Cytoplasmic mRNA regulation during cell differentation and proliferation during Drosophila development

我的研究项目旨在了解局部细胞质事件如何介导细胞分裂或分化。我们目前的重点是确定含有特定mrna和RNA结合蛋白的细胞质体如何局部调节参与中心体复制和分裂的mrna的翻译。***现在很清楚，mrna不仅仅是遗传信息从细胞核到细胞质翻译机制的被动传递者。我的实验室克隆并鉴定了果蝇gawky （gw）基因，该基因编码一种RNA结合蛋白，该蛋白构成细胞质mRNA调控复合物的核。众所周知，Gw在RNA加工(P)体介导的microRNA介导的mRNA调控中起着重要作用。我们的重点是第二个，p体独立，Gw定位调节mRNA翻译在特定细胞质域的作用。我们和其他人发现了独立于p体起作用的局部含gw RNA调节体（locgw - body）。我们还在胚胎中发现了几种与Gw结合但不被p体降解的mrna。这些gw结合/未降解mrna的很大一部分编码参与中心体合成或复制的蛋白质。中心体复制是细胞周期进程和建立细胞不对称性所必需的。我们认为这些特化的中心体locgw - body局部调控参与这些关键细胞过程的mRNA翻译。在接下来的5年里，我们将提出一些基本的问题：什么是核特化的locgw小体，以及与locgw小体结合的中心体特异性mrna的限制性定位和调控表达如何影响胚胎细胞的分裂或分化？***通过共免疫沉淀，我们发现了四种与Gw相互作用的中心体蛋白：Belle、Centrosomin、CG30122和Kinesin样蛋白61F。我们还发现微管星、中心体蛋白97kDa、中心体蛋白和纺锤体组装异常-6是与gw结合的mrna。这些mrna中的每一个都编码已知参与中心体分裂或复制的蛋白质。***我们的第一个目标是确定这些蛋白质相互作用物如何诱导核周（中心体）locgw -小体的形成，以及特异性mrna如何靶向果蝇细胞中这些特化的locgw -小体。我们的第二个目标是确定locgw小体如何通过在固定细胞或细胞组分中关联mRNA和蛋白质来局部调节蛋白质翻译。我们还在开发mRNA活细胞成像的新载体，以监测mRNA进入locgw小体的运动，以及这与局部蛋白质翻译的关系。这将允许细胞质mRNA定位与蛋白质翻译和后续加工的影响直接相关。我们的研究将为p体独立/ gw介导的mRNA调控的鲜为人知的作用以及它如何影响中心体功能提供基本的见解。

My research program seeks to understand how localized cytoplasmic events mediate cell division or differentiation. Our current focus is determining how cytoplasmic bodies containing specific mRNAs and RNA binding proteins locally regulate translation of mRNAs involved in centrosome duplication and division in Drosophila.***It is now clear that mRNAs are not simply passive transmitters of genetic information from the nucleus to the cytoplasmic translation machinery. My laboratory cloned and characterized the Drosophila gawky (gw) gene, encoding an RNA binding protein that nucleates cytoplasmic mRNA regulatory complexes. Gw has a well-known role during microRNA mediated mRNA regulation by RNA processing (P) bodies. Our focus is a second, P-body independent, role for Gw -localized regulation of mRNA translation in specific cytoplasmic domains. We, and others, discovered localized Gw-containing RNA regulatory bodies (locGw-bodies) that act independently of P-bodies. We also identified several mRNAs bound by Gw in embryos but not degraded by P-bodies. A large fraction of these Gw-bound/non-degraded mRNAs encode proteins involved in centrosome synthesis or duplication. Centrosome duplication is required for progression of the cell cycle and establishing cellular asymmetry. We propose that these specialized centrosome locGw-bodies locally regulate mRNA translation involved in these critical cellular processes. For the next 5 years we will ask the fundamental questions -what nucleates specialized locGw-bodies and how does restricted localization and regulated expression of centrosome specific mRNAs bound by locGw-bodies affect embryo cell division or differentiation?***By co-immunoprecipitation, we found four centrosome proteins that interact with Gw: Belle, Centrosomin, CG30122 and Kinesin like protein 61F. We also identified microtubule star, Centrosomal protein 97kDa, centrosomin and Spindle assembly abnormal-6 as Gw-bound mRNAs. Each of these mRNAs encodes proteins known to be involved in centrosome division or duplication.***Our first objective is to determine how these protein interactors induce formation of perinuclear (centrosome) locGw-bodies and how specific mRNAs are targeted into these specialized locGw-bodies in Drosophila cells. Our second objective is to determine how locGw-bodies regulate locally regulate protein translation by correlating mRNA and protein in fixed cells or cell fractions. We are also developing novel vectors for live-cell imaging of mRNAs to monitor mRNA movement into locGw-bodies and how this relates to local protein translation. This will allow direct correlation of cytoplasmic mRNA localization to the effects on protein translation and subsequent processing. Our studies will provide fundamental insight into the poorly understood roles of P-body independent/Gw-mediated mRNA regulation and how this affects centrosome function.