biostudies-literatureUnknown11(2)Li HHoward Hughes Medical InstitutePseudouridine (Ψ) is the most abundant RNA modification, yet studies of Ψ have been hindered by a lack of robust methods to profile comprehensive Ψ maps. Here we utilize bisulfite-induced deletion sequencing to generate transcriptome-wide Ψ maps at single-base resolution across various plant species. Integrating ribosomal RNA, transfer RNA and messenger RNA Ψ stoichiometry with mRNA abundance and polysome profiling data, we uncover a multilayered regulation of translation efficiency through Ψ modifications. rRNA pseudouridylation could globally control translation, although the effects vary at different rRNA Ψ sites. Ψ in the tRNA T-arm loop shows strong positive correlations between Ψ stoichiometry and the translation efficiency of their respective codons. We observed a general inverse correlation between Ψ level and mRNA stability, but a positive correlation with translation efficiency in Arabidopsis seedlings. In conclusion, our study provides critical resources for Ψ research in plants and proposes prevalent translation regulation through rRNA, tRNA and mRNA pseudouridylation.Nature plants234-247https://www.ebi.ac.uk/biostudies/studies/S-EPMC12273003biostudies-literatureQuantitative RNA pseudouridine maps reveal multilayered translation control through plant rRNA, tRNA and mRNA pseudouridylation.PMC12273003Li HGao BHe CZhang JYe CWang GHe KZhang LLiu SJu CYang FZou ZJiang BChen YfalseQuantitative RNA pseudouridine maps reveal multilayered translation control through plant rRNA, tRNA and mRNA pseudouridylation.Pseudouridine (Ψ) is the most abundant RNA modification, yet studies of Ψ have been hindered by a lack of robust methods to profile comprehensive Ψ maps. Here we utilize bisulfite-induced deletion sequencing to generate transcriptome-wide Ψ maps at single-base resolution across various plant species. Integrating ribosomal RNA, transfer RNA and messenger RNA Ψ stoichiometry with mRNA abundance and polysome profiling data, we uncover a multilayered regulation of translation efficiency through Ψ modifications. rRNA pseudouridylation could globally control translation, although the effects vary at different rRNA Ψ sites. Ψ in the tRNA T-arm loop shows strong positive correlations between Ψ stoichiometry and the translation efficiency of their respective codons. We observed a general inverse correlation between Ψ level and mRNA stability, but a positive correlation with translation efficiency in Arabidopsis seedlings. In conclusion, our study provides critical resources for Ψ research in plants and proposes prevalent translation regulation through rRNA, tRNA and mRNA pseudouridylation.2025-01-01T00:00:00Z2025 Feb2025-08-28T03:09:00.652Z2025-08-28T03:09:00.652ZS-EPMC122730033978909210.1038/s41477-024-01894-7