Simultaneous nanopore profiling of mRNA m⁶A and pseudouridine reveals translation coordination

N6-methyladenosine (m(6)A) and pseudouridine (psi) 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 psi in the human transcriptome. Applying NanoSPA to polysome profiling, we reveal opposing transcriptomic co-occurrence of m6A and psi and synergistic, hierarchical effects of m(6)A and psi on the polysome.