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

本研究计划旨在结合生物化学、结构和细胞为基础的方法，研究RNA解旋酶UPF1组装的多蛋白/RNA复合物或降解体在功能性mRNA转换途径中的组成、结构和功能。mRNA的衰变是转录后基因调控的关键步骤；它的错误调控通常会导致细胞病变。因此，毫不奇怪，细胞已经设计出机制，使mRNA的衰变过程尽可能高效和稳健。细胞使功能性mRNA的衰变流程化的方法之一是通过组装具有降解能力的rna -蛋白质复合物，即降解体。最小的降解体包含RNA解旋酶和核糖核酸酶，而更复杂的组装可能包括聚合酶和发出RNA衰变信号的“传感器”蛋白。解旋酶UPF1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UPF1在nmd组装中的分子机制已经被很好地理解，但相对而言，我们对其在mRNA转换途径中的募集和功能知之甚少。为此，我建议研究两种依赖upf1的mRNA转换途径——staufen介导的mRNA衰变（SMD）和复制依赖性组蛋白mRNA的衰变。先前的研究表明，UPF1通过与特定的反式作用蛋白因子（即SMD途径中的Staufen和组蛋白mRNA衰减中的茎环结合蛋白（SLBP））的直接相互作用而被招募到这些途径中。UPF1解绕结构RNA，从而促进目标mRNA转录物的衰变。我们最近的研究首次表明，除了UPF1， NMD因子UPF2在SMD中起着至关重要的作用，它通过形成一个支架来组装具有衰变能力的mrna -蛋白复合物（mRNP）。不同的反式作用因子参与SMD和组蛋白mRNA的衰变，指出每种情况下都有不同的诱导衰变的mRNP的组装。由于UPF1-mRNP的组装是SMD和组蛋白mRNA衰变中启动mRNA衰变的触发因素，因此有必要在分子水平上研究这一过程。因此，我们建议确定两种降解途径中upf1中心降解体的组成，并研究它们的分步组装和拆卸以及每种情况下导致目标mRNA衰变的分子机制。我们将采用结构（x射线晶体学和低温电子显微镜）和生化方法的组合，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，并将使我们更接近于定义真核生物中mRNP/降解体组装的原理。

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.