Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library.

Recent years have witnessed rapid progress in the field of epitranscriptomics. Functional interpretation of the epitranscriptome relies on sequencing technologies that determine the location and stoichiometry of various RNA modifications. However, contradictory results have been reported among studies, bringing the biological impacts of certain RNA modifications into doubt. Here, we develop a synthetic RNA library resembling the endogenous transcriptome but without any RNA modification. By incorporating this modification-free RNA library into established mapping techniques as a negative control, we reveal abundant false positives resulting from sequence bias or RNA structure. After calibration, precise and quantitative mapping expands the understanding of two representative modification types, N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We propose that this approach provides a systematic solution for the calibration of various RNA-modification mappings and holds great promise in epitranscriptomic studies.

Mapping single-nucleotide m6A by m6A-REF-seq.

The past few years have witnessed rapid progress in the field of RNA modifications. As the most prevailing modification on eukaryotic mRNA, m6A is characterized to play a vital role in various cellular activities. However, limitations of the detection method impede functional studies of m6A. Here we introduce m6A-REF-seq, a powerful and straightforward method to identify m6A at single-nucleotide resolution. m6A-REF-seq relies on the recognition of RNA endonuclease MazF towards m6A at the ACA motif, providing an orthogonal method independent of the m6A antibody being adopted by most of current methods. We describe a detailed protocol to perform m6A-REF-seq, including NGS library construction and sequencing data analysis. In particular, we describe an optimized assay to validate individual m6A sites identified by m6A-REF-seq, which can also be applied to detect any candidate m6A sites.

High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome.

N6-deoxyadenosine methylation (6mA) is the most widespread type of DNA modification in prokaryotes and is also abundantly distributed in some unicellular eukaryotes. However, 6mA levels are remarkably low in mammals. The lack of a precise and comprehensive mapping method has hindered more advanced investigations of 6mA. Here, we report a new method MM-seq (modification-induced mismatch sequencing) for genome-wide 6mA mapping based on a novel detection principle. We found that modified DNA bases are prone to form a local open region that allows capture by antibody, for example, via a DNA breathing or base-flipping mechanism. Specified endonuclease or exonuclease can recognize the antibody-stabilized mismatch-like structure and mark the exact modified sites for sequencing readout. Using this method, we examined the genomic positions of 6mA in bacteria (E. coli), green algae (C. reinhardtii), and mammalian cells (HEK239T, Huh7, and HeLa cells). In contrast to bacteria and green algae, human cells possess a very limited number of 6mA sites which are sporadically distributed across the genome of different cell types. After knocking out the RNA m6A methyltransferase METTL3 in mouse ES cells, 6mA becomes mostly diminished. Our results imply that rare 6mA in the mammalian genome is introduced by RNA m6A machinery via a non-targeted mechanism.