Following mRNA from birth to death at single-molecule resolution

以单分子分辨率追踪 mRNA 从出生到死亡的过程

• 批准号: 10265376
• 负责人: Robert H Singer
• 金额: $ 42万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-17 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10265376
• 关键词: Address; Binding Proteins; Birth; Cell Cycle; Cell Nucleus; Cell division; Cells; Cellular biology; Cessation of life; Crowding; Cytoplasm; Development; Funding; Goals; Grant; Image; Life Cycle Stages; Messenger RNA; Methods; Photobleaching; Process; Proteins; Resolution; Scheme; Time; Translating; Translations; Yeasts; cell growth; mRNA Transcript Degradation; new technology; novel strategies; real-time images; single molecule; tool

PROJECT SUMMARY/ABSTRACT: Over the quarter century of this grant funding, we have been developing tools to investigate the fate of single mRNA molecules in living cells. Most recently we have been able to image the real time translation of proteins in living cells, and the degradation of mRNAs at a precise moment during the cell cycle. We now would like to integrate all these processes into one coherent scheme, by imaging a single mRNA molecule from birth to death. This provides technical challenges, due to the photobleaching of fluorescent proteins and the difficulty of tracking mRNAs in a crowded cytoplasm. We propose in this application to address these obstacles by developing new approaches to reduce photobleaching in order to track mRNAs for longer times, equivalent to their lifetimes. We will reversibly photoactivate the mRNAs as they are transcribed in the nucleus, and then follow the few mRNAs as they export into the cytoplasm, translate and degrade. We will likewise simultaneously follow proteins bound to the mRNA in the nucleus that may regulate these processes. This will allow us to follow a single mRNA and proteins bound to it throughout its entire life cycle.

项目摘要/摘要： 在这笔赠款资金的四分之一个世纪里，我们一直在开发工具来调查 活细胞中的单个 mRNA 分子。最近我们已经能够对实时翻译进行成像 活细胞中蛋白质的降解，以及细胞周期中某个特定时刻 mRNA 的降解。我们 现在想通过对单个 mRNA 进行成像，将所有这些过程整合到一个连贯的方案中 分子从出生到死亡。由于荧光的光漂白，这带来了技术挑战 蛋白质以及在拥挤的细胞质中追踪 mRNA 的困难。我们在此申请中建议 通过开发新方法来减少光漂白以追踪 mRNA，从而解决这些障碍 更长的时间，相当于他们的一生。我们将按原样可逆地光激活 mRNA 在细胞核中转录，然后跟随少数 mRNA 输出到细胞质中，进行翻译 并降解。我们同样会同时追踪与细胞核中 mRNA 结合的蛋白质，这可能是 规范这些过程。这将使我们能够全程跟踪单个 mRNA 和与其结合的蛋白质 它的整个生命周期。