Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-protein Interactions

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 E. Coli 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 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.

RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

“…RNA-TAG utilizes a bacterial tRNA guanine transglycosylase (TGT) to exchange a guanine nucleobase within a specific 17-nucleotide RNA stem loop structure (Tag) with a modified analog of the natural substrate preqeuosine1 (preQ1). Covalent modification is site-specific, robust, irreversible, and versatile, and RNA-TAG technology has been adapted to image cellular RNA in fixed cells, regulate mRNA translation, and study RNA-protein interactions [18][19][20][21][22][23]. We were curious whether or not RNA-TAG could be utilized to cyclize RNA targets with synthetic linkers.…”