Dataset Information

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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Publications

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]

PMID: 32706237

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Project description:The general role of RNA in the cell beyond protein biosynthesis is only beginning to emerge with little knowledge about the structural or organizational functions of RNA. In turn, functional non-coding RNAs (ncRNAs) are often identified by their regulation or impact on cellular phenotypes. However, the huge number and diversity of ncRNAs highly complicate the task of their molecular characterization and rationalization into cellular processes. For the vast majority of ncRNAs as well as for non-coding functions of known mRNAs, the mechanisms underlying their modes of action as well as their impact on protein complexes are unknown. Thus, the greatest challenge in the field of RNA biology today is the elucidation of molecular mechanisms and functional interaction partners of RNA molecules. Since RNAs do not act alone, but often interact with specific protein partners, proteomic approaches have been developed to study RNA-binding proteins (RBPs) and aim at revealing the molecular mechanisms underlying RNA function. Large-scale studies aiming at globally identifying RNA-binding proteins principally focused on poly(A)-RNA transcripts. A notable limitation of such approaches is that many ncRNAs are not polyadenylated, so that their interaction partners were not detected in these screens. Most importantly however, the overlap between the numerous studies aiming to identify RBPs is limited and their specificity remains unclear. Moreover, the previous studies were based on the identification of RBPs omitting their participation in protein-protein complexes and more interestingly in RNA-dependent protein-protein complexes. Here, we introduce the concept of "RNA dependence" to overcome these challenges. We define a protein as RNA-dependent if its interactome (hence likely its function) depends on RNA without necessarily directly binding to RNA. Based on this new concept, we developed a proteome-wide screening approach to gain mechanistic insight into the function of RNAs - both coding and non-coding - in RNA-protein complexes and their impact on the function of such complexes.

Dataset Information

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

Publications

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

Similar Datasets

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