Mechanisms and regulation of localized mRNA translation

局部 mRNA 翻译的机制和调控

• 批准号: 10237161
• 负责人: Molly Marie Hannigan
• 金额: $ 5.94万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10237161
• 关键词: Address; Binding; Bioinformatics; Biological Models; Biology; Cell Fractionation; Cell Line; Cells; Cellular Stress; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Companions; Complex; Coupling; Data; Data Set; Disease; Endoplasmic Reticulum; Event; Foundations; Gene Expression; Genes; Genetic Transcription; Genetic Translation; Health; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Imaging technology; Knock-out; Leucine-Rich Repeat; Link; Malignant Neoplasms; Mediating; Membrane; Membrane Proteins; Messenger RNA; Methods; Modeling; Molecular; Neurodegenerative Disorders; Organelles; Pathway interactions; Peptide Initiation Factors; Peptide Signal Sequences; Peptides; Physiology; Play; Post-Transcriptional Regulation; Process; Protein Biosynthesis; Protein translocation; Proteins; Proteome; Proteomics; RNA; RNA Binding; RNA-Binding Proteins; Regulation; Reporting; Research; Ribosomal RNA; Ribosomes; Role; Signal Recognition Particle; Signal Transduction; Small Interfering RNA; Stress; Technology; Testing; Thapsigargin; Training; Translating; Translations; Tunicamycin; Work; base; cohort; comparative; deep sequencing; design; endoplasmic reticulum stress; genome-wide; human disease; imaging approach; in vivo; insight; knock-down; multidisciplinary; novel; optical imaging; polypeptide; polysome profiling; programs; protein expression; proteostasis; response; ribosome profiling; secretory protein; signal recognition particle receptor; spatiotemporal; transcriptome; transcriptome sequencing; translatome

ABSTRACT RNA localization is an evolutionarily conserved mechanism for the spatiotemporal regulation of protein synthesis. Secretory and membrane protein-encoding mRNAs are the largest cohort of localized mRNAs, comprising approximately 35% of protein-encoding genes, and are thought to undergo co-translational localization to the endoplasmic reticulum (ER) via the signal recognition particle (SRP) pathway. While SRP pathway function in protein translocation is well accepted, a companion role in mRNA localization on the ER has not been established. Strikingly, recent studies revealed that all mRNAs can be locally translated on the ER, indicating an unexpected diversity in ER-localized gene expression and suggesting that multiple, though as yet unknown, pathways function in RNA localization to the ER. To this end, work from our lab and others have provided new evidence that RNA binding proteins (RBPs) contribute key functions in mRNA localization to the ER. However, our understanding of how RBPs mediate mRNA transport to the ER for regulated translation is very limited. Here, we propose to uncover the molecular mechanisms of ER-specific mRNA regulation by RBPs, using leucine-rich repeat containing 59 (LRRC59) as a model. LRRC59 is a highly conserved and constitutively expressed ER-resident protein known to bind the ribosome and RNA. Using proximity proteomics, we recently discovered that LRRC59 also interacts with SRP factors and translation machinery. From these data, we hypothesize that LRRC59 functions in compartmentalized mRNA regulation by anchoring target mRNAs and ribosomes in close proximity to facilitate efficient protein synthesis. To test this hypothesis, we will combine advanced deep sequencing approaches with optimized subcellular fractionation methods on wild type and LRRC59 knockout models to: (1) identify the LRRC59- associated RNA transcriptome, and (2) investigate the molecular mechanism of LRRC59-regulated translation on the ER. Given the importance of ER stress and translational re-programming on human health, we further hypothesize that LRRC59 may have a role in maintaining cellular proteostasis. Using transcriptome-wide sequencing approaches and optical imaging technologies, we will be able to address how ER translation is regulated during ER stress, both globally and in a LRRC59-specific manner. Collectively, we expect this work to reveal new mechanisms that govern compartmentalized mRNA localization and translation in normal human physiology and disease. Importantly, the proposed studies will enhance my training in multiple aspects of RNA biology and RNA-related technologies, and provide me with the necessary foundation to build an independent research program.

抽象的 RNA定位是一种蛋白质时空调节的进化保守机制 合成。分泌和膜蛋白编码的mRNA是最大的局部mRNA队列， 占蛋白质编码基因的约35％，被认为经历了共同翻译 通过信号识别粒子（SRP）途径定位到内质网（ER）。而SRP 蛋白质易位中的途径功能被广泛接受，在ER上的mRNA定位中起作用 尚未建立。令人惊讶的是，最近的研究表明，所有mRNA都可以在局部翻译 ER，表明ER定位基因表达的意外多样性，并表明多个 尚不清楚，途径在RNA定位到ER中的作用。为此，我们的实验室和其他人工作 提供了新的证据，表明RNA结合蛋白（RBP）在mRNA定位中贡献了关键功能 到急诊室。但是，我们对RBP如何介导mRNA转运到ER的理解 翻译非常有限。在这里，我们提议揭示ER特异性mRNA的分子机制 通过RBPS进行调节，使用含有59（LRRC59）的富含亮氨酸重复作为模型。 LRRC59是高度 保守和组成型表达的ER居民蛋白已知可以结合核糖体和RNA。使用 接近蛋白质组学，我们最近发现LRRC59还与SRP因子和翻译相互作用 机械。从这些数据中，我们假设LRRC59在分隔的mRNA中起作用 通过固定靶标mRNA和核糖体密切接近来调节，以促进有效的蛋白质 合成。为了检验这一假设，我们将将高级深层测序方法与优化相结合 野生型和LRRC59敲除模型的亚细胞分级方法：（1）确定LRRC59- 相关的RNA转录组和（2）研究了LRRC59调节翻译的分子机制 在急诊室。鉴于ER压力和翻译对人类健康的重要性，我们进一步 假设LRRC59可能在维持细胞蛋白抑制剂中起作用。使用整个转录组 测序方法和光学成像技术，我们将能够解决ER翻译的方式 在全球和LRRC59特异性方式上，在ER应力下进行调节。总体而言，我们期望这项工作 揭示了针对正常人中分隔的mRNA定位和翻译的新机制 生理和疾病。重要的是，拟议的研究将增强我在多个方面的培训 RNA生物学和与RNA相关的技术，并为我提供必要的基础来建立 一个独立的研究计划。