Simultaneous nanopore profiling of mRNA m6A and pseudouridine reveals translation coordination

N6-methyladenosine (m 6 A) and pseudouridine (Ψ) are the two most abundant modifications in mammalian messenger RNA, but the coordination of their biological functions remains poorly understood. We develop a machine learning-based nanopore direct RNA sequencing method (NanoSPA) that simultaneously analyzes m 6 A and Ψ in the human transcriptome. Applying NanoSPA to polysome profiling, we reveal opposing transcriptomic co-occurrence of m 6 A and Ψ and synergistic, hierarchical effects of m 6 A and Ψ on the polysome. A machine learning pipeline for nanopore sequencing analyzes m 6 A and Ψ simultaneously.