A Kinase and a Glycosylase Catabolize Pseudouridine in the Peroxisome to Prevent Toxic Pseudouridine Monophosphate Accumulation

Pseudouridine, a frequent nucleoside modification in noncoding RNA and mRNA, is degraded in Arabidopsis peroxisomes to prevent the accumulation of the toxic intermediate pseudouridine monophosphate. Pseudouridine (psi) is a frequent nucleoside modification that occurs in both noncoding RNAs and mRNAs. In pseudouridine, C5 of uracil is attached to the Rib via an unusual C-glycosidic bond. This RNA modification is introduced on the RNA by site-specific transglycosylation of uridine (U), a process mediated by pseudouridine synthases. RNA is subject to constant turnover, releasing free pseudouridine, but the metabolic fate of pseudouridine in eukaryotes is unclear. Here, we show that in Arabidopsis (Arabidopsis thaliana), pseudouridine is catabolized in the peroxisome by (1) a pseudouridine kinase (PUKI) from the PfkB family that generates 5 '-pseudouridine monophosphate (5 '-psi MP) and (2) a psi MP glycosylase (PUMY) that hydrolyzes psi MP to uracil and ribose-5-phosphate. Compromising pseudouridine catabolism leads to strong pseudouridine accumulation and increased psi MP content. psi MP is toxic, causing delayed germination and growth inhibition, but compromising pseudouridine catabolism does not affect the psi/U ratios in RNA. The bipartite peroxisomal PUKI and PUMY are conserved in plants and algae, whereas some fungi and most animals (except mammals) possess a PUMY-PUKI fusion protein, likely in mitochondria. We propose that vacuolar turnover of ribosomal RNA produces most of the pseudouridine pool via 3 '-psi MP, which is imported through the cytosol into the peroxisomes for degradation by PUKI and PUMY, a process involving a toxic 5 '-psi MP intermediate.