1Medizinische Fakultät Mannheim der Universität Heidelberg Abteilung für Herz-Kreislauf-Forschung Mannheim, Deutschland; 2Bioscience Institute Faculty of Medical Sciences Vascular Biology and Mediceine Newcastle, Großbritannien; 3Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland; 4National and Kapodistrian University of Athens Department of Clinical Therapeutics Athens, Deutschland; 5University of Lethbridge Department of Chemistry and Biochemistry AB T1K 3M4, Kanada
Background
N6-methyladenosine (m6A) is the most prevalent, abundant and conserved internal cotranscriptional modification in eukaryotic RNAs. m6A modification is added by the m6A methyltransferases, or writers, such as METTL3/14 and WTAP, and, removed by the demethylases, or erasers, including FTO and ALKBH5. m6A RNA methylation plays diverse RNA-regulatory roles, but its role in cardiovasculuar and inflammatory diseases remains yet elusive.
Methods
Transcriptome-wide single nucleotide m6A RNA methylation profiles were generated by nanopore direct long RNA sequencing and MazF microarray array in peripheral blood mononuclear cells derived from 16 healthy individuals, 15 individuals with established chronic coronary syndrome (CCS), 14 patients with ST-elevation myocardial infarction (STEMI), 5 patients with rheumatoid arthritis (RA) and 8 patients with sepsis. Ingenuity pathway analysis, gene ontology enrichment analysis and protein-protein interaction network were used to identify common disease-related methylome signatures. Primary human endothelial cells were used to evaluate the endothelial-specific m6A RNA methylation effects on endothelial innate immune responses. Molecular studies involving RNA methylation-specific RNA immunoprecipitation, transcriptomics, stability assays, RNAi and gain- and loss-of-function assays provided mechanistic insights of the human disease-related methylome signatures.
Results
Analysis of m6A RNA methylome signatures at single nucleotide level, revealed seventy-seven common hypomethylated mRNAs in patients with CCS, STEMI, RA or sepsis which are related to processes associated with VEGF signaling, DNA damage response and integrin signaling. The mRNA molecules of these genes hardbor several AU-rich elements making them susceptible to the regulatory function of Human antigen R (HuR, gene name ELAVL1), an RNA-binding protein that controls the fate of several AU-enriched pro-inflammatory genes in a synchronised manner. Interestingly, HuR itself was among the 77 common hypomethylated genes in human cardiovascular and inflammatory diseases and its expression levels were inversely associated with its m6A RNA methylation levels in humans. Mechanistically, analysis of endothelial cell m6A RNA methylome and transcriptome revealed that HuR target molecules are among the most extensively methylated transcripts. Deficient m6A RNA methylation in endothelial cells led to an excessive auto- and TNF-a-induced inflammatory response characterised by increased expression of several HuR target molecules including ICAM-1 and IL-6. RNAi-mediated or pharmacological inhibition of HuR expression or binding to AU-rich elements, respectively, rescued the m6A RNA methylation-deficient endothelial autoinflammatory or TNF-a-mediated hyperinflammatory phenotype.
Conclusion
Dysregulation of m6A RNA methylation in human cardiovascular and inflammatory diseases contributes to increased expression levels of several HuR target molecules, comprising an additional epitranscriptional regulatory layer of inflammation in human disease.