β-actin mRNA interactome mapping by proximity biotinylation

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 endogenous MS2 tagged β-actin mRNA (MBS) 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. We found that FUBP3 binds to the 3'-UTR 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. Significance statementTransport of specific mRNAs to defined sites in the cytoplasm allows local protein production and contributes to cell polarity, embryogenesis, and neuronal function. These localized mRNAs contain signals (zipcodes) that help directing them to their destination site. Zipcodes are recognized by RNA-binding proteins that, with the help of molecular motor proteins and supplementary factors, mediate mRNA trafficking. To identify all proteins assembling with a localized mRNA we advanced a proximity labeling method (BioID) by tethering a biotin ligase to the 3' untranslated region of mRNA encoding the conserved beta-actin protein. We demonstrate that this method allows the identification of novel, functionally important proteins that are required for mRNA localization.

Recent advances in proximity-based labeling methods for interactome mapping

“…The technique was used to assess the effect of disease-related point mutations on protein binding and to identify proteins that associate with Zika virus RNA 29 . An article detailing a similar approach (RNA-BioID), based on the incorporation of MS2 stem loops into nascent RNA and the expression of BirA* fused to the MS2 coat protein (MCP) for proximal protein labeling, was recently deposited on bioRxiv 40 . Given the success of BioID in mapping multiprotein complexes, it is likely that these types of RNA-targeted proximity labeling approaches will become popular methods for characterizing RNPs.…”

The SARS-CoV-2 RNA interactome

“…FUBP3 is a nuclear protein with 4 KH domains and binds to the 3′ UTR of cellular mRNAs regulating mRNA localization (Blichenberg et al, 1999;Mukherjee et al, 2019). Its connection to the life cycle of coronavirus is unknown to our knowledge.…”

MS2-TRIBE Evaluates Both Protein-RNA Interactions and Nuclear Organization of Transcription by RNA Editing

“…Recently, proteins that interact with the β-actin-MBS were profiled using MCP fused to the biotin ligase BirA. After controlling for background, nuclear proteins as well as chromatin components were discovered to interact with β-actin-MBS, suggesting similar co-transcriptional RNA-protein interactions when the MBS array is used ( Mukherjee et al., 2019 ). However, TRIBE differs in the mechanism of transcriptional marking: the enzyme is affixed to the transcript rather than an interaction that occurs by chance diffusion of a reactive intermediate.…”