{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T02:55:30Z","timestamp":1792032930649,"version":"4.4.0"},"reference-count":56,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T00:00:00Z","timestamp":1642550400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002428","name":"FWF Austrian Science Fund","doi-asserted-by":"publisher","award":["M2578-B30"],"award-info":[{"award-number":["M2578-B30"]}],"id":[{"id":"10.13039\/501100002428","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002996","name":"Hartstichting","doi-asserted-by":"publisher","award":["2014T102"],"award-info":[{"award-number":["2014T102"]}],"id":[{"id":"10.13039\/501100002996","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>N-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser enzymes. Alteration in the levels of these enzymes can lead to changes in mRNA stability, alternative splicing or microRNA processing, depending on the m6A-binding proteins. Dynamic regulation of mRNA m6A methylation after ischemia and hypoxia influences mRNA stability, alternative splicing and translation, contributing to heart failure. In this study, we studied vasoactive microRNA m6A methylation in fibroblasts and examined the effect of hypoxia on microRNAs methylation using m6A immunoprecipitation. Of the 19 microRNAs investigated, at least 16 contained m6A in both primary human fibroblasts and a human fibroblast cell line, suggesting vasoactive microRNAs are commonly m6A methylated in fibroblasts. More importantly, we found that mature microRNA m6A levels increased upon subjecting cells to hypoxia. By silencing different m6A writer and eraser enzymes followed by m6A immunoprecipitation, we identified METTL4, an snRNA m6A methyltransferase, to be predominantly responsible for the increase in m6A modification. Moreover, by using m6A-methylated microRNA mimics, we found that microRNA m6A directly affects downstream target mRNA repression efficacy. Our findings highlight the regulatory potential of the emerging field of microRNA modifications.<\/jats:p>","DOI":"10.3390\/ijms23031057","type":"journal-article","created":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T08:20:57Z","timestamp":1642580457000},"page":"1057","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":40,"title":["N-6-Methyladenosine in Vasoactive microRNAs during Hypoxia; A Novel Role for METTL4"],"prefix":"10.3390","volume":"23","author":[{"given":"Daphne A. L.","family":"van den Homberg","sequence":"first","affiliation":[{"name":"Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"},{"name":"Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reginald V. C. T.","family":"van der Kwast","sequence":"additional","affiliation":[{"name":"Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"},{"name":"Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6853-5760","authenticated-orcid":false,"given":"Paul H. A.","family":"Quax","sequence":"additional","affiliation":[{"name":"Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"},{"name":"Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2155-4286","authenticated-orcid":false,"given":"A. Ya\u00ebl","family":"Nossent","sequence":"additional","affiliation":[{"name":"Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"},{"name":"Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands"},{"name":"Department for Laboratory Medicine, Medical University of Vienna, AT-1090 Vienna, Austria"},{"name":"Department of Internal Medicine II, Medical University of Vienna, AT-1090 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1093\/cvr\/cvw039","article-title":"The multifactorial nature of microRNAs in vascular remodelling","volume":"110","author":"Welten","year":"2016","journal-title":"Cardiovasc. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1042\/CS20130203","article-title":"MicroRNAs in the onset and development of cardiovascular disease","volume":"126","author":"Vickers","year":"2014","journal-title":"Clin. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1161\/CIRCRESAHA.114.304747","article-title":"Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 increases neovascularization and blood flow recovery after ischemia","volume":"115","author":"Welten","year":"2014","journal-title":"Circ. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1038\/nrm3838","article-title":"Regulation of microRNA biogenesis","volume":"15","author":"Ha","year":"2014","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Bokar, J.A. (2005). The biosynthesis and funtional roles of methylated nucleosides in eukaryotic mRNA. Fine-Tuning of RNA Functions by Modification and Editing, Springer. Topics in Current Genetics.","DOI":"10.1007\/b106365"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1038\/nchembio.1432","article-title":"A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation","volume":"10","author":"Liu","year":"2014","journal-title":"Nat. Chem. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1038\/cr.2014.3","article-title":"Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase","volume":"24","author":"Ping","year":"2014","journal-title":"Cell Res."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Nance, D.J., Satterwhite, E.R., Bhaskar, B., Misra, S., Carraway, K.R., and Mansfield, K.D. (2020). Characterization of METTL16 as a cytoplasmic RNA binding protein. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0227647"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"9250","DOI":"10.1093\/nar\/gkaa684","article-title":"METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing","volume":"48","author":"Goh","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.molcel.2012.10.015","article-title":"ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility","volume":"49","author":"Zheng","year":"2013","journal-title":"Mol. Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1038\/nchembio.687","article-title":"N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO","volume":"7","author":"Jia","year":"2011","journal-title":"Nat. Chem. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1007\/s11684-018-0654-8","article-title":"RNA m(6)A modification and its function in diseases","volume":"12","author":"Tong","year":"2018","journal-title":"Front. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1038\/nature12730","article-title":"N6-methyladenosine-dependent regulation of messenger RNA stability","volume":"505","author":"Wang","year":"2013","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1388","DOI":"10.1016\/j.cell.2015.05.014","article-title":"N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency","volume":"161","author":"Wang","year":"2015","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1016\/j.molcel.2016.01.012","article-title":"Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing","volume":"61","author":"Xiao","year":"2016","journal-title":"Mol. Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1038\/nature11112","article-title":"Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq","volume":"485","author":"Dominissini","year":"2012","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1635","DOI":"10.1016\/j.cell.2012.05.003","article-title":"Comprehensive analysis of mRNA methylation reveals enrichment in 3\u2019 UTRs and near stop codons","volume":"149","author":"Meyer","year":"2012","journal-title":"Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1038\/s41422-018-0040-8","article-title":"Dynamic transcriptomic m(6)A decoration: Writers, erasers, readers and functions in RNA metabolism","volume":"28","author":"Yang","year":"2018","journal-title":"Cell Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1007\/s12265-021-10108-w","article-title":"N6-Adenosine Methylation (m(6)A) RNA Modification: An Emerging Role in Cardiovascular Diseases","volume":"14","author":"Chen","year":"2021","journal-title":"J. Cardiovasc. Transl. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1161\/CIRCULATIONAHA.118.033794","article-title":"FTO-Dependent N(6)-Methyladenosine Regulates Cardiac Function During Remodeling and Repair","volume":"139","author":"Mathiyalagan","year":"2019","journal-title":"Circulation"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1161\/CIRCULATIONAHA.118.036146","article-title":"The N(6)-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy","volume":"139","author":"Dorn","year":"2019","journal-title":"Circulation"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1016\/j.bbrc.2020.09.057","article-title":"N6-methyladenosine in RNA of atherosclerotic plaques: An epitranscriptomic signature of human carotid atherosclerosis","volume":"533","author":"Gregersen","year":"2020","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2191","DOI":"10.1016\/j.ymthe.2020.07.022","article-title":"Role of METTL3-Dependent N(6)-Methyladenosine mRNA Modification in the Promotion of Angiogenesis","volume":"28","author":"Yao","year":"2020","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e402","DOI":"10.1002\/ctm2.402","article-title":"Loss of m6A demethylase ALKBH5 promotes post-ischemic angiogenesis via post-transcriptional stabilization of WNT5A","volume":"11","author":"Zhao","year":"2021","journal-title":"Clin. Transl. Med."},{"key":"ref_25","first-page":"e325","article-title":"METTL3 Regulates Angiogenesis by Modulating let-7e-5p and miRNA-18a-5p Expression in Endothelial Cells","volume":"41","author":"Sweaad","year":"2021","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1299","DOI":"10.1016\/j.cell.2015.08.011","article-title":"HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events","volume":"162","author":"Alarcon","year":"2015","journal-title":"Cell"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1038\/nature14281","article-title":"N6-methyladenosine marks primary microRNAs for processing","volume":"519","author":"Alarcon","year":"2015","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Berulava, T., Rahmann, S., Rademacher, K., Klein-Hitpass, L., and Horsthemke, B. (2015). N6-adenosine methylation in MiRNAs. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0118438"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"113","DOI":"10.3389\/fcvm.2019.00113","article-title":"miRMap: Profiling 14q32 microRNA Expression and DNA Methylation Throughout the Human Vasculature","volume":"6","author":"Goossens","year":"2019","journal-title":"Front. Cardiovasc. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"921","DOI":"10.1136\/heartjnl-2013-305402","article-title":"MicroRNAs in cardiovascular disease: An introduction for clinicians","volume":"101","author":"Romaine","year":"2015","journal-title":"Heart"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1038\/nrcardio.2015.101","article-title":"MicroRNAs in platelet function and cardiovascular disease","volume":"12","author":"McManus","year":"2015","journal-title":"Nat. Rev. Cardiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"703","DOI":"10.1161\/CIRCRESAHA.115.306300","article-title":"MicroRNA Regulation of Atherosclerosis","volume":"118","author":"Feinberg","year":"2016","journal-title":"Circ. Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1161\/CIRCRESAHA.116.308434","article-title":"Circulating Noncoding RNAs as Biomarkers of Cardiovascular Disease and Injury","volume":"120","author":"Viereck","year":"2017","journal-title":"Circ. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1097\/SLA.0000000000001466","article-title":"Inhibition of MicroRNA-494 Reduces Carotid Artery Atherosclerotic Lesion Development and Increases Plaque Stability","volume":"262","author":"Wezel","year":"2015","journal-title":"Ann. Surg."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"638","DOI":"10.1016\/j.omtn.2019.09.021","article-title":"Antisense Oligonucleotide Inhibition of MicroRNA-494 Halts Atherosclerotic Plaque Progression and Promotes Plaque Stabilization","volume":"18","author":"Foks","year":"2019","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Goossens, E.A.C., de Vries, M.R., Jukema, J.W., Quax, P.H.A., and Nossent, A.Y. (2020). Myostatin Inhibits Vascular Smooth Muscle Cell Proliferation and Local 14q32 microRNA Expression, But Not Systemic Inflammation or Restenosis. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21103508"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1016\/j.molcel.2019.06.018","article-title":"Time-Resolved Small RNA Sequencing Unravels the Molecular Principles of MicroRNA Homeostasis","volume":"75","author":"Reichholf","year":"2019","journal-title":"Mol. Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1038\/s41580-018-0059-1","article-title":"Regulation of microRNA biogenesis and its crosstalk with other cellular pathways","volume":"20","author":"Treiber","year":"2019","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1007\/s11481-014-9531-7","article-title":"Vascular endothelial growth factor signaling in hypoxia and inflammation","volume":"9","author":"Ramakrishnan","year":"2014","journal-title":"J. Neuroimmune Pharmacol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3888","DOI":"10.1038\/s41467-019-11826-1","article-title":"Distinct methylation levels of mature microRNAs in gastrointestinal cancers","volume":"10","author":"Konno","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.omtn.2020.08.010","article-title":"N6-Adenosine Methylation of miRNA-200b-3p Influences Its Functionality and Is a Theranostic Tool","volume":"22","author":"Briand","year":"2020","journal-title":"Mol. Ther.-Nucleic Acids"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1228","DOI":"10.1016\/j.omtn.2021.10.027","article-title":"Inhibition of microRNA-494-3p activates Wnt signaling and reduces proinflammatory macrophage polarization in atherosclerosis","volume":"26","author":"Foks","year":"2021","journal-title":"Mol. Ther.-Nucleic Acids"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Thalhammer, A., Bencokova, Z., Poole, R., Loenarz, C., Adam, J., O\u2019Flaherty, L., Schodel, J., Mole, D., Giaslakiotis, K., and Schofield, C.J. (2011). Human AlkB homologue 5 is a nuclear 2-oxoglutarate dependent oxygenase and a direct target of hypoxia-inducible factor 1alpha (HIF-1alpha). PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0016210"},{"key":"ref_44","first-page":"E2047","article-title":"Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m(6)A-demethylation of NANOG mRNA","volume":"113","author":"Zhang","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1080\/15476286.2020.1804697","article-title":"Reprogramming of m(6)A epitranscriptome is crucial for shaping of transcriptome and proteome in response to hypoxia","volume":"18","author":"Wang","year":"2021","journal-title":"RNA Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"5195","DOI":"10.1093\/nar\/gky156","article-title":"SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function","volume":"46","author":"Du","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"932","DOI":"10.1016\/j.omtn.2020.07.020","article-title":"Adenosine-to-Inosine Editing of Vasoactive MicroRNAs Alters Their Targetome and Function in Ischemia","volume":"21","author":"Parma","year":"2020","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"9949","DOI":"10.1002\/chem.201801640","article-title":"Existence of Diverse Modifications in Small-RNA Species Composed of 16-28\u2005Nucleotides","volume":"24","author":"Lan","year":"2018","journal-title":"Chemistry"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1161\/CIRCRESAHA.117.312345","article-title":"Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization","volume":"122","author":"Parma","year":"2018","journal-title":"Circ. Res."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"10867","DOI":"10.1021\/ja029742d","article-title":"Substrate analogues for an RNA-editing adenosine deaminase: Mechanistic investigation and inhibitor design","volume":"125","author":"Veliz","year":"2003","journal-title":"J. Am. Chem. Soc."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.molcel.2017.12.006","article-title":"N(6)-Methyladenosines Modulate A-to-I RNA Editing","volume":"69","author":"Xiang","year":"2018","journal-title":"Mol. Cell"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1858","DOI":"10.1038\/s41467-019-09712-x","article-title":"Excessive miR-25-3p maturation via N(6)-methyladenosine stimulated by cigarette smoke promotes pancreatic cancer progression","volume":"10","author":"Zhang","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"6322","DOI":"10.1093\/nar\/gkm657","article-title":"Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine","volume":"35","author":"Dai","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"560","DOI":"10.1038\/nature14234","article-title":"N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions","volume":"518","author":"Liu","year":"2015","journal-title":"Nature"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.omtn.2017.03.003","article-title":"Inhibition of Mef2a Enhances Neovascularization via Post-transcriptional Regulation of 14q32 MicroRNAs miR-329 and miR-494","volume":"7","author":"Welten","year":"2017","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1140","DOI":"10.1038\/nm.4172","article-title":"Adenosine-to-inosine RNA editing controls cathepsin S expression in atherosclerosis by enabling HuR-mediated post-transcriptional regulation","volume":"22","author":"Stellos","year":"2016","journal-title":"Nat. Med."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/3\/1057\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:03:55Z","timestamp":1760133835000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/3\/1057"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,19]]},"references-count":56,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["ijms23031057"],"URL":"https:\/\/doi.org\/10.3390\/ijms23031057","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,19]]}}}