Proteomics

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


ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Neal Devaraj  

PROVIDER: MSV000085100 | MassIVE | Fri Mar 13 18:26:00 GMT 2020

REPOSITORIES: MassIVE

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

Busby Kayla N KN   Fulzele Amitkumar A   Zhang Dongyang D   Bennett Eric J EJ   Devaraj Neal K NK  

ACS chemical biology 20200810 8


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 <i>E. coli</i> tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establi  ...[more]

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