PMID- 33788967 OWN - NLM STAT- MEDLINE DCOM- 20210719 LR - 20211204 IS - 1530-6860 (Electronic) IS - 0892-6638 (Linking) VI - 35 IP - 5 DP - 2021 May TI - Role of epigenetic m(6) A RNA methylation in vascular development: mettl3 regulates vascular development through PHLPP2/mTOR-AKT signaling. PG - e21465 LID - 10.1096/fj.202000516RR [doi] AB - N(6) -methyladenosine (m6A) methylation is the most prevalent RNA modification, and it emerges as an important regulatory mechanism of gene expression involved in many cellular and biological processes. However, the role of m(6) A methylation in vascular development is not clear. The m(6) A RNA methylation is regulated by dynamic interplay among methyltransferases, binding proteins, and demethylases. Mettl3 is a member of the mettl3-mettl14 methyltransferase complex, referred to as writers that catalyze m6A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl3(+/-) KO embryos show defective vascular development, which is directly visible in fli-EGFP and flk-EGFP zebrafish. Alkaline phosphatase staining and whole mount in situ hybridization with cdh5, and flk markers demonstrated defective development of intersegmental vessels (ISVs), subintestinal vessels (SIVs), interconnecting vessels (ICVs) and dorsal longitudinal anastomotic vessels (DLAV) in both heterozygous mettl3(+/-) and homozygous mettl3(-/-) KO zebrafish embryos. Similar phenotypes were observed in zebrafish embryos with morpholino knockdown (KD) of mettl3; however, the vascular defects were rescued fully by overexpression of constitutively active AKT1. KD of METTL3 in human endothelial cells inhibited cell proliferation, migration, and capillary tube formation. Mechanistically, mettl3 KO and KD significantly reduced the levels of m(6) A RNA methylation, and AKT phosphorylation (S473) by an increase in the expression of phosphatase enzyme PHLPP2 and reduction in the phosphorylation of mTOR (S2481), a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. These data suggest that m(6) A RNA methylation regulates vascular development via PHLPP2/mTOR-AKT signaling. CI - (c) 2021 Federation of American Societies for Experimental Biology. FAU - Parial, Ramendu AU - Parial R AD - Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China. FAU - Li, Hui AU - Li H AD - Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China. FAU - Li, Jia AU - Li J AD - Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China. FAU - Archacki, Stephen AU - Archacki S AD - Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. AD - Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA. FAU - Yang, Zhongcheng AU - Yang Z AD - Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China. FAU - Wang, Isabel Z AU - Wang IZ AD - Symbolic Systems Program, Stanford University, Stanford, CA, USA. FAU - Chen, Qiuyun AU - Chen Q AD - Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. AD - Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA. FAU - Xu, Chengqi AU - Xu C AD - Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China. FAU - Wang, Qing K AU - Wang QK AUID- ORCID: 0000-0002-2235-6572 AD - Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China. AD - Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. AD - Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA. AD - Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH, USA. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - United States TA - FASEB J JT - FASEB journal : official publication of the Federation of American Societies for Experimental Biology JID - 8804484 RN - 0 (Zebrafish Proteins) RN - CLE6G00625 (N-methyladenosine) RN - EC 2.1.1.- (Methyltransferases) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - EC 3.1.3.16 (Phosphoprotein Phosphatases) RN - K72T3FS567 (Adenosine) SB - IM MH - Adenosine/*analogs & derivatives/chemistry MH - Animals MH - Embryo, Nonmammalian/*cytology/metabolism MH - Methylation MH - Methyltransferases/genetics/*metabolism MH - *Neovascularization, Physiologic MH - Phosphoprotein Phosphatases/genetics/*metabolism MH - Proto-Oncogene Proteins c-akt/genetics/*metabolism MH - TOR Serine-Threonine Kinases/genetics/*metabolism MH - Zebrafish MH - Zebrafish Proteins/genetics/metabolism OTO - NOTNLM OT - mettl3 OT - PHLPP2 OT - m6A methylation OT - mTOR-AKT OT - vascular development OT - zebrafish EDAT- 2021/04/01 06:00 MHDA- 2021/07/20 06:00 CRDT- 2021/03/31 17:23 PHST- 2021/02/03 00:00 [revised] PHST- 2020/03/06 00:00 [received] PHST- 2021/02/05 00:00 [accepted] PHST- 2021/03/31 17:23 [entrez] PHST- 2021/04/01 06:00 [pubmed] PHST- 2021/07/20 06:00 [medline] AID - 10.1096/fj.202000516RR [doi] PST - ppublish SO - FASEB J. 2021 May;35(5):e21465. doi: 10.1096/fj.202000516RR.