@article{a74dc1b62e774f058ca4a52f2dbbe6d7,
title = "β-Actin mRNA interactome mapping by proximity biotinylation",
abstract = "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.",
keywords = "FUBP3, RNA-BioID, RNA-binding protein, mRNA localization",
author = "Joyita Mukherjee and Orit Hermesh and Carolina Eliscovich and Nicolas Nalpas and Mirita Franz-Wachtel and Boris Ma{\v c}ek and Jansen, {Ralf Peter}",
note = "Funding Information: We thank Jeff Chao (Friedrich Miescher Institute for Biomedical Research, Basel), Imre Gaspar (European Molecular Biology Laboratory, Heidelberg), Juli{\'e}n Bethune (Heidelberg University Biochemistry Center, Heidelberg), Dierk Niessing (University of Ulm), Michael Kiebler (University of Munich), Stefan Kindler (University of Hamburg), Stefan H{\"u}ttelmaier (University of Halle), and Ibrahim Muhammad Syed (Interfaculty Institute of Biochemistry, T{\"u}bingen), for plasmids, cell lines, antibodies, or spike RNA. We are grateful to Robert H. Singer for hosting J.M. during an imaging internship. We also thank Frank Essmann, Ruth Schmid (both at Interfaculty Institute of Biochemistry, T{\"u}bingen), and Silke Wahle (Proteome Center T{\"u}bingen) for technical support; Jeetayu Biswas (Albert Einstein College) for help with the polarization index scripts and the IGF2BP1 KO cell line; and Matthew Cheng (Interfaculty Institute of Biochemistry, T{\"u}bingen) for suggestions on the manuscript. The project was funded as a project of the Deutsche Forschungsgemeinschaft (DFG) Research Unit FOR2333 by a grant from the DFG (DFG JA696/11-1). C.E. was supported by an NIH grant (NS083085). Funding Information: ACKNOWLEDGMENTS. We thank Jeff Chao (Friedrich Miescher Institute for Biomedical Research, Basel), Imre Gaspar (European Molecular Biology Laboratory, Heidelberg), Juli{\'e}n Bethune (Heidelberg University Biochemistry Center, Heidelberg), Dierk Niessing (University of Ulm), Michael Kiebler (University of Munich), Stefan Kindler (University of Hamburg), Stefan H{\"u}ttelmaier (University of Halle), and Ibrahim Muhammad Syed (Interfaculty Institute of Biochemistry, T{\"u}bingen), for plasmids, cell lines, antibodies, or spike RNA. We are grateful to Robert H. Singer for hosting J.M. during an imaging internship. We also thank Frank Essmann, Ruth Schmid (both at Interfaculty Institute of Biochemistry, T{\"u}bingen), and Silke Wahle (Proteome Center T{\"u}bingen) for technical support; Jeetayu Biswas (Albert Einstein College) for help with the polarization index scripts and the IGF2BP1 KO cell line; and Matthew Cheng (Interfaculty Institute of Biochemistry, T{\"u}bingen) for suggestions on the manuscript. The project was funded as a project of the Deutsche Forschungsgemeinschaft (DFG) Research Unit FOR2333 by a grant from the DFG (DFG JA696/11-1). C.E. was supported by an NIH grant (NS083085). Publisher Copyright: {\textcopyright} 2019 National Academy of Sciences. All rights reserved.",
year = "2019",
doi = "10.1073/pnas.1820737116",
language = "English (US)",
volume = "116",
pages = "12863--12872",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "26",
}