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

这项研究计划旨在用生化和结构(X射线结晶学)的方法剖析ATP依赖的RNA解旋酶UPF1在细胞mRNA周转途径中的作用。MRNA的降解是转录后基因调控的重要步骤，因为它不仅调节基因表达的水平，而且还调节基因表达的质量。因此，信使核糖核酸的衰变途径与几种炎症性和自身免疫性疾病以及由信使核糖核酸转录本中的异常引起的遗传性疾病有关。虽然有不同的途径来调节信使核糖核酸的水平(信使核糖核酸的转换)和信使核糖核酸的质量控制(信使核糖核酸的监测)，某些蛋白质因子，如UPF1，已被发现参与了这两种降解途径。UPF1最初被认为是无意义介导的mRNA衰退(NMD)途径的关键组成部分，该途径是一种监测途径，降解含有过早终止密码子的mRNA转录本。随后的研究表明，UPF1还可以与其他RNA结合蛋白一起作用，调节特定靶向mRNAs的水平。尽管人们对UPF1在NMD中的作用机制和调控机制已有很好的了解，但对其在mRNA转运途径中的分子机制却知之甚少。为此，我将重点介绍UPF1介导的两条信使核糖核酸转换途径：组蛋白信使核糖核酸降解和史陶芬介导的信使核糖核酸衰变(SMD)。组蛋白mRNA和SMD的降解分别由RNA结合蛋白SLBP和Staufen介导。这些伙伴蛋白以翻译终止依赖的方式将UPF1招募到目标mRNA的3‘-UTR端。UPF1定位于mRNA的3‘-UTR区，随后引发降解。了解UPF1是如何被这些RNA结合蛋白招募的，并剖析它在这些途径中的功能，将有助于阐明它在细胞mRNA周转中的作用。这将使我们能够为UPF1功能建立一个全面的模型(在mRNA监测和mRNA周转中)，并提供对真核细胞中不同mRNA降解途径之间的串扰的洞察。这项拟议的研究也将是朝着将mRNA衰退视为一种全球现象而不是单个线性途径迈出的独特一步，并将增强我们对致病基因和转录后基因调控的总体知识。

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.