N6-methyladenosine (m6A) is the most abundant modification on mRNA, which plays a critical role in development, physiology, and disease. Studies in this area have been facing great challenges because of the lack of antibody-independent methodologies for interrogating m6A. Recently, the study 'Deciphering the "m6A Code" via Antibody-Independent Quantitative Profiling' was published in Cell , in which scientists from Weizmann Institute of Science and Medical University of Vienna developed an enzymatic approach MAZTER-seq for precise mapping and measurement of m6A within mRNAs.
This method combines ACA-specific MazF RNase and high-throughput sequencing/qPCR. MazF has an ability to cleave RNA immediately upstream of an "ACA" sequence, but not upstream of "m6A-CA". After MazF digestion, mRNA fragments are thentreated by end repair, reverse transcription, adaptor ligation, cDNA amplification and paired-end sequencing. Ideally, each fragment should begin with an ACA site (5’ ACA) and terminate immediately prior to a downstream ACA site (3’ ACA). The ACA motifs containing m6A are supposed to be inside the fragments. A computational pipeline MAZTER-MINE is used to quantify the number of reads that begin, terminate, and read through each transcriptomic ACA site. In this way, MazF cleave efficiency of each ACA site can be calculated, thus methylation is evaluated.
With MAZTER-seq, the authors succeeded in quantitatively measuring methylation levels and observed an excellent linear relation between m6A stoichiometries and cleavage efficiencies. Then optimized this method to detect and quantify m 6A levels at endogenous sites in yeast. Results showed that MAZTER-seq can not only measure known m6A sites that had been identified by other methods, but also detect novel m6A sites. They also confirmed the applicability of MAZTER-seq in mammals, demonstrating that the principles of the m6A code are to a large extent conserved between yeast and mouse. To decrease the cost and input amount, the authors developed MazF-qPCR to quantify m 6A levels at single loci with a low cost and as little as 25–50 ng of mRNA.
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