Integrative analysis of nanopore direct RNA sequencing data reveals a global impact of pseudouridylation on m6A and m5C modifications

RNA modifications play a crucial role in regulating cellular functions. Among the most abundant modifications in the human transcriptome are pseudouridine (Ψ), N6-methyladenosine (m⁶A), and 5-methylcytosine (m⁵C). However, the interplay between these modifications remains poorly understood due to limited integrative studies. To address the gap, we utilized nanopore direct RNA sequencing to quantify the stoichiometry of Ψ, m⁶A, and m⁵C after depleting the pseudouridine synthases PUS7 or DKC1. We used the custom tool NanoPsiPy to quantify pseudouridine by analyzing differential U-to-C base-calling errors in nanopore sequencing data. For m⁶A and m⁵C, we applied the established tool CHEUI to conduct stoichiometry differential analysis. Our investigation identified both known and novel pseudouridylation sites in tRNA, rRNA, and mRNA targeted by PUS7 or DKC1. Integrative analysis revealed that depletion of PUS7 or DKC1 reduced pseudouridylation levels while simultaneously increasing global m⁶A and m⁵C levels, with functional implications for mRNA translation regulation. These findings suggest that pseudouridylation may play an active role in repressing m⁶A and m⁵C modifications. This study demonstrates the analytical power of nanopore direct RNA sequencing for investigating co-regulation of RNA modifications.