title: Contribution of two conserved histidines to the dual activity of archaeal RNA guide-dependent and -independent pseudouridine synthase Cbf5.
abstract: In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)--the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U(55) in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems.
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Tillault
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Fourmann
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Loegler
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wieden
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Ute
authors.initials: U
authors.lastname: Kothe
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 54505 Vandœuvre-lès-Nancy, France bruno.charpentier@univ-lorraine.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Charpentier
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D006639:Histidine / Q000502:physiology*; D019751:Intramolecular Transferases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D019642:RNA, Archaeal; D006639:Histidine; D019751:Intramolecular Transferases; C096995:pseudouridine synthases
keywords: Archaea; Cbf5; H/ACA sRNP; RNA modification; pseudouridine