Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N-methyladenosine (mA), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of mA at single-nucleotide resolution. Within the cytosol, mA is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single mA site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. mA leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that mA on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over mA levels was adopted as a potential means of post-transcriptional regulation.

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N6-methyladenosine (m 6 A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m 6 A stoichiometry and the lack of antibodyindependent methodologies for interrogating m 6 A.Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m 6 A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m 6 A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is ''hard coded'' in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m 6 A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m 6 A regulation in subcellular fractions, diverse cell types, and disease states. (A) Distribution of cleavage efficiencies (y axis) in RNA extracted from WT ime4D/D strains with versus without m 6 A-IP treatment. (B) Clustered pairwise correlation of the samples in (A). (C) Empirical false-detection rates per confidence group. (D) Distribution of m 6 A-seq sites across the confidence groups defined via MAZTER-seq. (E) Distribution of m 6 A-seq scores from Schwartz et al. (2013) by MAZTER-seq confidence groups. (F) Sequence logos for sites identified via MAZTER-seq shown separately for the indicated confidence groups. (G) Higher-confidence sites are closer to the end of the transcript. Distributions of 3 0 end distances by confidence group. (H) SCARLET-based readouts of methylation levels at each of the indicated sites.

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Asymmetric messenger RNA (mRNA) localization facilitates efficient translation in cells such as neurons and fibroblasts. However, the extent and importance of mRNA polarization in epithelial tissues are unclear. Here, we used single-molecule transcript imaging and subcellular transcriptomics to uncover global apical-basal intracellular polarization of mRNA in the mouse intestinal epithelium. The localization of mRNAs did not generally overlap protein localization. Instead, ribosomes were more abundant on the apical sides, and apical transcripts were consequently more efficiently translated. Refeeding of fasted mice elicited a basal-to-apical shift in polarization of mRNAs encoding ribosomal proteins, which was associated with a specific boost in their translation. This led to increased protein production, required for efficient nutrient absorption. These findings reveal a posttranscriptional regulatory mechanism involving dynamic polarization of mRNA and polarized translation.