Mechanistic study of single mRNA translation dynamic in live cells

活细胞中单个mRNA翻译动态的机制研究

• 批准号: 1817447
• 负责人: Bin Wu
• 金额: $ 120万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-15 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817447&HistoricalAwards=false
• 关键词: Mechanistic; study; single; mRNA; translation

Translation is the process by which the genetic information in mRNAs is read and used by the ribosomes to generate proteins with specific cellular functions. A novel approach for monitoring translation of individual mRNAs has been developed for this project. Using this approach, a number of fundamental questions about translation will be addressed. For example, are single mRNAs translated constitutively or in a burst-like fashion and do mRNAs encoded by the same gene behave homogeneously or heterogeneously? This work has the potential to influence the current understanding of the central dogma of molecular biology. The research and educational activities associated with this project will provide students at various levels with training in cutting edge molecular techniques. This project also includes efforts to increase diversity in STEM and outreach to the local community. The current proposal aims to address temporal dynamics and control mechanism of translation at the single molecule level in live cells. mRNA transcribed from the same gene may be bound by different protein factors, non-coding RNAs, or may adapt different conformations, which results in heterogeneity or noise in their behaviors. In agreement with this concept, it was previously observed that translation appears to be regulated at the level of single-mRNAs, as indicated by idiosyncratic "bursting" behavior. Ensemble biochemical approaches cannot be used because they average millions of cells and molecules. An in vivo, single-molecule technique is the only way to uncover the origins of this previously unexplored facet of gene expression regulation. Researchers have previously developed ways to visualize single mRNAs and their nascent peptides. However, further developments in experiment and theory are needed to study the temporal translation dynamics. First, single mRNAs and their translation status will be imaged for an extended period of time to capture the long-term translation dynamics. Second, the binding of trans-acting factors to mRNAs will be imaged. It is generally accepted that the fate of mRNA is determined by factors associated with it, such as RNA binding proteins and microRNAs. By correlating the trans-factor binding with translation activity, the mechanism of translation regulation could be uncovered. Third, a quantitative biophysical model of translation will be established to extract physiological parameters from the proposed single-molecule imaging experiments. After careful measurement of steady state, translation dynamics will be perturbed chemically or genetically to systematically vary cis- or trans- acting factors on mRNA, and to monitor the intramolecular mRNA conformation. A mechanistic model of translation will be built to elucidate the innate temporal heterogeneity.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

翻译是核糖体读取mrna中的遗传信息并利用其产生具有特定细胞功能的蛋白质的过程。为此项目开发了一种监测单个mrna翻译的新方法。通过这种方法，我们将探讨一些关于翻译的基本问题。例如，单个mrna是组成性翻译还是以突发性方式翻译？由同一基因编码的mrna的行为是同质性还是异质性？这项工作有可能影响目前对分子生物学中心教条的理解。与此项目相关的研究和教育活动将为不同层次的学生提供尖端分子技术的培训。该项目还包括努力增加STEM的多样性，并向当地社区推广。本课题旨在研究活细胞中单分子水平翻译的时间动力学和控制机制。同一基因转录的mRNA可能被不同的蛋白因子、非编码rna结合，或适应不同的构象，从而导致其行为的异质性或杂音。与这一概念一致，先前观察到翻译似乎在单mrna水平上受到调节，如特殊的“破裂”行为所表明的那样。集合生化方法不能使用，因为它们平均数百万个细胞和分子。在体内，单分子技术是唯一的方法来揭示这个以前未被探索的基因表达调控方面的起源。研究人员先前已经开发出可视化单个mrna及其新生肽的方法。然而，对时间平移动力学的研究还需要进一步的实验和理论研究。首先，将对单个mrna及其翻译状态进行长时间成像，以捕获长期翻译动态。其次，将对交易因子与mrna的结合进行成像。人们普遍认为mRNA的命运是由与其相关的因素决定的，例如RNA结合蛋白和microRNAs。通过反式因子结合与翻译活性的关联，可以揭示翻译调控的机制。第三，建立翻译的定量生物物理模型，从所提出的单分子成像实验中提取生理参数。在仔细测量稳态后，翻译动力学将被化学或遗传干扰，以系统地改变mRNA上的顺式或反式作用因子，并监测分子内mRNA的构象。我们将建立一个翻译的机制模型来阐明这种内在的时间异质性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Cas9 deactivation with photocleavable guide RNAs.

• DOI: 10.1016/j.molcel.2021.02.007
• 发表时间: 2021-04-01
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Zou RS;Liu Y;Wu B;Ha T
• 通讯作者: Ha T

One Message, Many Translations: Heterogeneity Revealed with Multicolor Imaging

一条消息，多种翻译：多色成像揭示异质性

• DOI: 10.1016/j.molcel.2019.06.026
• 发表时间: 2019
• 期刊: Molecular Cell
• 影响因子: 16
• 作者: Blake, Lauren A.;Wu, Bin
• 通讯作者: Wu, Bin

Live-cell imaging reveals kinetic determinants of quality control triggered by ribosome stalling.

• DOI: 10.1016/j.molcel.2021.01.029
• 发表时间: 2021-04-15
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Goldman DH;Livingston NM;Movsik J;Wu B;Green R
• 通讯作者: Green R

Visualizing the translation and packaging of HIV-1 full-length RNA

• DOI: 10.1073/pnas.1917590117
• 发表时间: 2020-03-17
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Chen, Jianbo;Liu, Yang;Hu, Wei-Shau
• 通讯作者: Hu, Wei-Shau

Translation imaging of single mRNAs in established cell lines and primary cultured neurons

• DOI: 10.1016/j.ymeth.2019.03.021
• 发表时间: 2019-06-01
• 期刊: METHODS
• 影响因子: 4.8
• 作者: Latallo, Malgorzata J.;Livingston, Nathan M.;Wu, Bin
• 通讯作者: Wu, Bin