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Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-Protein Interactions. | LitMetric

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-Protein Interactions.

ACS Chem Biol

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.

Published: August 2020

AI Article Synopsis

Article Abstract

Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification of putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10198264PMC
http://dx.doi.org/10.1021/acschembio.0c00445DOI Listing

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