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Genome-wide Mapping of Ribosome Occupancies with Inhibitor-Induced mRNA Covalent Labeling

通过抑制剂诱导的 mRNA 共价标记进行核糖体占据的全基因组图谱

基本信息

批准号：
10575642
负责人：
Brian Liau
金额：
$ 19.85万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10575642
关键词：
Acylation Address Automobile Driving Benchmarking Binding Bypass Cell Line Cells Chemicals Chemistry Clinical Complex Cryoelectron Microscopy Data Set Digestion Disease Drops Elements Emerging Technologies Emetine Energy consumption Event Foundations Functional disorder Future Hydroxyl Radical In Vitro Label Mammalian Cell Maps Measures Messenger RNA Methods Mission Modification Movement Mutation National Institute of General Medical Sciences Natural Products Photoaffinity Labels Physiological Polyribosomes Positioning Attribute Process Protein Biosynthesis Proteins Protocols documentation RNA RNA Splicing RNA-Protein Interaction Relaxation Research Proposals Resolution Reverse Transcription Ribosomes Sampling Signal Transduction Site Structure System Technology Testing Transcript Transfer RNA Translating Translational Regulation Translational Repression Translations Variant adduct cell type crosslink design genome-wide high reward high risk improved inhibitor innovation insight meetings natural product derivative next generation sequencing novel preservation ribosome profiling single cell technology single molecule small molecule success tool transcriptome sequencing translation assay

项目摘要

Title: Genome-wide Mapping of Ribosome Occupancies with Inhibitor-Induced mRNA Covalent Labeling Ribosome profiling is a powerful method to study translation through genome-wide mapping of translating ribosomes. This breakthrough technology has illuminated many facets of protein synthesis, such as ribosomal pausing, alternative initiation sites, and protein synthesis rates across numerous cellular systems. Although recent innovations have reduced ribosome profiling to single cells, ribosome profiling typically requires large numbers of cells to conduct and therefore is not routinely used to study rare cell types and clinical samples. Furthermore, ribosome profiling fragments mRNA and hence cannot map multiple ribosomes along a single mRNA molecule and requires integration of separate mRNA sequencing datasets to account for the total number of transcripts. New efficient methods to simultaneously measure ribosome occupancies and mRNA abundance by preserving mRNA integrity would further enable the global study of translational regulation and provide new fundamental insight into translation dynamics. To bridge these technological gaps, here we propose to develop new small molecule tools based off the natural product ribosome inhibitors to chemically footprint ribosome positioning atop mRNA transcripts through covalent labeling. Our central hypothesis is that these mRNA chemical footprints can be detected through error prone reverse transcription and subsequent mRNA sequencing to map ribosome occupancies while maintaining the integrity of the mRNA. This approach is termed ribosome mutational profiling (Ribo-MaP). To develop Ribo-MaP, the first aim proposes the structure-based design of natural product derivatives that potently inhibit ribosome elongation while maintaining polysome stability. Critically, these derivatives are based off emetine, a ribosome inhibitor which contacts the mRNA template to block translation. Furthermore, photoaffinity labeling groups will be attached to these emetine derivatives and the ability to covalently label mRNA will be tested. Additionally, an alternative approach for mRNA covalent labeling is proposed, in which emetine derivatives will be functionalized with reactive acylating groups to label the proximal mRNA 2'- hydroxyl. In both approaches, mRNA labeling efficiency and the ability to readout covalent modifications as mutations in error prone reverse transcription will be measured. In the second aim, the ability of Ribo-MaP to map ribosome occupancies genome-wide will be evaluated and benchmarked against conventional ribosome profiling. We expect that Ribo-MaP will bypass inefficient steps in ribosome profiling and drop into the streamlined workflow of both robust mRNA sequencing methods and new emerging technologies for single-cell and long-read mRNA sequencing. Altogether, Ribo-MaP would provide a highly enabling method to broadly study protein synthesis and ribosome dynamics in cells.

标题：抑制物诱导的mRNA共价核糖体占据的全基因组图谱