Simultaneous nanopore profiling of mRNA m6A and pseudouridine reveals translation coordination

N6-methyladenosine (m6A) and pseudouridine (?) are the two most abundant modifications in mammalian mRNA, 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 m6A and ? in the human transcriptome. Applying NanoSPA to polysome profiling, we reveal opposing transcriptomic co-occurrence of m6A and ? and synergistic, hierarchical effects of m6A and ? on the polysome.