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bioRxiv · 10.1101/405589

β-actin mRNA interactome mapping by proximity biotinylation

Mukherjee, J.·Hermesh, O.·Nalpas, N.·Franz-Wachtel, M.·Macek, B.·Jansen, R.-P.
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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 3UTR of endogenously MS2 tagged {beta}-actin mRNA in mouse embryonic fibroblasts (MEFs). We demonstrate the dynamics of the {beta}-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 {beta}-actin associated RBPs by RNA-BioID, among them the KH-domain containing protein FUBP3/MARTA2 has shown to be required for {beta}-actin mRNA localization. This protein binds to the 3-untranslated region of {beta}-actin mRNA, is essential for {beta}-actin mRNA localization but does not interact with the characterized {beta}-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.

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Mukherjee, J., Hermesh, O., Nalpas, N., Franz-Wachtel, M., Macek, B., Jansen, R.-P.. 2018-08-31. β-actin mRNA interactome mapping by proximity biotinylation. https://doi.org/10.1101/405589

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