Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation

被引:99
作者
Charpentier, B [1 ]
Muller, S [1 ]
Branlant, C [1 ]
机构
[1] Univ Nancy 1, Lab Maturat ARN & Enzymol Mol, UHP, CNRS UMR 7567, F-54506 Vandoeuvre Les Nancy, France
关键词
D O I
10.1093/nar/gki630
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:3133 / 3144
页数:12
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