MassIVE MSV000085100

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Enzymatic Site-specific Biotinylation Enables Analysis of RNA-protein Interactions and Enrichment of Cellular RNA

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Description

The importance of RNA in regulating cellular processes has generated tremendous interest in methods to reliably characterize RNAs and their interactions within cells. Existing methodologies rely on noncovalent interactions to facilitate RNA affinity purification, hindering the purification of less abundant transcripts and limiting the stringency of the purification conditions. Here we demonstrate that enzymatic installation of a single covalent biotin modification directly onto an RNA of interest enables robust affinity purification of RNA-protein complexes via immunoblotting or quantitative mass spectrometry. Using this approach, known binding partners of 7SK snRNA, an important regulatory RNA, were successfully identified. This approach was further applied to the study of the long-noncoding RNA HOTAIR, using a modification of existing hairpin structures to allow for RNA-TAG labeling. A 4-nucleotide internal mutation enabled RNA-TAG mediated identification of putative HOTAIR binding partners in MCF7 breast cancer cells. Lastly, the selective and efficient labeling of an expressed RNA in mammalian cell lysate was demonstrated using a dimerized TGT enzyme through quantitative PCR and RNA-sequencing, enabling 145-fold direct enrichment from mammalian cell lysates. The flexibility and breadth of the RNA-TAG approach suggest that this methodology could be routinely applied to study RNA-protein complexes with minimal perturbation of the RNA sequence and to target less abundant RNA transcripts. [doi:10.25345/C5797F] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Reductive dimethyl labeling

Contact

Principal Investigators:
(in alphabetical order) 		Neal Devaraj, University of California San Diego, United States
Submitting User: Amit_Fulzele1803

Publications

Busby KN, Fulzele A, Zhang D, Bennett EJ, Devaraj NK.
Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-Protein Interactions.
ACS Chem Biol. 2020 Aug 21;15(8):2247-2258. Epub 2020 Aug 10.

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Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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