ABSTRACT
The molecular function and fate of mRNAs are controlled by RNA binding proteins (RBPs). However, identification of the interacting proteome of a specific mRNA in vivo is still very challenging. Based on the widely-used RNA tagging with MS2 aptamers for RNA visualization, we developed a novel RNA proximity biotinylation (RNA-BioID) method by tethering biotin ligase (BirA*) via MS2 coat protein (MCP) at the 3’UTR of endogenously MS2 tagged β-actin mRNA in mouse embryonic fibroblasts (MEFs). We demonstrate the dynamics of the β-actin mRNA interactome by characterizing its changes upon serum-induced localization of the mRNA. Apart from the previously known interactors, we identified over 60 additional β-actin associated RBPs by RNA-BioID, among them the KH-domain containing protein FUBP3/MARTA2 has shown to be required for β-actin mRNA localization. This protein binds to the 3’-untranslated region of β-actin mRNA, is essential for β-actin mRNA localization but does not interact with the characterized β-actin zipcode element. RNA-BioID provides a tool to identify new mRNA interactors and to study the dynamic view of the interacting proteome of endogenous mRNAs in space and time.
Footnotes
Figures 1, 4, and 5 have been updated and include new experimental data. Microscopic co-localization analysis has been updated with controls. Supplementary information and figures have been re-arranged or replaced.