RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting.
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 -methyladenosine (mA) demethylase ALKBH5 and showed that ALKBH5 is able to erase the mA 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.
|Year of Publication||
Proc Natl Acad Sci U S A
2020 09 08
|PubMed Central ID||
R01 DK121409 / DK / NIDDK NIH HHS / United States
U01 CA214125 / CA / NCI NIH HHS / United States
U24 CA210986 / CA / NCI NIH HHS / United States