N-6-Methyladenosine in Vasoactive microRNAs during Hypoxia; A Novel Role for METTL4.

Daphne A L Van Den Homberg,Paul H A Quax,A Yaël Nossent,Reginald V C T Van Der Kwast

doi:10.3390/ijms23031057

Daphne A L Van Den Homberg, Paul H A Quax + Show 2 more

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https://doi.org/10.3390/ijms23031057

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Abstract

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.

Highlights

MicroRNAs are a class of short (~22 nucleotides) non-coding RNA molecules, which regulate the translation of mRNAs into proteins
To investigate whether m6A is relevant to vasoactive microRNAs, we selected a subset of 19 vasoactive microRNAs to study in detail in fibroblasts (Table 1)
Cells cultured under 48 h of hypoxia showed similar m6A writer and eraser levels as 24 h of hypoxia (Supplemental Figure S2); we did observe a significant upregulation of mRNA levels of the m6A writer METTL3 after 48 h of hypoxia, which was not observed after 24 h

Summary

Introduction

MicroRNAs are a class of short (~22 nucleotides) non-coding RNA molecules, which regulate the translation of mRNAs into proteins. The role of microRNAs as cardiovascular modulators has been well-established in multiple vascular remodelling processes [1]. It has been shown that microRNAs are involved in the different stages of cardiovascular disease, where they can play a role in the development of dyslipidaemia, vascular inflammation, hypertension and atherosclerotic lesion development and stability [2]. The progression of cardiovascular diseases can lead to insufficient blood flow through tissues, causing them to become ischemic and resulting in oxygen deprivation or hypoxia. MicroRNAs can regulate the restoration of blood flow to these ischemic tissues by regulating the growth and maturation of blood vessels, a process called neovascularisation [3]. Like all RNAs, microRNAs are subject to post-transcriptional modifications [4]. The most well-known m6A ‘writers’, the m6A writer complex, composed of METTL3 and METTL14

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