Single molecule sequencing and imaging of pseudouridine modifications in the human transcriptome.

While the genomic makeup of cells is identical, cell specialization is partially achieved by chemical modification to their biomolecules, including RNA. Pseudouridine (psi) is among the most highly represented mRNA modifications but is extremely difficult to detect. Psi-modified mRNAs are more resistant to RNAse-mediated degradation and also have the potential to modulate immunogenicity and enhance translation in vivo. Psi has recently taken the spotlight due to the inclusion of a methylated analog in the Moderna and Pfizer mRNA vaccines for SARS CoV-2. We have recently developed an algorithm for identifying psi sites directly on mammalian mRNA transcripts using direct RNA nanopore sequencing and systematic base-calling errors at psi sites. We have verified psi sites identified previously by biochemical methods and uncovered new psi sites. We applied our synthetic standards to train a machine-learning algorithm that achieves precise quantification of psi at specific sites in the mRNA. We then use our algorithm to classify types of psi hyper-modification that may occur on mRNAs: Type 1 is mRNAs that have a high percentage of psi at a given site; type 2 is mRNAs that may have 2 or more pseudouridines on a single transcript.