A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation

Abstract

N6-methyladenosine (m6A) is the most common and abundant RNA modification. 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. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. 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. Using this technique, we have been able to confirm the recently described m6A methylation in the 3′UTR of SOCS1 and SOCS3 transcripts. 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.

Gluten-induced RNA methylation changes regulate intestinal inflammation via allele-specific XPO1 translation in epithelial cells

“…Using online available m 6 A individual-nucleotide-resolution cross-linking and immunoprecipitation from HCT116 cells 27 we observed that the three m 6 A consensus motifs in the 5’UTR are methylated (see figure 1B ) and confirmed these results using an RT-qPCR-based method (see online supplemental figure 1E ). 28 In addition, taking advantage of the heterozygosity of the HCT116 cell line for the CD-associated SNP, we observed that the mRNA transcript carrying the CD-risk allele ( XPO1 *T) is more methylated than its alternative, protective form ( XPO1 *C) (see figure 1C ). As m 6 A methylation is involved in diverse RNA metabolic processes, we wanted to elucidate the effects of this differential methylation on the XPO1 mRNA.…”

“…Downloaded from Coeliac disease 1E). 28 In addition, taking advantage of the heterozygosity of the HCT116 cell line for the CD-associated SNP, we observed that the mRNA transcript carrying the CD-risk allele (XPO1*T) is more methylated than its alternative, protective form (XPO1*C) (see figure 1C). As m 6 A methylation is involved in diverse RNA metabolic processes, we wanted to elucidate the effects of this differential methylation on the XPO1 mRNA.…”

Impact of the gut microbiota on the m6A epitranscriptome of mouse cecum and liver

“…Differentially methylated peaks between GF and CONV mice in the cecum were mainly linked to metabolism and immunological and inflammatory responses, functions known to be influenced by the microbiota. Additionally, changes in m 6 A modifications of specific transcripts have been linked to inflammatory intestinal disease in humans 34 , and also a T-cell specific conditional knock-out of Mettl3 led to intestinal inflammation in a mouse model 14 . Interestingly, we identified several risk genes for inflammatory bowel disease to be differentially methylated when comparing CONV and GF mice 64,65 , suggesting that microbiota-induced changes in m 6 A modifications might thus be linked to inflammatory conditions.…”

“…However, a complete understanding of mechanisms underlying gut-microbiota-host interactions still remains elusive. Recent reports have suggested that changes in m 6 A levels are associated with inflammatory states 14,34 and very recently, the presence of a microbiota has been suggested to induce changes in epitranscriptomic profiles in the brain, and several other tissues 35 .…”

Epitranscriptomics of Ischemic Heart Disease—The IHD-EPITRAN Study Design and Objectives

“…Several techniques, including untargeted epitranscriptomic sequencing methods, to measure m 6 A are available. Their strengths and weaknesses are listed in Supplementary Table S2 [ 20 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 ]. First, the IHD-EPITRAN study plans to utilize, based on competitive tendering, one of the various mainstay second generation sequencing platforms for detecting the epitranscriptomically modified transcripts for initial insight [ 117 , 118 , 120 , 121 ].…”