mRNA Localization in Organelle Biogenesis

描述(由申请人提供)：了解调控内膜细胞器维持和生物发生的分子原理是细胞生物学中的一个原则性问题。到目前为止，研究已经建立了支持内膜细胞器维持的蛋白质/膜运输途径，通过这些研究，已经确定了内质网作为内膜蛋白合成场所的核心重要性。然而，膜内细胞器生物发生的分子基础仍然是个谜。本研究的目的是明确内质网导向的mRNA定位在膜内细胞器生物发生中的作用。已有的研究表明，真核细胞的蛋白质转运途径也指导着转录组在细胞内的两个主要蛋白质合成区段--细胞质和内质网之间的分配。因此，编码分泌/整合膜蛋白的mRNAs通过信号识别颗粒(SRP)途径定位于内质网，而编码胞浆/核质蛋白的mRNAs缺乏编码的信号序列，在胞浆中保留默认定位。虽然这种正选择模型已经建立得很好，但我们最近报道：i)编码膜内细胞器驻留蛋白(MRNAendo)的mRNAs可以通过SRP和翻译不依赖的途径定位于ER；ii)mRNAendo队列通过直接的、非核糖体无关的相互作用与ER结合；以及iii)mRNAendo队列的特点是ER异常丰富。这些发现表明，mRNA定位在细胞器生物发生中发挥着不可或缺的作用，尽管还没有被探索过。我们假设，内膜驻留蛋白编码的mRNAs对内质网的自主定位，以及它们与内质网膜的直接、核糖体无关的结合，代表了细胞器生物发生中的一种自组织机制。这一假说将在以下特定目标中得到验证：1)在全基因组水平上建立编码内膜蛋白的mRNAs的亚细胞分配模式；2)确定将内膜驻留蛋白编码的mRNAs定向到内质网的分子信号；3)鉴定介导mRNAs与ER膜的核糖体非依赖性结合的RNA结合蛋白和内质网驻留蛋白；4)确定编码mRNAs的内膜蛋白与内质网的核糖体非依赖的结合是否对细胞器生物发生是必要的。全基因组真核细胞中的信使核糖核酸的分离研究揭示了信使核糖核酸定位的亚细胞模式出人意料的复杂性。在这一建议中，我们建议将最近发现的RNA定位的mRNA队列特异性模式扩展到内质网，以解决细胞器生物发生的问题。如果成功，这些研究将提供对细胞器生物发生的分子原理的基本见解，并支持开发治疗细胞器功能障碍疾病的方法。 与公共卫生相关：真核细胞的细胞器执行许多对细胞存活至关重要的功能。事实上，许多遗传性代谢性疾病以及老年性疾病都是由于细胞器功能的破坏而引起的。本研究的目的是确定细胞器是如何产生和修复的基本原理，以便更好地了解细胞器疾病，并确定治疗细胞器疾病的新方法。

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.