Following mRNA from birth to death at single-molecule resolution

项目总结/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解决这些障碍，以减少光漂白，以跟踪mRNA 更长的时间，相当于他们的寿命。我们将可逆地光激活mRNA 在细胞核中转录，然后跟随少数mRNA输出到细胞质， 并降解。我们将同样地同时跟踪与细胞核中的mRNA结合的蛋白质， 规范这些过程。这将使我们能够跟踪一个单一的mRNA和蛋白质结合到它的整个 它的整个生命周期

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.