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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 36; pp. 22068 - 22079
• Main Authors: Han, Shuo, Zhao, Boxuan Simen, Myers, Samuel A., Carr, Steven A., He, Chuan, Ting, Alice Y.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 08.09.2020
• Subjects: AlkB Homolog 5, RNA Demethylase - genetics; AlkB Homolog 5, RNA Demethylase - metabolism; Biological Sciences; Biotinylation; Cells (biology); CRISPR; CRISPR-Cas Systems; DNA Methylation; DNA-(Apurinic or Apyrimidinic Site) Lyase - genetics; DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism; Endonucleases - genetics; Endonucleases - metabolism; HEK293 Cells; Humans; Mass Spectrometry; Multifunctional Enzymes - genetics; Multifunctional Enzymes - metabolism; N6-methyladenosine; Peptide mapping; Peroxidase; Protein Binding; Protein interaction; Protein Interaction Mapping - methods; Proteins; Ribonucleic acid; RNA; RNA - genetics; RNA - metabolism; Telomerase; Telomerase - genetics; Telomerase - metabolism; proximity labeling; interactome; APEX; telomerase RNA; RNA binding protein
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2006617117

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 N⁶-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.