title: RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature.
abstract: The box H/ACA small ribonucleoprotein particles (H/ACA sRNPs) are RNP enzymes that isomerize uridines (U) into pseudouridines (Ψ) in archaeal RNAs. The RNA component acts as a guide by forming base-pair interactions with the substrate RNA to specify the target nucleotide of the modification to the catalytic subunit Cbf5. Here, we have analyzed association of an H/ACA sRNP enzyme from the hyperthermophilic archaeon Pyrococcus abyssi with synthetic substrate RNAs of different length and with target nucleotide variants, and estimated their turnover at high temperature. In these conditions, we found that a short substrate, which length is restricted to the interaction with RNA guide sequence, has higher turnover rate. However, the longer substrate with additional 5' and 3' sequences non-complementary to the guide RNA is better discriminated by the U to Ψ conversion allowing the RNP enzyme to distinguish the modified product from the substrate. In addition, we identified that the conserved residue Y179 in the catalytic center of Cbf5 is crucial for substrate selectivity.
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 9 avenue de la forêt de Haye, BP 184, 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, 9 avenue de la forêt de Haye, BP 184, 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, 9 avenue de la forêt de Haye, BP 184, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Loegler
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 9 avenue de la forêt de Haye, BP 184, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Blaud
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 9 avenue de la forêt de Haye, BP 184, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Christiane
authors.initials: C
authors.lastname: Branlant
authors.affiliation: Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 9 avenue de la forêt de Haye, BP 184, 54505 Vandœuvre-lès-Nancy, France. Electronic address: bruno.charpentier@univ-lorraine.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Charpentier
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011560:Pseudouridine / Q000096:biosynthesis*; D043903:Pyrococcus abyssi / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019642:RNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D019843:Archaeal Proteins; D019642:RNA, Archaeal; D012261:Ribonucleoproteins; D011560:Pseudouridine
keywords: Box H/ACA sRNP; Pseudouridine; RNA modification; RNA:Pseudouridine-synthase; Substrate specificity