Investigating early messenger ribonucleoprotein complex remodelling with RNA interactome capture

它是信使RNA转录本(MRNA)，将遗传信息传递给细胞的生物合成机制。从转录的那一刻起，所有的mRNAs都与RNA结合蛋白结合，并在其整个生命周期中一直是核糖核蛋白(RNP)复合体的一部分。RNP外衣将mRNA的状态与周围环境联系起来。这个信使核糖核酸有没有经过适当的加工？它能获得核出口许可吗？在mRNP形成的早期阶段，细胞决定一条mRNA是否适合输出到细胞质中，或者应该被丢弃，这些信息被写入RNP外壳，并随着处理的进展而更新。尽管通过纯化和染色质免疫沉淀方法获得了大量关于核mRNP稳态组成的信息，但对mRNP早期成熟的动力学仍然知之甚少。我的实验室想要了解RNA命运的决定是如何在RNP中被分子编码的--一方面，在什么条件下，哪些蛋白质的存在会引发核监视和RNA衰变；另一方面，关于许多不同的RNA加工事件的信息是如何被整合在一起的，以最终获得“良好”的RNP出口许可。在这个项目中，我们希望定义早期核反应堆改造事件的顺序，这些事件导致了一份“核出口许可证”。为此，我们将采用基于RNA相互作用组捕获技术的动力学比较蛋白质组学方法，其中我们用光交联型核苷类似物脉冲标记，然后以时间分辨的方式跟踪新合成的RNA成熟时的蛋白质相互作用。为了便于识别离散的mRNP状态和活跃的重塑事件，我们将包括在早期mRNP重塑中涉及的三种基本死盒螺旋酶之一缺乏的菌株，以及我们预计mRNP状态之间的转换受到损害的菌株。该项目将解决该领域的关键悬而未决的问题：mRNP的早期形成是一个定向的过程吗？我们可以观察到mRNP的哪些状态？必需的死盒重塑解旋酶是否调节确定的蛋白质周转事件？这些是否与早期的mRNP生物发生检查点有关？我们希望这项研究将是我们理解细胞如何能够有力地监控作为基因表达基础的复杂RNA加工事件的忠实完成的基础。

It is the messenger RNA transcript (mRNA) that conveys the genetic information to the cell’s biosynthetic machinery. From the moment of transcription, all mRNAs associate with RNA-binding proteins and remain part of ribonucleoprotein (RNP) complexes throughout their lifetime. The RNP coat communicates the status of the mRNA to the surrounding environment. Has this mRNA been properly processed? Can it be licensed for nuclear export? It is at the stage of early mRNP formation that cells determine whether an mRNA is fit for export into the cytoplasm or should be trashed, and that information is written into the RNP coat and updated as processing progresses. Despite a wealth of information on the steady state composition of nuclear mRNPs gained by purification and chromatin immunoprecipitation approaches, the dynamics of early mRNP maturation remain poorly understood. My lab wants to understand how RNA fate decisions are molecularly encoded in the RNP – on the one hand, under what conditions the presence of which proteins triggers nuclear surveillance and RNA decay; on the other hand, how information on many different RNA processing events is integrated to finally license “good” RNPs for export. In this project, we hope to define the sequence of early mRNP remodelling events that result in a “nuclear export license”. For this, we will employ a kinetic comparative proteomics approach based on the RNA interactome capture technique, where we pulse label with a photocrosslinkable nucleoside analogue, then chase and crosslink at intervals to follow protein interactions of newly synthesized RNAs in a time-resolved manner as they mature. To facilitate the identification of discrete mRNP states and active remodelling events, we will include strains that are deficient in one of the three essential DEAD-box helicases that have been implicated in early mRNP remodelling and where we would expect transitioning between mRNP states to be impaired. This project will address key outstanding questions in the field: Is early mRNP formation a directed process? What mRNP states can we observe? Do the essential DEAD-box remodelling helicases mediate defined protein turnover events? Can these be related to early mRNP biogenesis checkpoints? We expect that this study will be fundamental for our understanding of how cells are able to robustly monitor the faithful completion of the complex RNA processing events that underlie gene expression.