![]() More recently, m6A has been shown to affect the LPS-induced inflammatory response in human dental pulp cells 7 opening a new layer of investigation into the molecular mechanisms of inflammatory diseases. A recent study analyzing the effects of deleting writer Mettl3 in mouse T-cells found that, in the absence of Mettl3, mice develop a chronic inflammation of the intestine and that the mRNAs for STAT signaling inhibitory proteins SOCS1 and SOCS3, that generally present m6A methylation marks in their 3′UTR region, exhibited slower decay, resulting in increased mRNA and protein levels, and establishing a link between the m6A methylation of those transcripts and T-cell responses 5, 6. Genetic loss-of-function studies for m6A methyltransferases (m6A writers), m6A-binding proteins (m6A readers), and m6A demethylases (m6A erasers) have highlighted a critical role for m6A modifications in the control of gene expression in a range of physiological processes 3, 4. These modifications are generally dynamic and exhibit changes in distribution or frequency in response to certain stimuli, constituting a new layer of gene regulation termed epitranscriptomics 1, 2. N6-methyladenosine (m6A) is the most abundant internal modification on mammalian mRNAs and long-noncoding RNAs. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic β-cells exposed to inflammatory stimuli. Using this technique, we have been able to confirm the recently described m6A methylation in the 3′UTR of SOCS1 and SOCS3 transcripts. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. N6-methyladenosine (m6A) is the most common and abundant RNA modification. ![]()
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