Following mRNA from birth to death at single-molecule resolution

项目总结 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 活细胞中蛋白质的变化，以及在细胞周期的精确时刻的mRNAs的降解。我们 现在我想将所有这些过程整合到一个连贯的方案中，通过对单个mRNA进行成像 分子从出生到死亡。由于荧光的光漂白，这带来了技术挑战。 蛋白质和在拥挤的细胞质中追踪mRNAs的困难。我们在本申请中建议 通过开发新的方法来绕过光漂白来解决这些障碍 更长时间的mRNA，相当于它们的寿命。我们将使用稀疏标记来标记mRNA，这样一来 单个信使核糖核酸被标记到每个细胞，然后跟随少数信使核糖核酸输出到细胞质中， 翻译和降级。我们同样会同时追踪细胞核中与信使核糖核酸结合的蛋白质。 这可能会规范这些过程。这将使我们能够跟踪与之结合的单个mrna和蛋白质。 在其整个生命周期中。

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.