Abstract
It has become evident that epitranscriptome events, mediated by specific enzymes, regulate gene expression and, subsequently, cell differentiation processes. We show that methyltransferase-like proteins METTL3/METTL14 and N6-adenosine methylation (m6A) in RNAs are homogeneously distributed in embryonic hearts, and histone deacetylase (HDAC) inhibitors valproic acid and Trichostatin A (TSA) up-regulate METTL3/METTL14 proteins. The levels of METTL3 in mouse adult hearts, isolated from male and female animals, were lower in the aorta and pulmonary trunks when compared with atria, but METT14 was up-regulated in the aorta and pulmonary trunk, in comparison with ventriculi. Aging caused METTL3 down-regulation in aorta and atria in male animals. Western blot analysis in differentiated mouse embryonic stem cells (mESCs), containing 10–30 percent of cardiomyocytes, showed METTL3/METTL14 down-regulation, while the differentiation-induced increased level of METTL16 was observed in both wild type (wt) and HDAC1 depleted (dn) cells. In parallel, experimental differentiation in especially HDAC1 wild type cells was accompanied by depletion of m6A in RNA. Immunofluorescence analysis of individual cells revealed the highest density of METTL3/METTL14 in α-actinin positive cardiomyocytes when compared with the other cells in the culture undergoing differentiation. In both wt and HDAC1 dn cells, the amount of METTL16 was also up-regulated in cardiomyocytes when compared to co-cultivated cells. Together, we showed that distinct anatomical regions of the mouse adult hearts are characterized by different levels of METTL3 and METTL14 proteins, which are changed during aging. Experimental cell differentiation was also accompanied by changes in METTL-like proteins and m6A in RNA; in particular, levels and distribution patterns of METTL3/METTL14 proteins were different from the same parameters studied in the case of the METTL16 protein.
Highlights
Accurate gene expression, cell fate programs, or cell stress response require harmonious interplay between all regulatory levels, including epigenetic and epitranscriptomic mechanisms
We show that methyltransferase-like proteins METTL3/METTL14 and N6-adenosine methylation (m6A) in RNAs are homogeneously distributed in embryonic hearts, and histone deacetylase (HDAC) inhibitors valproic acid and Trichostatin A (TSA) up-regulate METTL3/METTL14 proteins
We studied the levels of METTL3, METTL14, methyltransferase-like 16 (METTL16) enzymes, and m6A RNA in the following regions of embryonic hearts: right atrium (RA), left atrium (LA), aorta (AO), right ventriculus (RV) and left ventriculus (LV) or intraventricular septum (IVS)
Summary
Cell fate programs, or cell stress response require harmonious interplay between all regulatory levels, including epigenetic and epitranscriptomic mechanisms. The orchestrated interplay among writers, erasers, and readers drives the dynamics and outcomes of the m6A modification of RNAs. The appearance of the methyl group in RNAs is a co-transcriptional event, occurring in nuclear speckles, and involves the recruitment of the METTL3 protein, characterized by the catalytic activity, in pre-mRNA. The appearance of the methyl group in RNAs is a co-transcriptional event, occurring in nuclear speckles, and involves the recruitment of the METTL3 protein, characterized by the catalytic activity, in pre-mRNA This regulatory process mostly appears in intron regions [5,6,7,8]. A significant eraser of m6A in mRNA is fat mass and obesity-associated protein (FTO) [16] and its homolog, alkB homolog 5 (ALKBH5) [17] These proteins can oxidatively reverse N6-adenosine methylation in nuclear RNA [16]. This process in DNA is mediated by ten-eleven translocation (TET) enzymes [22,23,24]
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