Reports ◥ STEM CELLS m 6 A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation Shay Geula, 1 * Sharon Moshitch-Moshkovitz, 2 * Dan Dominissini, 3 * Abed AlFatah Mansour, 1 * Nitzan Kol, 2 Mali Salmon-Divon, 2 Vera Hershkovitz, 2 Eyal Peer, 2 Nofar Mor, 1 Yair S. Manor, 1 Moshe Shay Ben-Haim, 2 Eran Eyal, 2 Sharon Yunger, 2 Yishay Pinto, 4 Diego Adhemar Jaitin, 5 Sergey Viukov, 1 Yoach Rais, 1 Vladislav Krupalnik, 1 Elad Chomsky, 1 Mirie Zerbib, 1 Itay Maza, 1 Yoav Rechavi, 1 Rada Massarwa, 1 Suhair Hanna, 1,6 Ido Amit, 5 Erez Y. Levanon, 4 Ninette Amariglio, 2,4 Noam Stern-Ginossar, 1 Noa Novershtern, 1 †‡ Gideon Rechavi, 2 †‡ Jacob H. Hanna 1 †‡ Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here,we identify Mettl3, an N 6 -methyladenosine (m 6 A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m 6 A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m 6 A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner. M urine pluripotent embryonic stem cells (ESCs) reside in a “naïve” molecular state that largely resembles that of the preim- plantation inner cell mass, whereas epi- blast stem cells (EpiSCs), derived from the postimplantation epiblast, resemble an advanced developmental stage and are already “primed” for differentiation (1, 2). Limited knowledge exists with regard to the molecular regulators that are critical for transitioning toward or for exclusively maintaining primed pluripotent EpiSCs. Thus, we conducted a small interfering RNA (siRNA) screen against selected transcriptional and ep- igenetic regulators previously evaluated in the context of naïve pluripotency modulation (1, 2) and tested whether primed EpiSCs, harboring a green fluorescent protein (GFP) reporter knock- in allele under the control of endogenous Oct4 promoter (Oct4-GFP +/+ ), might selectively rely on some of these factors (Fig. 1A and fig. S1). Reg- ulators that specifically inhibited the stability and viability of Oct4-GFP + primed cells includ- ed the epigenetic repressors Dnmt1, Eed, and Suz12 Polycomb components; Mbd3; and N 6 - adenosine methyltransferase Mettl3, a compo- nent of the N 6 -methyladenosine (m 6 A) mRNA methylating complex (3) (Fig. 1A and fig. S1). We subsequently focused on the role of m 6 A in pluripotency transitions, because the bio- logical role of RNA modifications is only starting to be unveiled (4). m 6 A is an RNA modification catalyzed by Mettl3 (methyl transferase–like 3) and Mettl14, components of a partially charac- terized multicomponent methyltransferase com- plex (3). This modification is removed by Fto and Alkbh5 demethylases. A role for m 6 A was RESEARCH | REPORTS 1002 27 FEBRUARY 2015 • VOL 347 ISSUE 6225 sciencemag.org SCIENCE 1 The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel. 2 Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel, and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. 3 Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA. 4 Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel. 5 The Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. 6 The Department of Pediatrics and the Pediatric Immunology Unit, Rambam Medical Center, and the B. Rappaport Faculty of Medicine, Technion, Haifa, Israel. *These authors contributed equally to this work. †These authors contributed equally to this work. ‡Corresponding author. E-mail: jacob.hanna@weizmann.ac.il (J.H.H.); noa.novershtern@weizmann. ac.il (N.N.); gidi.rechavi@sheba.health.gov.il (G.R.) Fig. 1. Derivation and characterization of Mettl3 KO ESCs. (A) siRNA screen for regulators that destabilize Oct4-GFP+ mouse naïve or primed pluripotent cells. Error bars indicate SD (n = 3). Student’ s t test *P < 0.05 relative to scrambled control. Gray boxes highlight EpiSC-specific regulators. (B) ESC derivation efficiency after mating of Mettl3 heterozygote mice. (C) Liquid chromatography–tandem mass spectrometry analysis of m 6 A percentage relative to adenosine in purified mRNA. Error bars indicate SD (n = 3). (D) ESC morphology and immunostaining.