但已发现某些蛋白质因子（例如UPF 1）参与了这两种降解途径。UPF 1最初被鉴定为无义介导的mRNA衰变（NMD）途径的关键组分，NMD途径是降解含有提前终止密码子的mRNA转录物的监视途径。随后的研究表明，UPF 1还可以与其他RNA结合蛋白一起调节特定靶mRNA的水平。虽然UPF 1在NMD中的作用机制和调控机制已经非常清楚，但对其在mRNA周转途径中的分子机制知之甚少。为此，我将重点介绍UPF 1介导的两种mRNA周转途径：组蛋白mRNA降解和施陶芬介导的mRNA衰变（SMD）。组蛋白mRNA和SMD的降解分别由RNA结合蛋白SLBP和Staufen介导。这些伴侣蛋白以翻译终止依赖性方式将UPF 1募集到靶mRNA的3 '-UTR。UPF 1定位于mRNA的3 '-UTR随后引发降解。了解UPF 1是如何被这些RNA结合蛋白招募并剖析其在这些途径中的功能将阐明其在细胞mRNA周转中的作用。这将使我们能够生成UPF 1功能的综合模型（mRNA监视和mRNA周转），并提供对真核细胞中不同mRNA降解途径之间的串扰的见解。拟议的研究也将是一个独特的一步，将mRNA衰变视为一个全球性的现象，而不是作为单独的线性途径，并将提高我们的知识，致病基因的调节和转录后基因调控一般。

This research proposal aims to dissect the role of the ATP-dependent RNA helicase UPF1 in cellular mRNA turnover pathways using a biochemical and structural (X-ray crystallography) approach. The degradation of mRNA is an important step in post-transcriptional gene regulation as it serves to regulate not only the level, but also the quality, of gene expression. As such, pathways of mRNA decay are implicated in several inflammatory and autoimmune disorders, as well as in genetic disorders that arise from aberrations in mRNA transcripts. Although there exist distinct pathways for the regulation mRNA levels (mRNA turnover) and mRNA quality control (mRNA surveillance), certain protein factors, such as UPF1, have been found to be involved in both kinds of degradation pathways. UPF1 was originally identified as a key component of the nonsense mediated mRNA decay (NMD) pathway, which is a surveillance pathway that degrades mRNA transcripts containing a premature stop codon. Subsequent studies showed that UPF1 can also act in conjunction with other RNA-binding proteins to regulate levels of specific target mRNAs. Although the mechanism and regulation of UPF1 in NMD is very well understood, little is known about its molecular mechanisms in mRNA turnover pathways. To this end, I will focus on two pathways of mRNA turnover that are mediated by UPF1: histone mRNA degradation and Staufen-mediated mRNA decay (SMD). Degradation of histone mRNA and SMD are mediated by the RNA binding proteins SLBP and Staufen respectively. These partner proteins recruit UPF1 in a translation termination-dependent manner to the 3'-UTR of the target mRNA. Localization of UPF1 to the 3'-UTR of the mRNA subsequently triggers degradation. Understanding how UPF1 is recruited by these RNA-binding proteins and dissecting its function in these pathways will elucidate its role in cellular mRNA turnover. This will enable us to generate a comprehensive model for UPF1 function (both in mRNA surveillance and mRNA turnover) and provide insight into the cross-talk between different mRNA degradation pathways in the eukaryotic cell. The proposed research will also be a distinct step towards viewing mRNA decay as a global phenomenon, rather than as individual linear pathways, and will enhance our knowledge of the regulation of disease-causing genes and of post-transcriptional gene regulation in general.