Understanding the Control of Processing Bodies by RhoA GTPase

了解 RhoA GTPase 对加工体的控制

• 批准号: RGPIN-2015-04882
• 负责人: Corcoran, Jennifer
• 金额: $ 2.33万
• 依托单位: University of Calgary
• 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=684478
• 关键词: Understanding; Control; Processing; Bodies; RhoA

Viruses are useful tools that often reveal previously unrecognized levels of control within a cell. By exploiting Kaposi's sarcoma-associated herpesvirus (KSHV), I identified that the cytoskeletal GTPase, RhoA is a key regulator of messenger RNA (mRNA) stability and cytoplasmic processing bodies (PBs). ****PBs are small ribonucleoprotein (RNP)-containing cytoplasmic granules and major sites of mRNA decay that significantly contribute to the precise post-transcriptional regulation of cellular gene expression. PBs are dynamic, responding to changes in mRNA accumulation or protein translation status by altering their size and number. PBs also respond to certain cellular stressors; however, many questions remain regarding the upstream signals controlling PB assembly and disassembly. ****The rho family of small GTPases are molecular switches that cycle between inactive GDP- and active GTP-bound forms and thereby control several fundamental cellular processes. RhoA regulates actin cytoskeleton dynamics to facilitate normal cell attachment, the formation of actin stress fibers, cell migration and angiogenesis. RhoA activation also positively impacts the transcription of genes containing serum-response elements (SREs), coupling changes to the actin cytoskeleton with increased transcription. RhoA-mediated control of other aspects of gene expression remains unclear. ****By exploiting KSHV, I showed that the viral protein Kaposin B (KapB) activates RhoA. RhoA activation is necessary for KapB to negatively regulate PB formation and decrease the turnover of labile cellular mRNA. Interestingly, KapB-mediated changes to the actin cytoskeleton can be uncoupled from its effect on PB assembly using an inhibitor of the RhoA downstream effector protein, Rho-associated kinase (ROCK). When ROCK is inhibited, KapB fails to promote actin polymerization yet it is still able to disrupt PBs. ****I hypothesize that RhoA mediates the dispersal of cellular PBs using a novel mechanism that is completely independent from its control of the actin cytoskeleton and microtubule network. Specific objectives are proposed to test this hypothesis: (1) to use time-lapse live cell microscopy to precisely track the changes that occur to PBs, the actin cytoskeleton, and microtubules immediately after RhoA activation (2) to determine if active RhoA modifies, degrades, or recruits PB component proteins (3) to use genetic approaches to identify the effector proteins downstream of active RhoA that are required for RhoA-mediated PB dispersal. My NSERC-funded research program will elucidate the upstream signals that link active RhoA to the control of PB formation and identify novel downstream effectors used by RhoA to mediate PB dispersion, providing insight into the poorly understood process of RhoA-controlled post-transcriptional gene expression.**

病毒是有用的工具，通常会揭示细胞内以前未被认识到的控制水平。通过利用Kaposi肉瘤相关疱疹病毒(KSHV)，我发现细胞骨架GTP酶RhoA是信使RNA(MRNA)稳定性和细胞质加工体(PBS)的关键调节因子。*PBS是含有小核糖核蛋白(RNP)的细胞质颗粒，也是mRNA衰变的主要部位，对细胞基因表达的精确转录后调控具有重要作用。PB是动态的，通过改变它们的大小和数量来响应mRNA积累或蛋白质翻译状态的变化。PB也对某些细胞应激源做出反应；然而，关于控制PB组装和拆卸的上游信号仍然存在许多问题。*Rho家族的小GTP酶是分子开关，在不活跃的GDP结合形式和活跃的GTP结合形式之间循环，从而控制几个基本的细胞过程。RhoA调节肌动蛋白细胞骨架的动力学，促进细胞的正常附着、肌动蛋白应激纤维的形成、细胞迁移和血管生成。RhoA的激活也积极地影响了含有血清反应元件(SRE)的基因的转录，使肌动蛋白细胞骨架的变化与转录的增加相结合。RhoA介导的对基因表达的其他方面的控制仍然不清楚。*通过利用KSHV，我证明了病毒蛋白Kaposin B(KapB)激活了RhoA。RhoA的激活是KapB负性调节PB形成和降低不稳定的细胞mRNA周转所必需的。有趣的是，KapB介导的肌动蛋白细胞骨架的变化可以通过RhoA下游效应蛋白的抑制剂Rho相关蛋白(ROCK)从其对PB组装的影响中分离出来。当ROCK被抑制时，KapB不能促进肌动蛋白聚合，但它仍然能够扰乱PBS。*我假设RhoA使用一种完全独立于其对肌动蛋白细胞骨架和微管网络的控制的新机制来介导细胞PBS的扩散。提出了检验这一假说的具体目标：(1)使用延时活细胞显微镜精确跟踪RhoA激活后立即发生的PBS、肌动蛋白细胞骨架和微管的变化(2)确定活性RhoA是否修饰、降解或招募PB组分蛋白(3)使用遗传学方法确定RhoA介导的PB扩散所需的活性RhoA下游的效应蛋白。我的NSERC资助的研究计划将阐明将活性RhoA与PB形成控制联系起来的上游信号，并确定RhoA用来调节PB扩散的新的下游效应物，从而深入了解RhoA控制转录后基因表达的鲜为人知的过程。**