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Nucleic Acids Research, 2003, Vol. 31, No. 3 869-877
© 2003 Oxford University Press

Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea

Timofey S. Rozhdestvensky, Thean Hock Tang, Inna V. Tchirkova, Jürgen Brosius, Jean-Pierre Bachellerie1 and Alexander Hüttenhofer*

Institut für Experimentelle Pathologie/Molekulare Neurobiologie (ZMBE), Universität Münster, D-48149 Münster, Germany and 1 Laboratoire de Biologie Moleculaire Eucaryote du CNRS, Universite Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse Cedex 04, France

*To whom correspondence should be addressed. Tel: +49 251 8352136; Fax: +49 251 8352134; Email: huttenh{at}uni-muenster.de Correspondence may also be addressed to Jean-Pierre Bachellerie. Tel. +33 5 61 33 59 34; Fax: +33 5 61 33 58 86; Email: bachel{at}ibcg.biotoul.fr

Small nucleolar RNAs (designated as snoRNAs in Eukarya or sRNAs in Archaea) can be grouped into H/ACA or C/D box snoRNA (sRNA) subclasses. In Eukarya, H/ACA snoRNAs assemble into a ribonucleoprotein (RNP) complex comprising four proteins: Cbf5p, Gar1p, Nop10p and Nhp2p. A homolog for the Nhp2p protein has not been identified within archaeal H/ACA RNPs thus far, while potential orthologs have been identified for the other three proteins. Nhp2p is related, particularly in the middle portion of the protein sequence, to the archaeal ribosomal protein and C/D box protein L7Ae. This finding suggests that L7Ae may be able to substitute for the Nhp2p protein in archaeal H/ACA sRNAs. By band shift assays, we have analyzed in vitro the interaction between H/ACA box sRNAs and protein L7Ae from the archaeon Archaeoglobus fulgidus. We present evidence that L7Ae forms specific complexes with three different H/ACA sRNAs, designated as Afu-4, Afu-46 and Afu-190 with an apparent Kd ranging from 28 to 100 nM. By chemical and enzymatic probing we show that distinct bases located within bulges or loops of H/ACA sRNAs interact with the L7Ae protein. These findings are corroborated by mutational analysis of the L7Ae binding site. Thereby, the RNA motif required for L7Ae binding exhibits a structure, designated as the K-turn, which is present in all C/D box sRNAs. We also identified four H/ACA RNAs from the archaeal species Pyrococcus which exhibit the K-turn motif at a similar position in their structure. These findings suggest a triple role for L7Ae protein in Archaea, e.g. in ribosomes as well as H/ACA and C/D box sRNP biogenesis and function by binding to the K-turn motif.


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