Investigating early messenger ribonucleoprotein complex remodelling with RNA interactome capture

通过 RNA 相互作用组捕获研究早期信使核糖核蛋白复合物重塑

• 批准号: 427447926
• 负责人: Dr. Cornelia Kilchert
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/427447926?language=en
• 关键词: Investigating; early; messenger; ribonucleoprotein; complex

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.

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mRNA都与RNA结合蛋白结合，并在其整个生命周期中保持核糖核蛋白（RNP）复合物的一部分。 RNP外壳将mRNA的状态传达给周围环境。这些mRNA是否经过了正确的处理？它能获得核出口许可吗？正是在mRNP形成的早期阶段，细胞决定mRNA是否适合输出到细胞质中或应该被丢弃，并且该信息被写入RNP外壳并随着加工的进行而更新。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的稳态组成的信息，但早期mRNP成熟的动力学仍然知之甚少。我的实验室想了解RNA命运决定是如何在RNP中进行分子编码的-一方面，在什么条件下哪些蛋白质的存在会触发核监视和RNA衰变;另一方面，如何整合许多不同RNA加工事件的信息，最终许可“好”RNP出口。在这个项目中，我们希望定义导致“核出口许可证”的早期mRNP重塑事件的顺序。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，其中我们用可光交联的核苷类似物脉冲标记，然后间隔追逐和交联，以时间分辨的方式跟踪新合成RNA的蛋白质相互作用，因为它们成熟。为了便于识别离散的mRNP状态和主动重塑事件，我们将包括缺乏三种必需的DEAD盒解旋酶之一的菌株，这些DEAD盒解旋酶与早期mRNP重塑有关，并且我们预计mRNP状态之间的转换会受损。该项目将解决该领域的关键未决问题：早期mRNP形成是一个定向过程吗？我们可以观察到哪些mRNP状态？必需的DEAD盒重构解旋酶介导确定的蛋白质周转事件吗？这些可能与早期mRNP生物发生检查点有关吗？我们希望这项研究将为我们理解细胞如何能够稳健地监测基因表达基础的复杂RNA加工事件的忠实完成奠定基础。