The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). Identification of the interacting proteome of a specific mRNA in vivo remains very challenging, however. Based on the widely used technique of RNA tagging with MS2 aptamers for RNA visualization, we developed a RNA proximity biotinylation (RNA-BioID) technique by tethering biotin ligase (BirA*) via MS2 coat protein at the 3′ UTR of endogenous MS2-tagged β-actin mRNA in mouse embryonic fibroblasts. We demonstrate the dynamics of the β-actin mRNA interactome by characterizing its changes on serum-induced localization of the mRNA. Apart from the previously known interactors, we identified more than 60 additional β-actin–associated RBPs by RNA-BioID. Among these, the KH domain-containing protein FUBP3/ MARTA2 has been shown to be required for β-actin mRNA localization. We found that FUBP3 binds to the 3′ UTR of β-actin mRNA and is essential for β-actin mRNA localization, but does not interact with the characterized β-actin zipcode element. RNA-BioID provides a tool for identifying new mRNA interactors and studying the dynamic view of the interacting proteome of endogenous mRNAs in space and time.
|Original language||English (US)|
|Number of pages||10|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 2019|
- RNA-binding protein
- mRNA localization
ASJC Scopus subject areas