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

ABSTRACT: 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.

SUBMITTER: Han S 

PROVIDER: S-EPMC7486720 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature