期刊
FASEB JOURNAL
卷 35, 期 5, 页码 -出版社
WILEY
DOI: 10.1096/fj.202000516RR
关键词
mettl3; m6A methylation; PHLPP2; mTOR-AKT; vascular development; zebrafish
资金
- China National Natural Science Foundation [81630002, 31430047]
- Hubei Province Natural Science Program Innovative Team [2017CFA014]
m6A RNA methylation plays a crucial role in vascular development by regulating the AKT signaling pathway.
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 m6A 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 m(6)A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl(3+/-) 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 mettl(3+/-) and homozygous mettl(3-/-) 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.
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