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: 10797742
• 负责人: Robert H Singer
• 金额: $ 24.99万
• 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/10797742
• 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; Label; Life Cycle Stages; Messenger RNA; Methods; Photobleaching; Process; Proteins; Research; Resolution; Scheme; Time; Translating; Translations; Visualization; Yeasts; cell growth; mRNA Transcript Degradation; mRNA tagging; new technology; novel strategies; parent grant; real-time images; single molecule; tool

PROJECT SUMMARY 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 circumvent photobleaching in order to track mRNAs for longer times, equivalent to their lifetimes. We will tag mRNAs with sparse labeling, so that a single mRNA is labeled per cell, 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 在这项赠款资助的四分之世纪里，我们一直在开发工具来调查 活细胞中的单个mRNA分子。最近我们已经能够想象真实的时间平移 活细胞中的蛋白质，以及在细胞周期中的精确时刻mRNA的降解。我们 现在，我想通过对单个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。这提供了技术挑战，由于荧光的光漂白， 蛋白质和在拥挤的细胞质中追踪mRNA的困难。在本申请中，我们建议 通过开发新的方法来克服这些障碍，以避免光漂白， mRNA的时间更长，相当于它们的寿命。我们将用稀疏标记标记mRNA， 每个细胞标记单个mRNA，然后随着少量mRNA输出到细胞质中， 翻译和降级。我们也将同时跟踪细胞核中与mRNA结合的蛋白质 可以调节这些过程。这将使我们能够跟踪单个mRNA和与之结合的蛋白质 在它的整个生命周期中。

项目成果

An improved imaging system that corrects MS2-induced RNA destabilization.

• DOI: 10.1038/s41592-022-01658-1
• 发表时间: 2022-12
• 期刊: Nature methods
• 影响因子: 48
• 作者: Li W;Maekiniemi A;Sato H;Osman C;Singer RH
• 通讯作者: Singer RH