m6A RNA methylation is integral for endothelial plasticity

Giorgia Ciliberti (Mannheim)1, S. Tual-Chalot (Newcastle)2, M. Polycarpou-Schwarz (Mannheim)1, M. Amponsah-Offeh (Mannheim)1, A. Turchinovich (Mannheim)1, A. Gatsiou (Newcastle)2, K. Stellos (Mannheim)1

1Medizinische Fakultät Mannheim der Universität Heidelberg Abteilung für Herz-Kreislauf-Forschung Mannheim, Deutschland; 2Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK. Biosciences Institute Newcastle, Großbritannien



N6-methyladenosine (m6A) RNA methylation is the most prevalent, abundant and conserved internal co-transcriptional modification in eukaryotic RNAs. m6A RNA modification is added by the m6A methyltransferase core complex, METTL3/14 and WTAP, and removed by the demethylases, FTO and ALKBH5. Whether m6A RNA methylation controls vascular homeostatic processes remain yet elusive.



We designed and generated two new mouse genetic models (WTAP flox mice and inducible vascular endothelial cell (iEC)-restricted WTAP mice) to study the role of m6A RNA methylation in vascular endothelial cell function. Primary human and murine vascular EC monolayers, 2D and 3D assays followed by confocal imaging were used to evaluate the EC-specific m6A effects in endothelial pro-angiogenic function. Molecular studies involving RNA methylation-specific RNA immunoprecipitation, transcriptomics, stability assays, RNAi and gain- and loss of function assays in primary ECs provided mechanistic insights.



Comparative m6A RNA methylation levels following silencing of each one of the m6A machinery components revealed that WTAP is the determinant of m6A RNA methylation in vascular endothelial cells. Strikingly, EC-specific WTAP deletion in mice resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-WTAP in life. WTAP-deficient ECs exhibited impaired angiogenic potential and reduced levels of crucial junctional molecules on EC membrane. Characterisation of the endothelial cell methylome transcriptome revealed that VE-cadherin, an essential gene of the endothelial-to-mesenchymal transition process, is among the most extensively methylated transcripts. VE-cadherin transcript exhibited decreased methylation levels in WTAP-deficient ECs compared to control ECs. Validation gene expression experiments confirmed VE-cadherin is among the highly downregulated targets after WTAP silencing. Mechanistically, WTAP regulates VE-cadherin mRNA stability and expression through the interaction with the RNA-binding protein Human Antigen R (HuR). Silencing of HuR phenocopied the reduced VE-cadherin expression levels initially observed in WTAP-deficient ECs. Conversely, overexpression of HuR was sufficient to restore the normal VE-cadherin expression levels in WTAP-deficient ECs.



m6A RNA methylation is essential for the maintenance of the endothelial fate in a WTAP/HuR-dependent manner.

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