Mechanisms of mRNA Anchoring and Translation Regulation on the Endoplasmic Reticulum

内质网mRNA锚定及翻译调控机制

• 批准号: 9752327
• 负责人: Christopher V. Nicchitta
• 金额: $ 30.68万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752327
• 关键词: Active Biological Transport; Animal Model; Binding; Binding Sites; Biological Assay; Cell Fractionation; Cell Surface Proteins; Cell physiology; Cells; Clip; Code; Complex; Cytosol; Data; Destinations; Diffusion; Disease; Disseminated Malignant Neoplasm; Dissociation; Endoplasmic Reticulum; Family; Gene Expression; Genes; Genetic Translation; Health; Immunoprecipitation; Integral Membrane Protein; Knowledge; Label; Locales; Maintenance; Membrane; Membrane Proteins; Messenger RNA; Methods; Modeling; Molecular; Molecular Analysis; Molecular Motors; Mutation Analysis; Organelles; Pathway interactions; Physiological; Polyribosomes; Portraits; Protein Biosynthesis; Proteins; Proteomics; RNA; RNA Binding; RNA Recognition Motif; RNA-Binding Proteins; Regulation; Reporting; Research; Ribosomes; Role; Signal Recognition Particle; Signal Transduction; Site; Small Interfering RNA; Testing; Tissue Model; Translating; Translation Initiation; Translations; Transport Process; Variant; candidate validation; cohort; crosslink; in vivo; knock-down; knockout animal; novel; novel strategies; predictive modeling; protein expression; protein function; recruit; secretory protein; tissue/cell culture; trafficking; transcriptome; transcriptome sequencing; validation studies

RNA localization, a ubiquitous cellular strategy for regulating the subcellular site of mRNA translation, operates via a common, staged mechanism. First, a cis-encoded localization sequence (“zipcode”) is recognized by RNA-binding proteins and the mRNA assembled into a translationally-silenced RNP transport complex. The RNP complex is then localized to the appropriate subcellular destination, either by diffusion or by active transport, and anchored. Lastly, translation of the mRNA is derepressed and local protein synthesis ensues. Although substantial progress has been made in identifying zipcode signals, trans-acting RNA binding proteins, molecular motors and transport mechanisms, very little is known regarding molecular mechanisms of mRNA anchoring, which is critical to the maintence of localized protein synthesis. In our research into mechanisms of mRNA localization and anchoring on the endoplasmic reticulum (ER), we discovered that organelle protein-encoding mRNAs are directly anchored to the ER membrane. In contrast, secretory protein-encoding mRNAs, which also localize to the ER, and are anchored indirectly, via translation on ER-bound ribosomes. We hypothesize that a direct RNA anchoring mechanism acts to spatially coordinate the synthesis of functionally related genes. To identify the mechanism of direct mRNA anchoring to the ER, we performed proteomic interactor screens of ER-bound polyribosomes and identified candidate ER integral membrane RNA anchoring proteins. In a first aim, functional validation studies of candidate RNA anchoring proteins will be performed. mRNA identities, cis-ER anchoring motifs, and RNA binding domains for candidate interactors will be identified via photocrosslinking and immunoprecipitation/RNA- Seq (CLIP-Seq) and PAR-CLIP approaches. Candidate ER-RNA anchoring protein function will be further validated through assays of target mRNA translation and localization, using siRNA knockdown and where available, knockout animal models, to determine roles for direct ER-mRNA anchoring in gene expression. The finding that mRNAs can be directly anchored to the ER suggests a novel mechanism of ribosome trafficking to the ER, where membrane-anchored mRNAs directly recruit ribosomes for de novo translation. In support of this model, we reported previously that ER-bound ribosomes function in de novo translation initiation and remain ER-associated following translation termination. Extending from these observations, we hypothesize that translation on the ER is functionally compartmentalized from cytosolic translation. A primary prediction of this model is that the ER translation cycle operates without an obligatory exchange of ribosomal subunits with a cytosolic pool. We propose to test this hypothesis in a second aim, where we will determine the subcellular site(s) of de novo translation initiation and the role of translation in the regulation of ribosome exchange on the ER. We expect that the proposed research will reveal new paradigms for the subcellular organization of mRNA translation and its regulation in health and disease.

RNA 定位是一种普遍存在的调节 mRNA 翻译亚细胞位点的细胞策略， 通过共同的、分阶段的机制运作。首先，顺式编码的本地化序列（“zipcode”）是 被 RNA 结合蛋白识别，mRNA 组装成翻译沉默的 RNP 运输 复杂的。然后，RNP 复合物通过扩散或通过扩散定位到适当的亚细胞目的地。 主动运输和锚定。最后，mRNA 的翻译被解除抑制，局部蛋白质合成被抑制 随后发生。尽管在识别邮政编码信号方面已经取得了实质性进展，但反式作用 RNA 结合 蛋白质、分子马达和运输机制，人们对分子知之甚少。 mRNA 锚定机制，这对于维持局部蛋白质合成至关重要。在 我们对 mRNA 定位和锚定在内质网 (ER) 机制的研究，我们 发现细胞器蛋白编码 mRNA 直接锚定于 ER 膜上。相比之下， 分泌蛋白编码 mRNA，也定位于 ER，并通过翻译间接锚定 位于 ER 结合核糖体上。我们假设直接的 RNA 锚定机制作用于空间 协调功能相关基因的合成。确定直接 mRNA 的机制 锚定到 ER，我们对 ER 结合的多核糖体进行了蛋白质组相互作用子筛选，并鉴定了 候选内质网整合膜RNA锚定蛋白。第一个目标是功能验证研究 将执行候选RNA锚定蛋白。 mRNA 身份、顺式 ER 锚定基序和 RNA 候选相互作用物的结合域将通过光交联和免疫沉淀/RNA-进行鉴定 Seq (CLIP-Seq) 和 PAR-CLIP 方法。候选ER-RNA锚定蛋白功能将进一步 通过使用 siRNA 敲除和其中的靶 mRNA 翻译和定位分析进行验证 可用敲除动物模型来确定 ER-mRNA 直接锚定在基因表达中的作用。 mRNA 可以直接锚定到 ER 的发现表明了核糖体的一种新机制 转运至内质网，膜锚定的 mRNA 直接招募核糖体进行从头翻译。在 为了支持该模型，我们之前报道过内质网结合核糖体在从头翻译起始中发挥作用 并在翻译终止后保持 ER 相关性。从这些观察出发，我们 假设内质网翻译在功能上与胞质翻译是分开的。一个 该模型的主要预测是 ER 翻译周期的运行无需强制交换 具有胞质池的核糖体亚基。我们建议在第二个目标中检验这个假设，我们将 确定从头翻译起始的亚细胞位点以及翻译在调节中的作用 ER 上的核糖体交换。我们期望所提出的研究将揭示新的范式 mRNA 翻译的亚细胞组织及其在健康和疾病中的调节。