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: 10612902
• 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 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612902
• 关键词: 3-Dimensional; Active Biological Transport; Address; Advisory Committees; Authorization documentation; Binding; Biochemical; Biological Assay; Biology; Biophysics; Caenorhabditis elegans; Cell physiology; Cells; Chromatin; Cis-Acting Sequence; Code; Collaborations; Communities; Complement; Cytoplasm; DNA; DNA Library; Data; Data Set; Development; Developmental Biology; Disease; Dynein ATPase; Elements; Embryo; Environment; Enzymes; Fractionation; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomics; Goals; Grant; Heterochromatin; Human; Human body; Image; Institution; Kinesin; Knowledge; Knowledge acquisition; 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; 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; model organism; multidisciplinary; nanometer; neuropathology; overexpression; programs; ribosome profiling; skill acquisition; 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 人体由万亿个细胞组成，这些细胞是生命的基石。每个细胞都是高度组织化的， 在特定区域内有不同的成分。细胞内的位置是一个基本的组织 基因控制和功能的原理，特别是对编码蛋白质或作为调节作用的RNA， 非编码RNA。每种细胞类型都有其独特的RNA种类谱，并且这些RNA位于其中。 细胞可以决定它们的折叠、编辑、剪接、翻译、降解、结合伴侣、催化活性， 甚至是它们编码的蛋白质的命运。不出所料，RNA定位的扰动导致疾病 包括癌症和神经病理学在内的病症。尽管亚细胞RNA定位至关重要， 在基因表达中，同时研究许多RNA种类的这种现象是具有挑战性的。 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技术， APEX-seq可以追踪人类活细胞中数千种RNA的位置， 纳米空间分辨率。APEX-seq具有变革性的潜力，可以提供高分辨率的快照， 成千上万的内源性细胞RNA种类，没有过度表达、细胞固定或 重组标记这份提案概述了法扎尔博士在研究原理时的五年职业计划 RNA亚细胞定位，并达到里程碑式的目标，将他转变为一个独立的 调查员通过改进APEX-seq技术并将其与一些尖端工具合并， 包括并行报告分析和基于深度学习的方法，Fazal博士将剖析如何 RNA内的序列将它们引导到不同的位置，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， 接受新基因组学工具开发先驱霍华德·张博士和新基因组学工具开发先驱乔安娜·威索卡博士的指导 脊椎动物发育和非编码基因组调控元件的权威。两位导师都 非常成功的跟踪记录，将博士后研究员安置到领先的独立学术职位， 全国和世界各地的机构。将通过与沈博士的合作提供额外的支持， Boettiger和Kundaje将提供遗传学、成像、统计和计算方面的培训和专业知识。 一个由世界领先的研究人员丁博士、库尔博士、李博士、布莱恩特博士和巴西克博士组成的咨询委员会也将 监测进展并提供专门知识。将进一步获得科学和专业技能 通过利用斯坦福大学医学院和办公室提供的教育资源， 博士后事务。斯坦福大学提供了一个出色的智力刺激环境， 成功所需的资源，所有拟议的培训将补充博士。 基因组学和生物物理学，并促进他转变为空间领域的多学科RNA生物学家 转录组学