RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested “exclusion-based” purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP–RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro.
|Journal||Journal of Biological Chemistry|
|State||Published - Mar 1 2022|
Bibliographical noteFunding Information:
Acknowledgments—Our appreciation to Ildiko Kasza and Josh Martin for their technical assistance and Gilson, Inc (Madison, WI) for their gift of the EXTRACTMAN device. This work was also supported in part by pilot funding from the National Institutes of Health/National Cancer Institute (grant no.: P30 CA014520)—UW Comprehensive Cancer Center Support. More information on the application of these SLIDE devices is presented in video form at https://www.gilson.com/extractman-starter-kit.html.
Funding and additional information—S. A. F. was supported by the Kuwait Foundation for the Advancement of Sciences under project no.: 2013-6302-03. This project was also supported by a Department of Defense Scholar Award (grant no.: W81XWH-06-1-0491; to C. M. A.), National Institute of Neurological Disorders and Stroke (grant no.: R21NS095187 [to A. R.], National Cancer Institute (grant no.: RO1CA186134 [to D. J. B.]), and National Institute of Arthritis and Musculoskeletal and Skin Diseases (grant nos.: RO1AR063361 and National Cancer Institute RO1CA121851 [to V. S. S.]). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
© 2022 THE AUTHORS.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology