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RNA protein interaction mapping via MS2 or Cas13 based APEX targeting

ABSTRACT: APEX2 fused to RNA binding proteins MS2 or an engineered Cas13

INSTRUMENT(S): Q Exactive Plus

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

SUBMITTER: Steven A. Carr

PROVIDER: MSV000086581 | MassIVE | Fri Dec 11 12:39:00 GMT 2020

REPOSITORIES: MassIVE

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RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting.

Project description:RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N 6-methyladenosine (m6A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA-protein interactions in living cells.

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