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Nucleic Acids Research Advance Access published online on February 27, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn077
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs

Sébastien Muller, Fabrice Leclerc, Isabelle Behm-Ansmant, Jean-Baptiste Fourmann, Bruno Charpentier and Christiane Branlant*

Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Nancy Université, Faculté des Sciences et Techniques, 54506 Vandoeuvre-lès-Nancy, France

*To whom correspondence should be addressed. Tel: +33 3 83 68 43 03; Fax: +33 3 83 68 43 07; Email: christiane.branlant{at}maem.uhp-nancy.fr

Received January 15, 2008. Revised February 7, 2008. Accepted February 8, 2008.

How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 {Psi} residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 {Psi} residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining {Psi} residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3'-guide sequence is more important than that formed with the 5'-guide sequence. Surprisingly, we showed that a sRNA–rRNA interaction with the targeted uridine in a single-stranded 5'-UNN-3' trinucleotide instead of the canonical 5'-UN-3' dinucleotide is functional.


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