Investigating early messenger ribonucleoprotein complex remodelling with RNA interactome capture
Cornelia Kilchert
Institute for Biochemistry
University of Giessen
PhD student on the project: Jacqueline Böhme
E-mail: cornelia.kilchert(at)chemie.bio.uni-giessen.de
For more information and contact visit the Kilchert lab.
Abstract
The messenger RNA transcript (mRNA) 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 complexes (RNPs) throughout their lifetime. The composition of the RNP communicates the status of the RNA to the surrounding environment. Has this mRNA completed processing? Can it be licensed for nuclear export? It is at the stage of early RNP formation that cells determine whether an mRNA is fit for export to the cytoplasm or whether it 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. We want to understand how RNA fate decisions are molecularly encoded in the mRNP - On the one hand, under what condition does the presence of which protein trigger nuclear surveillance and RNA decay? On the other hand, how is information on many different RNA processing events 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 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 include strains that are deficient in various essential DEAD-box helicases that have been implicated in early mRNP remodelling. This project addresses 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 fundamentally advance our understanding of how cells are able to robustly monitor the faithful completion of the complex RNA processing events that underlie gene expression.
Project-related publications
Kilchert, C., Kecman, T., Priest, E., Hester, S., Aydin, E., Kus, K., Rossbach, O., Castello, A., Mohammed, S., Vasiljeva, L.
System-wide analyses of the fission yeast poly(A)+RNA interactome reveal insights into organization and function of RNA-protein complexes. Genome Res. 2020 Jul;30(7):1012-1026. doi: 10.1101/gr.257006.119.
Kilchert, C., Sträßer, K., Kunetsky, V., Änkö, M.L.
From parts lists to functional significance—RNA–protein interactions in gene regulation. Wiley Interdiscip Rev RNA. 2020 May;11(3):e1582. doi: 10.1002/wrna.1582.