Interferon inducible pseudouridine modification in human mRNA by quantitative nanopore profiling

Sihao Huang,Devin Dersh,Wen Zhang,Karen Lolans,Tao Pan,Jonathan Yewdell,Qing Dai,A Murat Eren,Christopher D Katanski

doi:10.1186/s13059-021-02557-y

Abstract

Pseudouridine (Ψ) is an abundant mRNA modification in mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome-wide mapping. We develop a nanopore native RNA sequencing method for quantitative Ψ prediction (NanoPsu) that utilizes native content training, machine learning modeling, and single-read linkage analysis. Biologically, we find interferon inducible Ψ modifications in interferon-stimulated gene transcripts which are consistent with a role of Ψ in enabling efficacy of mRNA vaccines.

Highlights

Pseudouridine (Ψ) is the second most abundant mRNA modification in the mammalian transcriptome as measured by quantitative mass spectrometry [1] and may exert many cellular functions
In Illumina sequencing of the bisulfite method, Ψ sites are found by RT deletions which enable identification and quantitative assessment of closely spaced rRNA Ψ sites; these sites are more difficult to assess using the more commonly used carbodiimide method that identifies Ψ sites by RT stops
Sequencing the remaining sample via direct RNA nanopore sequencing, we found that 640 of these Ψ sites passed our filter of 20 read coverage for further analysis (Additional file 1: Fig. S1b)

Summary

Introduction

Pseudouridine (Ψ) is the second most abundant mRNA modification in the mammalian transcriptome as measured by quantitative mass spectrometry [1] and may exert many cellular functions. Ψ incorporation in synthetic, transfected reporter mRNA increases translation [2] through decreased activation of the RNAdependent protein kinase (PKR) [3]. Functional exploration and mechanistic investigation of mRNA Ψ modification requires appropriate mapping methods. Illumina sequencing of Ψ in mRNA relies on chemical RNA treatments that induce stop, mutation, or deletion signatures in cDNA synthesis [1, 5–8]. Many computational methods have been developed to map mRNA Ψ sites [9–20]. MRNA Ψ mapping is inconsistent among these studies, in part due to the high false positives and negatives generated by the chemical treatments

Methods

Results

Conclusion