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N6-Methyladenosine Modification Destabilizes Developmental

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N6-Methyladenosine Modification Destabilizes Developmental LETTERS N 6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells Yang Wang1, Yue Li2, Julia I. Toth1, Matthew D. Petroski1, Zhaolei Zhang2,3 and Jing Crystal Zhao1,4 N6-methyladenosine (m6A) has been identified as the most An NCBI Blast Protein sequence analysis revealed greater than 35% abundant internal modification of messenger RNA in sequence homology of the MTase domain between Mettl3 and Mettl14 eukaryotes1. m6A modification is involved in cell fate (Fig. 1a), suggesting that both are MTases. To investigate this possibility determination in yeast2,3 and embryo development in plants4,5. we constructed short hairpin RNAs (shRNAs) targeting Mettl3 or Its mammalian function remains unknown but thousands of Mettl14 and generated mESC lines harbouring efficient KD of each mammalian mRNAs and long non-coding RNAs (lncRNAs) (Fig. 1b). We then used two independent methods to determine m6A show m6A modification6,7 and m6A demethylases are required levels in KD mESCs. First, immunoblotting of RNA samples using a for mammalian energy homeostasis and fertility8,9. We identify highly specific α-m6A antibody6,7 indicated decreased m6A levels in two proteins, the putative m6A MTase, methyltransferase-like 3 both Mettl3 KD and Mettl14 KD versus control cells (Fig. 1c). We then (Mettl3; ref. 10), and a related but uncharacterized protein used liquid chromatography–tandem mass spectrometry to quantify Mettl14, that function synergistically to control m6A formation m6A=A ratios and observed a 60–70% decrease in m6A levels in each KD in mammalian cells. Knockdown of Mettl3 and Mettl14 in line relative to controls (Fig. 1d), suggesting that both proteins mediate mouse embryonic stem cells (mESCs) led to similar m6A formation in vivo. mESCs expressing additional shRNAs targeting phenotypes, characterized by lack of m6A RNA methylation and Mettl3 or Mettl14 were generated to control for shRNA off-target lost self-renewal capability. A large number of transcripts, effects (Supplementary Fig. 1A) and similarly decreased m6A levels were including many encoding developmental regulators, exhibit detected in all KD clones (Supplementary Fig. 1B). We then carried out m6A methylation inversely correlated with mRNA stability and a direct methylation assay by incubating an RNA probe exhibiting four gene expression. The human antigen R (HuR) and microRNA repeats of the canonical m6A methylation motif (GGACU) with equal pathways were linked to these effects. This gene regulatory amounts of luciferase, Mettl3 and/or Mettl14 proteins (Fig. 1e and mechanism operating in mESCs through m6A methylation is Supplementary Fig. 1C) purified from HEK293 cells in the presence required to keep mESCs at their ground state and may be of [3H]SAM and assessed [3H]methyl transfer. The RNA probe relevant to thousands of mRNAs and lncRNAs in various cell incubated with control luciferase protein showed no increase in [3H] types. levels. However, 1.5-, 3.5- and >27-fold increases in [3H] levels were detected in probes incubated with Mettl3, Mettl14 and Mettl3CMettl14 RNA and DNA MTases share structural motifs required to transfer (Fig. 1f), respectively. Thin-layer chromatography analysis confirmed methyl groups from S-adenosyl-l-methionine (SAM) to nucleic that methylated nucleotides were m6A(Fig. 1g and Supplementary acid. Previously, two mammalian m6A MTases, Mettl3 and Mettl14, Fig. 1D). To exclude the possibility that the enzymatic activity was were predicted computationally on the basis of conservation of the due to promiscuously co-purified mammalian proteins, we performed SAM-binding domain and phylogenetic analysis11,12. Mettl3 purified methylation assays using proteins purified from baculovirus-infected from HeLa cell nuclear extracts functions as a putative MTase (ref. 10). Sf9 insect cells. We found Mettl3 and Mettl14 difficult to purify Mettl14 remains uncharacterized. A recent study reported altered RNA alone but they were easily co-purified when co-expressed. As shown splicing in Mettl3 knockdown (KD) HeLa cells7, but as yet no direct in Supplementary Fig. 1E, Mettl3 was specifically pulled down by evidence links Mettl3 to m6A formation. Mettl14 but not luciferase, suggesting a strong interaction between 1Tumor Initiation and Maintenance Program, NCI-designated Cancer Center, Sanford Burnham Medical Research Institute, La Jolla, California 92037, USA. 2Department of Computer Science, The Donnelly Centre, University of Toronto, Toronto M5S 3G4, Canada. 3Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto M5S 3G4, Canada. 4Correspondence should be addressed to J.C.Z. (e-mail: [email protected]) Received 24 May 2013; accepted 29 November 2013; published online 7 January 2014; DOI: 10.1038/ncb2902 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 © 2014 Macmillan Publishers Limited. All rights reserved. LETTERS a SAM-binding domain Mettl3 1 580 Mettl14 1 456 Mettl3 AVVMADPPWD I HME LPYGT L TDDEMRRLN I PVLQDD–GF L FLWVT– GRAME LGRECLNLW 5 8 Mettl14 – – – – – – – – – ETG I TANEKCWTWDD I MKLE I DE I AAPRSF I FLWCGSGEGLDLGRVCLRKW 5 1 Mettl3 GYERVDE I I WVKTNQLQR – I I RTGRTGHWLNHGKEHCL VGVKGNPQGFNQG– – – – – –LDC 1 11 Mettl14 GYRRCED I CW I KTNKNNPGKT KTLDPKAVFQRTKEHCLMG I KG TVKRSTDGDF I HANVD I 1 11 Mettl3 DV I VAEVRS TS– –HKPDE I YGM I ERLSPGTRK I EL FGRPHNVQPNW I T LGNQ 1 6 1 Mettl14 D L I I TEEPE I GN I EKPVE I FH I I EHFCLGRRRLHL FGRDST I R PGWLTVGPT 1 6 3 b c Mettl3 Mettl14 M Scr shM3 shM14 Scr shM3 shM14 1.0 1.0 bp 6,583 Scr shM3 4,981 Mettl3 Scr shM14 3,638 0.5 Mettl14 Gapdh 0.5 2,604 Gapdh ∗∗∗ 1,908 Relative expression 1,383 Relative mRNA levels ∗∗∗∗ 955 0 0 623 shM3 shM14 281 Scramble Scramble d 0.20 e Replicate 1 LucM3 M14 M3 + M14 0.15 Replicate 2 Anti-flag 0.10 Anti-Mettl3 A/A (%) 6 m Anti-Mettl14 0.05 0 Scr shM3 shM14 fg∗∗∗∗ ∗∗∗∗ 400 1,000 ∗∗∗∗ A 300 6 ) ∗∗ m A 10 100 c.p.m. 200 10 c.p.m. (log 100 ∗∗∗ ∗ 1 0 Luc M3 Luc M3 Mock M14 M14 M3 + M14 M3 + M14 Figure 1 Mettl3 and Mettl14 are required for m6A formation in vitro and KD, Mettl14 KD and control cells. (d) Measurement of percentage of m6A=A in vivo.(a) Schematic drawing showing predicted MTase domains of mouse ratio by MS. (e) Flag, Mettl3 or Mettl14 western immunoblots of proteins Mettl3 (Accession: NP_062695:2) and Mettl14 (Accession: NP_964000:2) purified from lysates of HEK293 cells overexpressing flag-tagged Mettl3, proteins. Numbers represent amino acid numbers. (b) RT–qPCR (left) and Mettl14, or luciferase using M2 beads. Luc, luciferase; M3, Mettl3; M14, western blot (right) showing Mettl3 (left panel) and Mettl14 (right panel) Mettl14. The original gel is shown in Supplementary Fig. 5. (f) c.p.m. of RNA expression in mESC clones stably expressing shRNAs targeting each. Gapdh probes extracted after an in vitro methylation assay. (g) c.p.m. of excised m6A serves as a loading control in the western blot. Scr: scramble; shM3, shRNA spots from digested RNA probes used in the methylation assay. Error bars against Mettl3; shM14, shRNA against Mettl14. The original gel is shown in in b,f,g represent means ± s.e.m. from three separate experiments, except Supplementary Fig. 5. (c) Ethidium bromide staining (left panel) and m6A M3 in g where n D 7. One-tailed Student's t -test, ∗P < 0:05, ∗∗P < 0:01, immunoblot (right panel) of DNA-free, rRNA-free Poly(A)C RNA from Mettl3 ∗∗∗P <0:001, ∗∗∗∗P <0:0001 versus scramble control. Scr: scramble control. 2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION © 2014 Macmillan Publishers Limited. All rights reserved. LETTERS a Sox17 Scr shM14 shM3 b Mettl3 KD Mettl14 KD cdMettl3 IP Mettl14 IP Scr shM14 shM3 1% 1% Anti-Mettl14 input M3-IP IgG input M14-IP IgG Anti-Mettl3 371 4,395 354 Anti-Mettl3 Anti-Mettl14 Anti-Gapdh ef 754 P × –50 Scr Regulation of transcription < 1.91 10 1.5 shM3 Alternative splicing 1,386 P < 2.82 × 10–48 shM14 Chromatin modification 94 P < 1.56 × 10–7 1.0 ∗ Programmed cell death 145 P < 1.37 × 10–3 Cell morphogenesis 102 P < 2.00 × 10–3 0.5 In utero 90 P < 3.46 × 10–3 ∗∗ embryonic development ∗∗∗ Neuronal differentiation 122 P < 1.30 × 10–2 Relative RNA levels P × –2 0 Blood vessel development 81 < 2.25 10 P × –2 Heart development 75 < 2.96 10 Mettl3 Mettl14 0204060 P Hypergeometric enrichment (–log10( value)) Figure 2 Mettl3 and Mettl14 interact and regulate each other's stability. in Supplementary Fig. 5. (d) Western blot analysis of Mettl3 and Mettl14 (a) meRIP-seq analysis of a Sox17 transcript showing loss of m6A protein levels in Mettl3 KD and Mettl14 KD mESCs, respectively. The methylation in both Mettl3 KD and Mettl14 KD cells. Yellow highlighting, original gel is shown in Supplementary Fig. 5. (e) RT–qPCR analysis of peak location. Scr: scramble; shM3, shRNA against Mettl3; shM14, shRNA Mettl3 and Mettl14 RNA levels in Mettl3 KD and Mettl14 KD mESCs, against Mettl14.(b) Venn diagram showing overlap of Mettl3 and Mettl14 respectively. (f) GO analysis of 4,395 shared Mettl3 and Mettl14 targets. targets. (c) Mettl3 or Mettl14 western immunoblots of endogenous proteins Error bars in e represent means ± s.e.m. from three separate experiments. co-immunoprecipitated using antibodies against Mettl3 or Mettl14, or IgG One-tailed Student's t -test, ∗P < 0:05, ∗∗P < 0:01, ∗∗∗P < 0:001 versus in mESC lysates. M3, Mettl3; M14, Mettl14. The original gel is shown scramble control. Scr: scramble. Mettl3 and Mettl14. Importantly, Mettl3 C Mettl14 exhibited high 4,766 genes in Mettl3 KD and 4,749 in Mettl14 KD cells showed m6A MTase activity (Supplementary Fig. 1F,G), demonstrating that significantly decreased transcript m6A levels relative to controls.
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