Cytoplasmic mRNA regulation during cell differentation and proliferation during Drosophila development

果蝇发育过程中细胞分化和增殖过程中细胞质 mRNA 的调节

• 批准号: RGPIN-2017-05885
• 负责人: Simmonds, Andrew
• 金额: $ 2.04万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=682205
• 关键词: Cytoplasmic; mRNA; regulation; during; cell

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.

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果蝇中心体复制和分裂的mRNA的翻译。现在很清楚，mRNA不仅仅是从细胞核到细胞质翻译机器的遗传信息的被动传递者。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，该蛋白使细胞质mRNA调控复合物成核。Gw在通过RNA加工（P）体的microRNA介导的mRNA调节期间具有众所周知的作用。我们的重点是第二个，P-体独立的，Gw本地化的特定胞质结构域中的mRNA翻译的调节作用。我们和其他人，发现了本地化的Gw含RNA调控机构（locGw机构）的行为独立于P-机构。我们还确定了几个mRNA结合的Gw在胚胎中，但不降解的P-机构。这些GW结合/非降解的mRNA的大部分编码参与中心体合成或复制的蛋白质。中心体复制对于细胞周期的进展和建立细胞不对称性是必需的。我们建议，这些专门的中心体locGw-机构本地调节mRNA的翻译参与这些关键的细胞过程。在接下来的5年里，我们将提出基本问题-是什么使专门的locGw体成核，以及由locGw体结合的中心体特异性mRNA的限制性定位和调节性表达如何影响胚胎细胞分裂或分化？通过免疫共沉淀的方法，我们发现了四个与Gw相互作用的中心体蛋白：贝儿、中心体蛋白、CG 30122和驱动蛋白样蛋白61 F。我们还鉴定了微管星星、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mRNA。这些mRNA中的每一种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形成核周（中心体）locGw体和特定的mRNA是如何靶向到这些专门的locGw体在果蝇细胞。我们的第二个目标是确定locGw体如何通过将固定细胞或细胞组分中的mRNA和蛋白质相关联来调节局部调节蛋白质翻译。我们还在开发新的载体，用于mRNA的活细胞成像，以监测mRNA进入locGw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定位与对蛋白质翻译和后续加工的影响直接相关。我们的研究将提供基本的洞察P体独立/GW介导的mRNA调控的作用知之甚少，这是如何影响中心体功能。