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Molecular and Cellular Biology, October 2002, p. 6663-6668, Vol. 22, No. 19
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.19.6663-6668.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2'-O-Methylation of Target RNA In Vitro

Silvia Galardi,1,{dagger} Alessandro Fatica,1,{dagger} Angela Bachi,2,{dagger} Andrea Scaloni,3 Carlo Presutti,1 and Irene Bozzoni1*

Department of Genetics and Molecular Biology, Cenci-Bolognetti Foundation, Institute Pasteur, University of Rome "La Sapienza," 00185 Rome,1 IABBAM, Centro Nazionale di Ricerca, Naples,2 DIBIT, San Raffaele Scientific Institute, Milan,3 Italy3

Received 7 May 2002/ Returned for modification 5 June 2002/ Accepted 20 June 2002

Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2'-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D' of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-L-methionine-binding region of Nop1p is responsible for the catalytic activity.

* Corresponding author. Mailing address: Department of Genetics and Molecular Biology, University of Rome La Sapienza, P.le A. Moro 5, 00185 Rome, Italy. Phone: 39-06-49912202. Fax: 39-06-49912500. E-mail: irene.bozzoni{at}uniroma1.it.

{dagger} Present address: Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, United Kingdom.

Molecular and Cellular Biology, October 2002, p. 6663-6668, Vol. 22, No. 19
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.19.6663-6668.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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