Multimodal profiling reveals cell type-specific pseudouridine modification and density-dependent translational regulation.

Pseudouridine (psi) is one of the most abundant mRNA modifications, yet its impact on translation is unclear, in part because existing modification maps are inconsistent, curated comparisons across cell types are lacking, and paired analyses with translation are limited. Using direct RNA nanopore sequencing coupled with our Mod-p ID analytical framework, we mapped psi at single-nucleotide resolution across six immortalized human cell lines. Nanopore sequencing provided single-molecule resolution, enabling quantification of relative modification occupancy and detection of co-occurring modifications. Integrating these psi maps with matched proteomic and ribosome profiling datasets revealed that conserved psi sites installed by the psi synthase TRUB1 are associated with increased protein production. TRUB1 knockout experiments demonstrated a motif-specific reduction in protein abundance, providing direct causal evidence that pseudouridylation enhances protein output. In contrast, transcripts bearing clustered psi sites exhibited reduced protein abundance despite elevated translation efficiency. Controlled in vitro translation experiments confirmed that increasing pseudouridine density within a physiologically relevant range directly reduces protein output, demonstrating a density-dependent effect of pseudouridylation on translation. Together, these findings establish a mechanistic framework in which single-site pseudouridylation enhances protein production, whereas hypermodification impairs translational throughput, revealing pseudouridine density and enzyme specificity as key determinants of proteome output across human cell types.