Understanding the Control of Processing Bodies by RhoA GTPase

病毒是一种有用的工具，它经常揭示细胞内以前未被识别的控制水平。通过利用卡波济肉瘤相关疱疹病毒（KSHV），我确定了细胞骨架GT3，RhoA是信使RNA（mRNA）稳定性和细胞质加工体（PB）的关键调节剂。* PB是含有小核糖核蛋白（RNP）的细胞质颗粒和mRNA衰变的主要位点，显著有助于细胞基因表达的精确转录后调控。PB是动态的，通过改变其大小和数量来响应mRNA积累或蛋白质翻译状态的变化。PB也对某些细胞应激源作出反应;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。* 小GTP酶的rho家族是在非活性GDP结合形式和活性GTP结合形式之间循环的分子开关，从而控制几个基本的细胞过程。RhoA调节肌动蛋白细胞骨架动力学以促进正常细胞附着、肌动蛋白应力纤维的形成、细胞迁移和血管生成。 RhoA激活也对含有血清反应元件（SRE）的基因的转录产生积极影响，将肌动蛋白细胞骨架的变化与增加的转录偶联。RhoA介导的基因表达的其他方面的控制仍不清楚。* 通过利用KSHV，我证明了病毒蛋白Kaposin B（KapB）激活RhoA。RhoA激活是KapB负调节PB形成和降低不稳定细胞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从其对PB组装的影响中解偶联，使用RhoA下游效应蛋白Rho相关激酶（ROCK）的抑制剂。当ROCK被抑制时，KapB不能促进肌动蛋白聚合，但它仍然能够破坏PB。* 我假设RhoA使用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介导细胞PB的分散。为检验这一假设提出了具体目标：（1）使用延时活细胞显微镜来精确地跟踪在RhoA活化后立即发生在PB、肌动蛋白细胞骨架和微管上的变化（2）以确定活性RhoA是否修饰、降解，或招募PB组分蛋白（3）以使用遗传方法来鉴定活性RhoA下游的效应蛋白，所述效应蛋白是RhoA所需的。介导的PB分散。我的NSERC资助的研究计划将阐明上游信号，将活性RhoA与PB形成的控制联系起来，并确定RhoA用于介导PB分散的新型下游效应物，从而深入了解RhoA控制的转录后基因表达过程。

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.**