mRNA Localization in Organelle Biogenesis

描述（由申请人提供）：了解调节细胞膜细胞器维持和生物发生的分子原理是细胞生物学的一个基本问题。迄今为止的研究已经建立了支持膜细胞器维持的蛋白质/膜运输途径，通过这些研究，内质网作为常驻膜蛋白合成位点的核心重要性已经被确定。然而，膜细胞器生物发生的分子基础仍然是一个谜。本研究的目的是确定内质网定向mRNA定位在膜细胞器生物发生中的作用。已经确定的是，真核细胞的蛋白质运输途径也指导mRNA转录组在细胞中两个主要蛋白质合成区室之间的分配，即细胞质和内质网（ER）。因此，编码分泌/整体膜蛋白的mrna通过信号识别颗粒（SRP）途径定位于内质网，编码胞质/核质蛋白的mrna缺乏编码信号序列，在胞质中保留默认定位。虽然这种正选择模型已经建立，但我们最近报道了i)编码内膜细胞器驻留蛋白（mRNAendo）的mrna可以通过SRP和翻译无关的途径定位到内质网；ii) mRNAendo队列通过直接的、不依赖核糖体的相互作用与内质网结合；iii) mRNAendo队列的特点是其异常高的ER富集。这些发现表明，mRNA定位在细胞器生物发生中起着不可或缺的作用，尽管尚未被探索。我们假设，编码内质网的内质膜驻留蛋白mrna的自主定位，以及它们与内质网膜的直接、不依赖核糖体的结合，代表了一种在细胞器生物发生中起作用的自组织机制。这一假设将在以下具体目标中得到验证：1)在全基因组范围内建立膜驻留蛋白编码mrna的亚细胞分配模式；2)识别将编码膜驻留蛋白的mrna引导至内质网的分子信号；3)鉴定介导mrna与内质网膜不依赖核糖体结合的RNA结合蛋白和内质网驻留蛋白；4)确定编码mrna的膜蛋白与内质网的核糖体非依赖性结合是否对细胞器生物发生至关重要。真核细胞中mRNA分配的全基因组研究揭示了mRNA定位亚细胞模式的意想不到的复杂性。在本提案中，我们建议将最近发现的mRNA队列特异性RNA定位模式扩展到ER的细胞器生物发生问题。如果成功，这些研究将为细胞器生物发生的分子原理提供基本的见解，并支持细胞器功能障碍疾病的治疗方法的发展。

DESCRIPTION (provided by applicant): Understanding the molecular principles that regulate endomembrane organelle maintenance and biogenesis stands as a principle question in cell biology. Research to date has established the protein/membrane trafficking pathways that support endomembrane organelle maintenance and through these studies, the central importance of the ER as the site of resident endomembrane protein synthesis has been identified. The molecular basis for endomembrane organelle biogenesis remains, however, mysterious. The objective of the proposed research is to define the role of ER-directed mRNA localization in endomembrane organelle biogenesis. It is well established that the protein trafficking pathways of eukaryotic cells also direct the partitioning of the mRNA transcriptome between the two primary protein synthesis compartments in the cell, the cytoplasm and the endoplasmic reticulum (ER). Thus, mRNAs encoding secretory/integral membrane proteins are localized to the ER via the Signal Recognition Particle (SRP) pathway and mRNAs encoding cytosolic/nucleoplasmic proteins, which lack encoded signal sequences, retain default localization in the cytosol. Although this positive selection model is well established, we have recently reported that i) mRNAs encoding resident proteins of the endomembrane organelles (mRNAendo) can be localized to the ER via a SRP- and translation-independent pathway; ii) the mRNAendo cohort is bound to the ER via direct, ribosome-independent interactions; and iii) the mRNAendo cohort is distinguished by its exceptionally high ER enrichment. These findings suggest that mRNA localization serves an integral, though unexplored, role in organelle biogenesis. We hypothesize that the autonomous localization of endomembrane resident protein-encoding mRNAs to the ER, and their direct, ribosome-independent association with the ER membrane, represents a self-organization mechanism functioning in organelle biogenesis. This hypothesis will be tested in the following specific aims: 1) Establish, at a genome-wide scale, the subcellular partitioning patterns of endomembrane resident protein-encoding mRNAs; 2) Identify the molecular signals that direct endomembrane resident protein-encoding mRNAs to the ER; 3) Identify the RNA binding proteins and ER resident proteins that mediate ribosome-independent association of mRNAs with the ER membrane; 4) Determine if ribosome-independent binding of endomembrane protein encoding mRNAs to the ER is essential for organelle biogenesis. Genome-wide studies of mRNA partitioning in eukaryotic cells have revealed an unexpected complexity in the subcellular patterns of mRNA localization. In this proposal, we propose to extend recent discoveries of mRNA cohort-specific patterns of RNA localization to the ER to the question of organelle biogenesis. If successful, these studies will provide fundamental insights into the molecular principles of organelle biogenesis and support the development of therapeutic approaches to diseases of organelle dysfunction. PUBLIC HEALTH RELEVANCE: The organelles of eukaryotic cells perform numerous functions that are essential for cell viability. Indeed, numerous inherited metabolic diseases as well as diseases of ageing arise through disruptions in organelle function. The goal of this research is to identify the basic principles of how organelles are created and repaired, so that organelle-based diseases can be better understood and new therapies for organelle- based disease identified.