Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation.
Pseudouridine (Psi) are frequently modified residues in RNA. In Eukarya, their formation is catalyzed by enzymes or by ribonucleoprotein complexes (RNPs) containing H/ACA snoRNAs. H/ACA sRNA and putative ORFs for H/ACA sRNP proteins (L7Ae, aCBF5, aNOP10 and aGAR1) were found in Archaea. Here, by using Pyrococcus abyssi recombinant proteins and an in vitro transcribed P.abyssi H/ACA sRNA, we obtained the first complete in vitro reconstitution of an active H/ACA RNP. Both L7Ae and the aCBF5 RNA:Psi synthase bind directly the sRNA; aCBF5 also interacts directly and independently with aNOP10 and aGAR1. Presence of aCBF5, aNOP10 and a U residue at the pseudouridylation site in the target RNA are required for RNA target recruitment. In agreement, we found that the aCBF5-aNOP10 pair is the minimal set of proteins needed for the formation of a particle active for pseudouridylation. However, particles more efficient in targeted pseudouridylation can be formed with the addition of proteins L7Ae and/or aGAR1. Although necessary for optimal activity, the conserved ACA motif in the sRNA was found to be not essential.
Charpentier
Bruno
B
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 UHP-CNRS, Université des Sciences et Techniques Henri Poincaré Nancy I 54506 Vandoeuvre-Lès-Nancy cedex, France. bruno.charpentier@maem.uhp-nancy.fr
Muller
Sébastien
S
Branlant
Christiane
C
D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D017124:Conserved Sequence; D006836:Hydro-Lyases / Q000378:metabolism; D008969:Molecular Sequence Data; D011560:Pseudouridine / Q000378:metabolism*; D043903:Pyrococcus abyssi / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism*; D014529:Uridine / Q000378:metabolism
D019843:Archaeal Proteins; D019642:RNA, Archaeal; D020537:RNA, Small Nucleolar; D020718:Ribonucleoproteins, Small Nucleolar; D011560:Pseudouridine; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase; D014529:Uridine