Revealing Principles of Subcellular RNA Localization by Proximity Labeling

通过邻近标记揭示亚细胞 RNA 定位原理

• 批准号: 10441618
• 负责人: Furqan Fazal
• 金额: $ 24.9万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441618
• 关键词: 3-Dimensional; Active Biological Transport; Address; Advisory Committees; Animal Model; Binding; Biochemical Genetics; Biological Assay; Biology; Biophysics; Caenorhabditis elegans; Cell physiology; Cells; Chromatin; Cis-Acting Sequence; Code; Collaborations; Communities; Complement; DNA; DNA Library; Data; Data Set; Development; Developmental Biology; Disease; Dynein ATPase; Elements; Embryo; Environment; Enzymes; Fractionation; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genomics; Goals; Grant; Heterochromatin; Human; Human body; Image; Institution; Kinesin; Knowledge; Label; Life; Location; Machine Learning; Malignant Neoplasms; Maps; Mentors; Messenger RNA; Mitochondria; Modeling; Modification; Molecular Motors; Monitor; Morphologic artifacts; Myosin ATPase; Neurons; Nuclear; Nuclear Envelope; Nuclear Lamina; Nuclear Pore; Organelles; Outer Mitochondrial Membrane; Pattern; Peroxidases; Pharmaceutical Preparations; Phase; Positioning Attribute; Postdoctoral Fellow; Process; Proteins; Protocols documentation; RNA; RNA Splicing; RNA Transport; RNA-Binding Proteins; Recombinants; Records; Regulation; Regulatory Element; Repetitive Sequence; Reporter; Research Personnel; Resolution; Resources; Retrotransposon; Role; Single Nucleotide Polymorphism; Small Nuclear RNA; Structure; System; Techniques; Technology; Testing; Tissues; Training; Transcript; Translating; Translations; Universities; Untranslated RNA; Variant; Work; Writing; active control; authority; base; career; cell fixation; cell type; clinically significant; deep learning; design; education resources; experience; gene function; genomic tools; improved; in vitro Assay; insertion/deletion mutation; insight; medical schools; multidisciplinary; nanometer; neuropathology; overexpression; programs; ribosome profiling; skills; statistics; success; tool; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY The human body comprises of trillion of cells, which are the building blocks of life. Each cell is highly organized, with various constituents within localizing to specific regions. Location within a cell is a cardinal organizing principle of gene control and function, especially for RNAs that code for proteins or serve regulatory roles as non-coding RNAs. Each cell type has its distinct RNA-species profile, and where these RNAs are located within the cell can dictate their folding, editing, splicing, translation, degradation, binding partners, catalytic activity, and even the fate of the proteins that they encode. Unsurprisingly, perturbations in RNA localization result in disease conditions including cancers and neuropathologies. In spite of the vital importance of subcellular RNA localization in gene expression, investigating such phenomena simultaneously for many RNA species has been challenging. To address these long-standing issues, Dr. Furqan Fazal and others have recently developed a technique called APEX-seq that can track the location of thousands of RNAs in living human cells with high-temporal and nanometer-spatial resolution. APEX-seq has the transformative potential to provide high-resolution snapshots of tens of thousands of endogenous cellular RNA species, free from artifacts of overexpression, cell fixation or recombinant tagging. This proposal outlines a five-year career program for Dr. Fazal as he investigates principles of RNA subcellular localization, and reaches milestone goals that will transition him into an independent investigator. By improving the APEX-seq technique and amalgamating it with a number of cutting-edge tools including massively-parallel reporter assays and deep-learning-based approaches, Dr. Fazal will dissect how sequences within RNAs direct them to different locations, and how cells actively control RNA subcellular localization to regulate gene function. This work will be carried out at Stanford University, where Dr. Fazal will be mentored by Dr. Howard Chang, a pioneer in developing new genomics tools, and Dr. Joanna Wysocka, a leading authority in vertebrate development and non-coding genomic regulatory elements. Both mentors have highly-successful track records of placing postdoctoral fellows into independent academic positions at leading institutions around the nation and world. Additional support will be available through collaboration with Drs. Shen, Boettiger and Kundaje who will provide training and expertise in genetics, imaging, statistics and computation. An advisory committee comprising of world-leading investigators Drs. Ting, Kool, Li, Bryant and Bassik will also monitor progress and provide expertise. Further acquisition of scientific and professional skills will be achieved by utilizing the educational resources available through the Stanford University School of Medicine and Office of Postdoctoral Affairs. Stanford provides an outstanding intellectually-stimulating environment with all facilities and resources necessary for success, and all proposed training will complement Dr. Fazal’s previous knowledge in genomics and biophysics and facilitate his transition into a multidisciplinary RNA biologist in the field of spatial transcriptomics.

项目摘要 人体由数万亿个细胞组成，这些细胞是生命的基础。每个细胞都有高度组织 在本地化到特定区域内有各种宪法。牢房内的位置是基地组织 基因控制和功能的原理，尤其是针对蛋白质编码的RNA或发挥调节作用 非编码RNA。每种单元格类型都有其独特的RNA物种曲线，并且这些RNA位于 细胞可以决定其折叠，编辑，拼接，翻译，降解，结合伴侣，催化活性和 甚至它们编码的蛋白质的命运。毫不奇怪，RNA定位的扰动导致疾病 包括癌症和神经病理的疾病。尽管亚细胞RNA定位至关重要 在基因表达中，仅针对许多RNA物种研究这种现象已受到挑战。 为了解决这些长期存在的问题，Furqan Fazal博士和其他人最近开发了一种称为 可以跟踪数千个RNA在具有高速阶段和高时空的人类细胞中的位置 纳米空间分辨率。 Apex-Seq具有提供高分辨率快照的变革潜力 成千上万的内源性细胞RNA物种，没有过表达，细胞固定或 重组标记。该提案在调查原理时为法扎尔博士概述了一项为期五年的职业计划 RNA亚细胞本地化，并达到里程碑的目标，将他转变为独立的目标 研究者。通过改进顶点序列技术并使用许多尖端工具将其合并 包括大规模并行的记者分析和基于深度学习的方法，Fazal博士将剖析如何剖析 RNA中的序列将它们引导到不同位置，以及细胞如何主动控制RNA亚细胞 定位以调节基因功能。这项工作将在斯坦福大学进行，法扎尔博士将在那里 由开发新基因组工具的先驱霍华德·张博士（Howard Chang）和乔安娜·韦索卡（Joanna Wysocca） 脊椎动物发育和非编码基因组调节元素的领先权威。两位导师都有 高度成功的记录记录，将博士后研究员置于领先的独立学术职位 全国和世界各地的机构。将通过与DRS的合作提供额外的支持。沉， Boettiger和Kundaje将提供遗传学，成像，统计和计算的培训和专业知识。 咨询委员会涵盖了世界领先的调查员Drs。 Ting，Kool，Li，Bryant和Bassik也将 监视进度并提供专业知识。将进一步获得科学和专业技能 通过使用斯坦福大学医学院和办公室的教育资源 博士后事务。斯坦福大学为所有设施提供了杰出的智力刺激环境， 成功所必需的资源，所有拟议的培训将完成Fazal博士以前的知识 基因组学和生物物理学，并促进他向空间领域的多学科RNA生物学家的过渡 转录组学。