- Writers, readers, and erasers have now been discovered for many mRNA modifications.
- Global topographic candidate maps have been generated for many modifications, but high error rates need to be addressed by technical improvements in detection and validation using orthogonal methods that apply rigid selection criteria.
- Nanopore single-molecule direct RNA sequencing is progressing towards reliable detection of modified nucleotides in mRNA.
Modified nucleotides in mRNA are an essential addition to the standard genetic code of four nucleotides in animals, plants, and their viruses. The emerging field of epitranscriptomics examines nucleotide modifications in mRNA and their impact on gene expression. The low abundance of nucleotide modifications and technical limitations, however, have hampered systematic analysis of their occurrence and functions. Selective chemical and immunological identification of modified nucleotides has revealed global candidate topology maps for many modifications in mRNA, but further technical advances to increase confidence will be necessary. Single-molecule sequencing introduced by Oxford Nanopore now promises to overcome such limitations. Researchers at the Ontario Institute for Cancer Research summarize current progress with a particular focus on the bioinformatic challenges of this novel sequencing technology.
(A) Schematic of the library preparation procedure for Nanopore direct RNA sequencing. PolyA RNA is enriched using oligo-dT primers and a reverse transcription (RT) adaptor is ligated. After second-strand synthesis, the sequencing adapter RMX, which is preloaded with motor protein and tether protein, is then ligated. (B) Schematic of Nanopore direct RNA sequencing. The motor protein feeds the RNA molecule through the nanopore in the 3′–5′ direction. The five bases passing through a nanopore cause a characteristic disruption in the current which is stored as raw signal. (C) A current trace (squiggle plot) showing the raw signal generated by nanopore sequencing of a single mRNA molecule. (D) The two features recorded by Oxford Nanopore Technologies (ONT) sequencers are the current signal (in arbitrary units, AU) and the time that a given k-mer takes to transverse the pore (signal length, retention time or ‘dwell’).