Skip to Content
Merck
  • ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

Cell stem cell (2020-05-13)
Diana Guallar, Alejandro Fuentes-Iglesias, Yara Souto, Cristina Ameneiro, Oscar Freire-Agulleiro, Jose Angel Pardavila, Adriana Escudero, Vera Garcia-Outeiral, Tiago Moreira, Carmen Saenz, Heng Xiong, Dongbing Liu, Shidi Xiao, Yong Hou, Kui Wu, Daniel Torrecilla, Jochen C Hartner, Miguel G Blanco, Leo J Lee, Miguel López, Carl R Walkley, Jianlong Wang, Miguel Fidalgo, Diana Guallar, Alejandro Fuentes-Iglesias, Yara Souto, Cristina Ameneiro, Oscar Freire-Agulleiro, Jose Angel Pardavila, Adriana Escudero, Vera Garcia-Outeiral, Tiago Moreira, Carmen Saenz, Heng Xiong, Dongbing Liu, Shidi Xiao, Yong Hou, Kui Wu, Daniel Torrecilla, Jochen C Hartner, Miguel G Blanco, Leo J Lee, Miguel López, Carl R Walkley, Jianlong Wang, Miguel Fidalgo
ABSTRACT

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editing induces aberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity during cell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Polyethylenimine, branched, average Mw ~25,000 by LS, average Mn ~10,000 by GPC, branched
Sigma-Aldrich
Guanosine, BioReagent, suitable for cell culture
Sigma-Aldrich
Thapsigargin, ≥98% (HPLC), solid film
Millipore
Benzonase® Nuclease, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
Sigma-Aldrich
(Z)-4-Hydroxytamoxifen, ≥98% Z isomer
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Adenosine, suitable for cell culture, BioReagent
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
GenElute Plasmid Miniprep Kit, sufficient for 350 purifications
Sigma-Aldrich
Normal Mouse IgG, Normal Mouse IgG Polyclonal Antibody control validated for use in Immunoprecipitation & Western Blotting.
Sigma-Aldrich
GenElute Gel Extraction Kit, sufficient for 70 purifications
Sigma-Aldrich
PD 0325901, ≥98% (HPLC)
Sigma-Aldrich
Doxycycline hyclate
Sigma-Aldrich
Thymidine, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
RPMI-1640 Medium, With sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Cytidine, BioReagent, suitable for cell culture, powder, ≥99%
Sigma-Aldrich
(+)-Sodium L-ascorbate, BioXtra, ≥99.0% (NT)
Sigma-Aldrich
Tunicamycin from Streptomyces sp.
Sigma-Aldrich
2-Mercaptoethanol, ≥99.0%
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Sodium pyruvate solution, 100 mM, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Staurosporine from Streptomyces sp., for molecular biology, ≥95% (HPLC)
Sigma-Aldrich
Insulin human, recombinant, expressed in yeast (proprietary host)
Ficoll®-Paque PREMIUM 1.084, Cytiva 17-5446-02, pack of 6 × 100 mL
Sigma-Aldrich
CHIR99021, ≥98% (HPLC)