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Proc Natl Acad Sci U S A DOI:10.1073/pnas.2006617117

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

Publication TypeJournal Article
Year of Publication2020
AuthorsHan, S, Zhao, BSimen, Myers, SA, Carr, SA, He, C, Ting, AY
JournalProc Natl Acad Sci U S A
Volume117
Issue36
Pages22068-22079
Date Published2020 09 08
ISSN1091-6490
KeywordsAlkB Homolog 5, RNA Demethylase, Biotinylation, CRISPR-Cas Systems, DNA Methylation, DNA-(Apurinic or Apyrimidinic Site) Lyase, Endonucleases, HEK293 Cells, Humans, Mass Spectrometry, Multifunctional Enzymes, Protein Binding, Protein Interaction Mapping, RNA, Telomerase
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 -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.

DOI10.1073/pnas.2006617117
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/32839320?dopt=Abstract

Alternate JournalProc Natl Acad Sci U S A
PubMed ID32839320
PubMed Central IDPMC7486720
Grant ListR01 DK121409 / DK / NIDDK NIH HHS / United States
U01 CA214125 / CA / NCI NIH HHS / United States
U24 CA210986 / CA / NCI NIH HHS / United States