Kartierung der Interaktome von lokalisierten mRNAs mittels Umgebung-Biotinylierung

01/05/2019 to 30/04/2022

Function and fate of localized mRNAs are controlled by RNA binding proteins (RBPs) forming higher-order messenger ribonucleoprotein particles (mRNPs). Determining the proteome of a specific mRNP in vivo is still challenging. We have introduced a novel tool to study the interactome of mRNAs in vivo, RNA proximity biotinylation (RNA-BioID). RNA-BioID tethers a modified biotin ligase to an MS2-tagged RNA of choice that biotinylates proteins in the vicinity (about 20 nm diameter), and allows their affinity-based purification. We have demonstrated its practicality by mapping the dynamic proteome of the beta-actin mRNP in mouse embryonic fibroblasts (MEFs). We showed that this mRNP contains > 60 associated RBPs, including all six previously known interactors. In the second funding period, we will address three key questions of transport-competent mRNPs. Firstly, we will deepen the functional analysis of newly identified beta-actin interactors and their contribution to the local translation of beta-actin in MEFs. Secondly, we will characterize the interactome of beta-actin mRNA in differentiating neurons and compare it to the interactome in MEFs to map changes in RNA-associated proteins of a localizing mRNP. Thirdly, we aim to identify, characterize, and compare the interactome of an additional localized mRNA in MEFs (arp2) with that of beta-actin mRNA. Interactome mapping of mRNPs in cultured cells will be highly useful to study the dynamic interactions of proteins and mRNAs during their localization process.