β-Actin mRNA interactome mapping by proximity biotinylation.

Area of Science:

• Molecular Biology
• Proteomics
• RNA Biology

Background:

• RNA-binding proteins (RBPs) regulate mRNA function and fate.
• Identifying mRNA-interacting proteomes in vivo is technically challenging.
• Existing methods lack the resolution to capture dynamic mRNA-protein interactions.

Purpose of the Study:

• To develop a novel technique for identifying endogenous mRNA interactomes in vivo.
• To investigate the dynamic changes in the β-actin mRNA interactome.
• To identify novel RBPs associated with β-actin mRNA and their functional roles.

Main Methods:

• Developed RNA proximity biotinylation (RNA-BioID) using MS2 aptamer tagging and biotin ligase (BirA*).
• Tethered BirA* to the 3' UTR of endogenous MS2-tagged β-actin mRNA in mouse embryonic fibroblasts.
• Analyzed changes in the interactome upon serum-induced mRNA localization.

Main Results:

• Successfully identified the β-actin mRNA interactome in vivo.
• Discovered over 60 novel β-actin-associated RBPs beyond known interactors.
• Identified FUBP3/MARTA2 as a novel RBP essential for β-actin mRNA localization, binding the 3' UTR but not the zipcode element.

Conclusions:

• RNA-BioID is an effective tool for identifying novel mRNA interactors.
• The study provides insights into the dynamic nature of mRNA-protein interactions in space and time.
• FUBP3 plays a crucial role in β-actin mRNA localization, independent of the zipcode element.