RNA Interactome Identification via RNA-BioID in Mouse Embryonic Fibroblasts

Cytoplasmic localization of mRNAs is common to all organisms and serves the spatial expression of genes. Cis-acting RNA signals (mostly found in the mRNA's 3'-UTR), called zipcodes recruit trans-acting RNA-binding proteins that facilitate the localization of the mRNA. UV-cross-linking or affinity purification has been applied to identify such proteins but suffer from the need for stable RNA-protein binding or direct contact of protein and RNA. To identify stably or transiently interacting proteins that directly or indirectly associated with the localization elements and the body of the mRNA, we developed an in vivo proximity labeling method we call RNA-BioID. In RNA-BioID, we tether a fusion of the BirA* biotin ligase and the MS2 coat protein (MCP) at the 3'-UTR of MS2-tagged β-actin mRNA in vivo. Exposing BirA* expressing cells to biotin in the media and induces biotinylation of β-actin mRNA-associated proteins that can be isolated with streptavidin beads. This technique allowed us to identify by mass spectrometry analysis the β-actin mRNA 3'-UTR-interacting proteome in fibroblasts. The protocol can be useful to identify the interacting proteome of any mRNA in mammalian cells.