RNA exists in cells as ribonucleoprotein complexes (RNPs), containing RNA and associated RNA binding proteins (RBPs). RBPs play a critical role in determining the cellular function of RNA, and therefore methods to identify RNA-protein interactions are of tremendous interest. Here, we demonstrate the site-specific labeling of RNA transcripts using the RNA-TAG (transglycosylation at guanosine) methodology. This technology takes advantage of a bacterial tRNA transglycosylase (TGT) capable of incorporating nucleobase derivatives bearing functional probes into an encodable RNA stem loop recognition motif. Using this methodology, we have successfully labeled mRNA transcripts with a wide variety of probes including biotin, BODIPY, and Cy7 in a single enzymatic step. Endogenously transcribed and processed RNAs containing the RNA-TAG recognition hairpin in their 3’ UTR can be selectively visualized in cell imaging studies. Furthermore, we have demonstrated that an RNA of interest can be selectively biotinylated and affinity purified from a complex mixture of RNAs and cell lysate. Through the development of RNA-TAG, we aim to create a robust, convenient platform for the elucidation of RNA function via cellular localization studies and affinity purification of RNA-protein complexes.

RNA-TAG as a platform for the identification of RNA-protein interactions

251st National Meeting (2016)

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