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: 10405893
• 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/10405893
• 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.

项目总结 人体由万亿个细胞组成，这些细胞是生命的基石。每个单元都是高度组织的， 不同的成员在特定地区进行本地化。细胞内的位置是最基本的组织方式 基因控制和功能的原理，尤指编码蛋白质或起调节作用的核糖核酸 非编码RNA。每种细胞类型都有其不同的RNA物种分布，以及这些RNA位于 细胞可以决定它们的折叠、编辑、剪接、翻译、降解、结合伙伴、催化活性和 甚至是它们编码的蛋白质的命运。毫不奇怪，RNA定位的扰动会导致疾病 包括癌症和神经病理学在内的疾病。尽管亚细胞RNA定位至关重要 在基因表达方面，同时研究许多RNA物种的这种现象一直是具有挑战性的。 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名为 APEX-SEQ，可以跟踪活着的人类细胞中数千个RNA的位置，具有高时间和 纳米级空间分辨率。APEX-SEQ具有提供高分辨率快照的变革性潜力 数以万计的内源性细胞RNA物种，没有过度表达、细胞固定或 重组标签。这份提案为法扎尔博士勾勒出了一个五年的职业计划，因为他正在研究原理 ，并达到了里程碑式的目标，这将使他成为一名独立的 调查员。通过改进APEX-SEQ技术并将其与一些尖端工具相结合 包括大量平行的记者分析和基于深度学习的方法，法扎尔博士将剖析 RNA中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，法扎尔博士将在那里 由开发新基因组学工具的先驱霍华德·张博士和乔安娜·维索卡博士指导 脊椎动物发育和非编码基因组调控元件方面的领先权威。两位导师都有 将博士后研究员安排在Leading大学担任独立学术职位的非常成功的记录 国家和世界各地的机构。其他支持将通过与沈博士的合作提供， BoetTiger和Kundaje将提供遗传学、成像、统计学和计算方面的培训和专业知识。 由世界领先的调查人员丁博士、库尔博士、李博士、科比博士和巴西克博士组成的咨询委员会也将 监控进度并提供专业知识。将实现进一步获得科学和专业技能 通过利用通过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激发智力的环境，所有设施和 成功所需的资源，以及所有拟议的培训将补充Fazal博士之前在 基因组学和生物物理学，并促进他过渡到空间领域的多学科RNA生物学家 转录学。