A structural and biochemical approach to investigating the composition, architecture and functionality of UPF1-centric degradosomes assembled during turnover of functional mRNA in eukaryotes.

一种结构和生化方法，用于研究真核生物中功能 mRNA 周转期间组装的以 UPF1 为中心的降解体的组成、结构和功能。

• 批准号: 463158693
• 负责人: Professorin Dr. Sutapa Chakrabarti, Ph.D.
• 金额: --
• 依托单位: Chakrabarti Group - mRNA-Metabolism
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/463158693?language=en
• 关键词: structural; biochemical; approach; investigating; composition

This research proposal aims to investigate the composition, architecture and function of multi-protein/RNA complexes or degradosomes assembled by the RNA helicase UPF1 in pathways of functional mRNA turnover, using a combination of biochemical, structural and cell-based methods. The decay of mRNA is a crucial step in post-transcriptional gene regulation; its mis-regulation typically leads to cellular pathologies. Not surprisingly therefore, cells have devised mechanisms to make the process of mRNA decay as efficient and robust as possible. One of the ways by which cells streamline the decay of functional mRNA is by assembling decay-competent RNA-protein complexes known as degradosomes. The minimal degradosome contains an RNA helicase and a ribonuclease, while more complex assemblies might include a polymerase and a “sensor” protein that signals for RNA decay. The helicase UPF1, best known for its function in nonsense-mediated mRNA decay (NMD), is also involved in several pathways that mediate turnover of functional cellular mRNA. While the molecular mechanisms of UPF1 in context of an NMD-assembly are very well understood, comparatively little is known about its recruitment and functionality in pathways of mRNA turnover. To this end, I propose to investigate two UPF1-dependent mRNA turnover pathways – Staufen-mediated mRNA decay (SMD) and decay of replication-dependent histone mRNAs. Previous work suggested that UPF1 is recruited to these pathways by direct interactions with specific trans-acting protein factors, namely Staufen in the SMD pathway and the stem-loop binding protein (SLBP) in histone mRNA decay. UPF1 unwinds the structured RNA and, as a result, facilitates decay of the target mRNA transcript. Our recent studies showed, for the first time, that in addition to UPF1, the NMD factor UPF2 plays a crucial role in SMD by forming a scaffold for assembling a decay-competent mRNA-protein complex (mRNP). The involvement of different trans-acting factors in SMD and histone mRNA decay points to the assembly of a distinct decay-inducing mRNP in each case. Since assembly of a UPF1-mRNP is the trigger for initiating mRNA decay both in SMD and in histone mRNA decay, it is imperative to investigate this process at a molecular level. Therefore, we propose to determine the composition of the UPF1-centric degradosomes in the two decay pathways, and investigate their step-wise assembly and disassembly and the molecular mechanisms that lead to target mRNA decay in each case. We will employ a combination of structural (X-ray crystallography and cryo-electron microscopy) and biochemical methods, together with cell-based tools to address these aspects of mRNA decay. In addition to deciphering the mechanisms of these important cellular decay pathways, our studies will also provide insight into the crosstalk among different mRNA degradation pathways and will bring us closer towards defining the principles of mRNP/degradosome assembly in eukaryotes.

本研究计划旨在研究多蛋白质/RNA复合物或降解体的组成，结构和功能，通过RNA解旋酶UPF 1组装在功能mRNA周转途径中，使用生物化学，结构和细胞为基础的方法相结合。mRNA的降解是转录后基因调控的关键步骤;其错误调控通常导致细胞病理学。因此，毫不奇怪，细胞已经设计出机制，使mRNA衰变过程尽可能有效和稳健。细胞使功能性mRNA的衰变流线化的方法之一是组装有衰变能力的RNA-蛋白质复合物，称为降解体。最小的降解体包含RNA解旋酶和核糖核酸酶，而更复杂的组件可能包括聚合酶和“传感器”蛋白质，该蛋白质发出RNA降解的信号。解旋酶UPF 1以其在无义介导的mRNA衰变（NMD）中的功能而闻名，也参与介导功能性细胞mRNA周转的几种途径。虽然在NMD组装背景下UPF 1的分子机制非常清楚，但对其在mRNA周转途径中的募集和功能知之甚少。为此，我建议调查两个UPF 1依赖的mRNA周转途径-施陶芬介导的mRNA衰变（SMD）和复制依赖的组蛋白mRNA衰变。以前的工作表明，UPF 1通过与特定的反式作用蛋白因子直接相互作用而被招募到这些通路中，即SMD通路中的Staufen和组蛋白mRNA衰变中的茎环结合蛋白（SLBP）。UPF 1解旋结构化的RNA，因此促进靶mRNA转录物的衰变。我们最近的研究首次表明，除了UPF 1之外，NMD因子UPF 2通过形成组装衰变能力mRNA-蛋白质复合物（mRNP）的支架在SMD中起着至关重要的作用。SMD和组蛋白mRNA衰变中不同的反式作用因子的参与指向每种情况下不同衰变诱导mRNP的组装。由于UPF 1-mRNP的组装是SMD和组蛋白mRNA衰变中启动mRNA衰变的触发器，因此必须在分子水平上研究这一过程。因此，我们建议确定UPF 1为中心的降解体在两个衰变途径的组成，并研究其逐步组装和拆卸和分子机制，导致在每种情况下的目标mRNA衰变。我们将采用结构（X射线晶体学和冷冻电子显微镜）和生物化学方法的组合，以及基于细胞的工具来解决mRNA衰变的这些方面。除了破译这些重要的细胞衰变途径的机制外，我们的研究还将深入了解不同mRNA降解途径之间的串扰，并使我们更接近于定义真核生物中mRNP/降解体组装的原理。