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Molecular and Cellular Biology, December 2000, p. 9028-9040, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Human H/ACA Small Nucleolar RNPs and Telomerase Share Evolutionarily Conserved Proteins NHP2 and NOP10

Vanda Pogacic', François Dragon,dagger and Witold Filipowicz*

Friedrich-Miescher Institut, CH-4058 Basel, Switzerland

Received 27 July 2000/Returned for modification 30 August 2000/Accepted 12 September 2000

The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. In the yeast Saccharomyces cerevisiae, four common proteins are associated with H/ACA snoRNAs: Gar1p, Cbf5p, Nhp2p, and Nop10p. In vitro reconstitution studies showed that four proteins also specifically interact with H/ACA snoRNAs in mammalian cell extracts. Two mammalian proteins, NAP57/dyskerin (the ortholog of Cbf5p) and hGAR1, have been characterized. In this work we describe properties of hNOP10 and hNHP2, human orthologs of yeast Nop10p and Nhp2p, respectively, and further characterize hGAR1. hNOP10 and hNHP2 complement yeast cells depleted of Nhp2p and Nop10p, respectively. Immunoprecipitation experiments with extracts from transfected HeLa cells indicated that epitope-tagged hNOP10 and hNHP2 specifically associate with hGAR1 and H/ACA RNAs; they also interact with the RNA subunit of telomerase, which contains an H/ACA-like domain in its 3' moiety. Immunofluorescence microscopy experiments showed that hGAR1, hNOP10, and hNHP2 are localized in the dense fibrillar component of the nucleolus and in Cajal (coiled) bodies. Deletion analysis of hGAR1 indicated that its evolutionarily conserved core domain contains all the signals required for localization, but progressive deletions from either the N or the C terminus of the core domain abolish localization in the nucleolus and/or the Cajal bodies.

* Corresponding author. Mailing address: Friedrich Miescher Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Phone: 41 61 697 4128. Fax: 41 61 697 3976. E-mail: Witold.Filipowicz{at}fmi.ch.

dagger Present address: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520-8040.

Molecular and Cellular Biology, December 2000, p. 9028-9040, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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