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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629486
• 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.

基因表达的调控对生物体的健康至关重要。近年来，很明显，这种调节大部分发生在转录后，通过调节mRNA的加工，翻译和降解。在它们的转录、剪接、加帽和多聚腺苷酸化之后，mRNA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（例如在应激或病毒感染后），mRNA可以被翻译、储存到称为应激颗粒（SG）的颗粒中，或转移到称为“加工”或P体（PB）的其他致密RNA-蛋白质结构中，这些结构含有RNA降解机制的组分。虽然降解长期以来被认为是一个相对非特异性的过程，但对内源性表达的微小RNA（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，并重新引起了人们对理解胞质mRNA循环及其与颗粒和身体相互作用的兴趣。我们建议对不同RNA相关体的蛋白质组分进行详尽的蛋白质组学表征。在过去，对这些区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。在低严格性的缓冲液中进行标准生化富集，然后进行高速离心，也在很大程度上排除了通过亲和纯化偶联质谱法（AP-MS）对给定蛋白质的“不溶性”部分（例如与应激颗粒相关的翻译因子群体）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）引入了一种有吸引力的方法BioID，该方法有可能规避这些问题。作者将感兴趣的蛋白质融合到他们引入哺乳动物细胞中的修饰的细菌生物素连接酶（BirA*）。在添加生物素延长时间后，靠近诱饵的蛋白质被生物素化。这允许苛刻的裂解（我的实验室进一步优化了非常低溶解度的隔室）和回收链霉亲和素柱上的生物素化蛋白质，并通过质谱法进行鉴定。我的团队中的一名研究生Christopher Go在技术员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区室的组分，包括SG和PB（初步数据）。我们提出了三个相互关联的具体目标：1）表征的组成，在稳态下，PB和SG的系统分析已知的标记物，这些“细胞器”的BioID耦合到质谱。这将产生“部件列表”。2）阐明结构组织和蛋白质向PB和SG的募集模式。通过消耗或过度表达PB或SGs相关蛋白并监测其他因子的募集（显微镜下和蛋白质组学），我们将能够重建颗粒和体的形成。这将通过进行结构域映射实验在分子水平上进行细化。3）提供SG和PB形成的动态视图，并进一步表征特异性元素。我们将设计方法来定量监测过程或组装（或蛋白质分解成PB和SG），并评估新发现的组分的耗尽/过度表达对mRNA调控的影响。总之，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。