Deciphering the ‘m⁶A code’ via quantitative profiling of m⁶A at single-nucleotide resolution

Abstract

N6-methyladenosine (m 6 A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. The ability to map m 6 A using immunoprecipitation-based approaches has played a critical role in dissecting m 6 A functions and mechanisms of action. Yet, these approaches are of limited specificity, unknown sensitivity, and unable to quantify m6A stoichiometry. These limitations have severely hampered our ability to unravel the factors determining where m6A will be deposited, to which levels (the 'm6A code'), and to quantitatively profile m6A dynamics across biological systems. Here, we used the RNase MazF, which cleaves specifically at unmethylated RNA sites, to develop MASTER-seq for systematic quantitative profiling of m6A sites at 16-25% of all m6A sites at single nucleotide resolution. We established MASTER-seq for orthogonal validation and de novo detection of m6A sites, and for tracking of m6A dynamics in yeast gametogenesis and in early mammalian differentiation. We discover that antibody-based approaches severely underestimate the number of m6A sites, and that both the presence of m6A and its stoichiometry are 'hard-coded' via a simple and predictable code within the extended sequence composition at the methylation sites. This code accounts for~50% of the variability in methylation levels across sites, allows excellent de novo prediction of methylation sites, and predicts methylation acquisition and loss across evolution. We anticipate that MASTER-seq will pave the path towards a more quantitative investigation of m6A biogenesis and regulation in a wide variety of systems, including diverse cell types, stimuli, subcellular components, and disease states.

Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development

“…In mammalian cells, m 6 A levels are mostly static even during key developmental stages such as stem cell differentiation 39,44 , with deregulation of METTL3 and increases in global m 6 A of only 25% being sufficient to inhibit differentiation, promote cell growth, and maintain tumorigenicity 25 . Although the m 6 A-demethylases Alkbh5 and FTO may modulate methylation levels in some cases 17 , m 6 A is generally deposited early and maintained throughout the life of an mRNA transcript 45 .…”

In Vitro Selection with a Site-Specifically Modified RNA Library Reveals the Binding Preferences of N6-Methyladenosine Reader Proteins

“…Indeed, single-nucleotide m 6 A sequencing approaches have revealed the presence of such sequences and have indicated that the m 6 A-modified transcriptome is more diverse than previously appreciated. 48,51 Finally, we envision that site-specifically modified random RNA libraries can be applied to probe the effect of diverse epitranscriptomic marks and sequence context on fundamental nucleic acid-related processes including protein−RNA binding, catalysis by modification writer and eraser enzymes, and templated polymerization. Combined with nucleic acid index-…”

“…Nevertheless, the finding that YTHDF1/2 can bind tightly to noncanonical m 6 A-containing motifs suggests that related m 6 A sequences may exist in the transcriptome. Indeed, single-nucleotide m 6 A sequencing approaches have revealed the presence of such sequences and have indicated that the m 6 A-modified transcriptome is more diverse than previously appreciated. , …”