Interferon Inducible Pseudouridine Modification in Human Transcriptome by Quantitative Nanopore Sequencing

Abstract

Pseudouridine (Ψ) is an abundant mRNA modification in the mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome‐wide mapping. The Ψ derivative, N1‐methyl‐Ψ, substitutes every U residue in the Pfizer/BioNTech and Moderna COVID mRNA vaccines. Previous research revealed that Ψ increases translation of reporter genes and this effect depends on Protein kinase R (PKR), an interferon inducible gene. Thus, we hypothesize that endogenous Ψ modification in mRNA may be associated with interferon induced cellular response. In order to map Ψ in the human transcriptome, we developed a nanopore direct RNA sequencing method for quantitative Ψ mapping (NanoPsu, https://github.com/sihaohuanguc/Nanopore_psU) that utilizes native content training on 1329 Ψ and U sites, machine learning modeling and prediction, and single read linkage analysis. We validated our method using quantitative RT‐PCR of Ψ sites in specific mRNAs and through the observation of global Ψ level decrease upon knockdown of TRUB1, a known Ψ writer for mRNA. We apply the nanopore direct RNA sequencing method to interferon β and γ treated human cells and reveal a widespread differential distribution of Ψ in the human transcriptome. Biologically we find interferon inducible Ψ modifications in interferon stimulated gene transcripts which is consistent with a role of Ψ in enabling efficacy of mRNA vaccines. Single read analysis of the nanopore data reveals that multiple Ψ sites within the same transcript can be linked with each other. In summary, our results reveal a potential role of mRNA Ψ modification in interferon induced response and provide a new aspect in the research of mechanism of immune response.