Charpentier Bruno, Muller Sébastien, Branlant Christiane
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.
Nucleic Acids Res. 2005 Jun 2;33(10):3133-44. doi: 10.1093/nar/gki630. Print 2005.
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.
假尿苷（Ψ）是RNA中常见的修饰残基。在真核生物中，它们的形成由酶或含有H/ACA小核仁RNA（snoRNA）的核糖核蛋白复合物（RNP）催化。在古细菌中发现了H/ACA sRNA以及H/ACA sRNP蛋白（L7Ae、aCBF5、aNOP10和aGAR1）的推定开放阅读框。在这里，通过使用深渊热球菌重组蛋白和体外转录的深渊热球菌H/ACA sRNA，我们首次在体外完全重建了活性H/ACA RNP。L7Ae和aCBF5 RNA：假尿苷合酶都直接与sRNA结合；aCBF5也直接且独立地与aNOP10和aGAR1相互作用。在靶RNA的假尿苷化位点存在aCBF5、aNOP10和一个U残基是招募RNA靶标的必要条件。与此一致，我们发现aCBF5-aNOP10对是形成具有假尿苷化活性的颗粒所需的最小蛋白质组。然而，添加蛋白质L7Ae和/或aGAR1可以形成在靶向假尿苷化方面更有效的颗粒。虽然对于最佳活性是必需的，但发现sRNA中保守的ACA基序并非必不可少。
