Investigating early messenger ribonucleoprotein complex remodelling with RNA interactome capture

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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.