Pseudouridine metabolism in bacteria and mammals

Pseudouridine is the most common modified nucleoside in RNA. It is found in ribosomal RNAs, transfer RNAs, small nuclear RNAs and small nucleolar RNAs of ail species. Pseudouridine results from the isomerization of uridine at the polynucleotide level. It is characterized by a glycosidic C-C bond between the ribose and uracil moieties. Pseudouridine is known to be degraded by Escherichia coli and many other species but not in mammals, where it is excreted intact in urine. This thesis is devoted to the study of the metabolism of pseudouridine. Two enzymes are responsible for the dégradation of pseudouridine in bacteria. The first one is a kinase that phosphorylâtes pseudouridine to pseudouridine 5’-phosphate. The second one is a glycosidase that cleaves the glycosidic C-C bond of pseudouridine 5’-phosphate. The purpose of the first part of this thesis was to carry out the molecular identification of these two enzymes. To this end, we took advantage of the observation that yeiC is the most overexpressed gene when a uropathogenic strain of E. coli is grown on urine compared to a synthetic medium. This gene, which encodes an enzyme of the ribokinase family, is found in an operon in association with a gene coding for a putative nucleoside transporter and a gene coding for a protein of unknown function (YeiN). As pseudouridine is an abundant nucleoside in urine, these observations led us to hypothesize that YeiC encodes pseudouridine kinase, and YeiN, pseudouridine-5’-phosphate glycosidase. YeiC and YeiN were overexpressed in E. coli, purified and shown to catalyze the expected reaction. The reaction catalyzed by the latter is métal dépendent and easily réversible (AG°5 = + 5 kcal/mol), indicating the stability of the glycosidic C-C bond. BLAST searches indicate that homologues of YeiC and YeiN are présent in many bacterial and most eukaryotic species, with the exception of mammals. This accounts for the fact that pseudouridine is not degraded in humans. Pseudouridine 5’-phosphate is most likely an intermediate in the breakdown of RNAs. It was therefore of interest to détermine what kind of phosphatase is involved in its conversion to pseudouridine in mammalian cells. Using E. coli YeiC, we prepared radiolabeled pseudouridine 5’-phosphate and could show that human érythrocytes contain a spécifie pseudouridine-5’-phosphatase with high affinity for its substrate (Km < lpM). Characterization of the partially purified enzyme suggested that it belonged the ‘haloacid dehalogenase’ family of phosphatases and that it had a molecular mass of ~ 26 kDa. Based on this information we expressed four candidate proteins of the HAD family and could show that one of them, HDHD1A corresponded to pseudouridine-5’-phosphatase. This enzyme displayed a catalytic efficiency for pseudouridine 5’-phosphate that was more than 104-fold higher than that for its second best substrate. HDHD1A is encoded by a région of chromosome X that is not subject to inactivation in women and is deleted in most cases of X- linked ichthyosis. Accordingly, the pseudouridine phosphatase activity was found to be significantly higher in extracts of cells from women than of men and to be absent from cells of 4 patients with X-linked ichthyosis. These findings therefore confirm the identification of pseudouridine-5’-phosphatase as HDHD1A. In conclusion, this work clarifies the metabolism of pseudouridine in eukaryotes and prokaryotes and provides also insights in the function of IndA, a YeiN homologue thought to be involved in the synthesis of indigoidine, a compound secreted by several phytopathogens.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL,