Cytoplasmic mRNA regulation during cell differentation and proliferation during Drosophila development

我的研究项目试图了解局部细胞质事件如何调节细胞分裂或分化。我们目前的重点是确定含有特定mRNAs和RNA结合蛋白的细胞质小体如何在局部调节参与中心体复制和分裂的果蝇mRNAs的翻译。 现在很清楚，mRNAs不仅仅是遗传信息从细胞核到细胞质翻译机制的被动传送器。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，可形成细胞质mRNA调控复合体。GW在microRNA通过RNA加工(P)小体介导的mRNA调控中具有众所周知的作用。我们的重点是第二个，P-小体独立的，作用于GW对特定细胞质区域的mRNA翻译的局部调节。我们和其他人发现了局部的含有GW的RNA调节机构(LocGw-Body)，它们独立于P-Body发挥作用。我们还在胚胎中发现了几个与GW结合但不被P小体降解的mRNAs。这些与GW结合的/非降解的mRNAs中有很大一部分编码与中心体合成或复制有关的蛋白质。中心体复制是细胞周期进展和建立细胞不对称性所必需的。我们认为，这些特殊的中心体定位体在局部调节参与这些关键细胞过程的mRNA翻译。在接下来的5年里，我们将提出基本的问题--是什么形成了专门的LocGw小体，以及LocGw小体结合的中心体特定mRNAs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？ 通过免疫共沉淀，我们发现了四种与GW相互作用的中心体蛋白：Belle、CentroSomin、CG30122和类Kinesin蛋白61F。我们还鉴定了微管星形蛋白、中心体蛋白97 kDa、中心体蛋白和纺锤体组装异常-6为GW结合的mRNAs。这些mRNAs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如何诱导果蝇细胞中核周(中心体)LocGw小体的形成，以及特定的mRNAs如何靶向这些特殊的LocGw小体。我们的第二个目标是确定LocGw-Body如何通过关联固定细胞或细胞部分中的mRNA和蛋白质来调节局部调节蛋白质翻译。我们还在开发新的载体，用于对mRNAs进行活细胞成像，以监测mRNAs向LocGw小体的移动以及这与局部蛋白质翻译的关系。这将使细胞质mRNA定位与蛋白质翻译和后续加工的影响直接相关。我们的研究将为了解P-小体非依赖/GW介导的mRNA调控以及这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.