Granules and bodies: specificity in RNA storage and regulation

颗粒和物体：RNA 储存和调节的特异性

• 批准号: RGPIN-2014-06434
• 负责人: Gingras, AnneClaude
• 金额: $ 4.44万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=589002
• 关键词: Granules; bodies; specificity; RNA; storage

Regulation of gene expression is essential for organismal health. In recent years, it has become apparent that much of this regulation occurs post-transcriptionally, through the regulation of mRNA processing, translation and degradation. After their transcription, splicing, capping and polyadenylation, mRNAs are transported in the cytoplasm where they can meet different fates. mRNA can be translated, stored into granules known as stress granules, SGs, when translation needs to be paused (e.g. after stress or viral infections), or transferred to other dense RNA-protein structures known as “processing” or P-bodies, PBs, that contain components of the RNA degradation machinery. While degradation was long thought to be a relatively non-specific process, the identification of RNA silencing pathways driven by endogenously expressed micro RNAs (miRNAs) has revealed an intricate specificity in this process, and revived interest in understanding the cytosolic mRNA cycle and its interaction with granules and bodies. Building on our expertise in interaction proteomics, we propose to perform an exhaustive proteomics characterization of the protein components of different RNA-associated bodies. In-depth proteomics characterization of the composition of these compartments has not been possible in the past, since they are not easily amenable to biochemical purification. Standard biochemical enrichment in buffers of low stringencies followed by high-speed centrifugation also largely preclude the identification of protein-protein interactions by affinity purification coupled to mass spectrometry (AP-MS) for the “insoluble” portion of a given protein (e.g. the population of the translation factors that are associated to stress granules). However, in 2012, a study by Roux et al. (J Cell Biol, PMID 22412018) introduced an attractive approach, BioID, that has the potential to circumvent these issues. The authors fused a protein of interest to a modified bacterial biotin ligase (BirA*) which they introduced in mammalian cells. Following addition of biotin for extended periods, proteins which come in the vicinity of the bait become biotinylated. This permits harsh lysis (which my lab further optimized for compartments of very low solubility) and recovery of the biotinylated proteins on streptavidin columns and identification by mass spectrometry. A graduate student in my group, Christopher Go, with the help of technician Wade Dunham, has demonstrated that this approach could be applied – alongside quantitative proteomics and proper negative controls – to discriminate between components of adjacent cellular compartments, including SGs and PBs (preliminary data). We propose three interrelated specific aims: 1) Characterize the composition, at steady-state, of PBs and SGs by systematically analyzing known markers of these “organelles” by BioID coupled to mass spectrometry. This will generate a “parts list”. 2) Elucidate the structural organization and mode of recruitment of proteins to PBs and SGs. By depleting or overexpressing PBs or SGs associated proteins and monitoring the recruitment of other factors (microscopically and by proteomics) we will be able to reconstruct the formation of granules and bodies. This will be refined at the molecular level by performing domain-mapping experiments. 3) Provide a dynamic view of the formation of the SGs and PBs and further characterize elements of specificity. We will devise approaches to quantitatively monitor the process or assembly (or disassembly of proteins into PBs and SGs), and evaluate the impact of depletion/overexpression of newly identified components on mRNA regulation. Taken together, our studies will set the stage for a much better understanding of RNA regulation in eukaryotes.

基因表达调控对生物体健康至关重要。近年来，很明显，这种调控大部分发生在转录后，通过调节mRNA的加工、翻译和降解。MRNAs在转录、剪接、封端和多聚腺苷作用后，在细胞质中转运，在那里它们可以遇到不同的命运。当翻译需要暂停时(如在应激或病毒感染之后)，可将mRNA翻译、储存成称为应激颗粒(SGS)的颗粒，或转移到其他称为“加工”或P-体的致密RNA-蛋白质结构，该结构包含RNA降解机制的组件。虽然降解一直被认为是一个相对非特异性的过程，但内源表达的microRNAs(MiRNAs)驱动的RNA沉默途径的鉴定揭示了这一过程中复杂的特异性，并重新唤起了人们对理解细胞质mRNA循环及其与颗粒和小体相互作用的兴趣。 基于我们在相互作用蛋白质组学方面的专业知识，我们建议对不同RNA相关小体的蛋白质成分进行详尽的蛋白质组学表征。过去，对这些隔室的组成进行深入的蛋白质组学表征是不可能的，因为它们不容易进行生化纯化。在低要求的缓冲液中进行标准的生化浓缩，然后进行高速离心，也在很大程度上排除了通过亲和纯化结合质谱仪(AP-MS)对给定蛋白质的“不溶”部分(例如，与应激颗粒有关的翻译因子的群体)进行蛋白质-蛋白质相互作用的鉴定。然而，在2012年，Roux等人的一项研究。(J Cell Biol，PMID 22412018)介绍了一种有吸引力的方法，生物ID，它有可能绕过这些问题。作者将感兴趣的蛋白质与他们引入哺乳动物细胞的改良细菌生物素连接酶(BIRA*)融合在一起。在长时间添加生物素后，诱饵附近的蛋白质会发生生物素化。这允许苛刻的裂解(我的实验室进一步优化了极低溶解度的隔室)，并在链霉亲和素柱上回收生物素化蛋白质，并通过质谱学进行鉴定。我团队中的一名研究生Christopher Go在技术人员Wade Dunham的帮助下，证明了这种方法可以与定量蛋白质组学和适当的阴性对照一起应用于区分相邻细胞隔间的成分，包括SGS和PBS(初步数据)。 我们提出了三个相互关联的具体目标： 1)用BioID与质谱仪联用，系统分析PBS和SGS细胞器的已知标志物，表征PBS和SGS在稳态时的组成。这将生成一个“明细栏”。 2)阐明蛋白质在PBS和SGS中的结构组织和募集方式。通过耗尽或过度表达PBS或SGS相关蛋白，并监测其他因子的招募(显微镜和蛋白质组学)，我们将能够重建颗粒和小体的形成。这将通过进行结构域映射实验在分子水平上进行改进。 3)提供SGS和PBS形成的动态视图，并进一步表征专属性要素。我们将设计方法来定量监测蛋白质的过程或组装(或将蛋白质分解成PBS和SGS)，并评估新发现的成分的耗尽/过度表达对mRNA调控的影响。 综上所述，我们的研究将为更好地理解真核生物中的RNA调控奠定基础。