A kinase and a glycosylase catabolize pseudouridine in the peroxisome to prevent toxic pseudouridine monophosphate accumulation

Abstract

Pseudouridine (C) 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 59-pseudouridine monophosphate (59-CMP) and (2) a CMP glycosylase (PUMY) that hydrolyzes CMP to uracil and ribose-5-phosphate. Compromising pseudouridine catabolism leads to strong pseudouridine accumulation and increased CMP content. CMP is toxic, causing delayed germination and growth inhibition, but compromising pseudouridine catabolism does not affect the C/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 39-CMP, which is imported through the cytosol into the peroxisomes for degradation by PUKI and PUMY, a process involving a toxic 59-CMP intermediate.