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Molecular and Cellular Biology, April 2005, p. 3295-3304, Vol. 25, No. 8
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.8.3295-3304.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cotranscriptional Recruitment of the Pseudouridylsynthetase Cbf5p and of the RNA Binding Protein Naf1p during H/ACA snoRNP Assembly

Pok Kwan Yang,1 Coralie Hoareau,2 Carine Froment,3 Bernard Monsarrat,3 Yves Henry,2 and Guillaume Chanfreau1*

Department of Chemistry and Biochemistry and Molecular Biology Institute, UCLA, Los Angeles, California,1 Laboratoire de Biologie Moléculaire Eucaryote, UMR5099 CNRS-Université Paul Sabatier,2 Plate-forme protéomique, Institut de Pharmacologie et de Biologie Structurale (CNRS UMR 5089), Toulouse, France3

Received 13 December 2004/ Returned for modification 14 January 2005/ Accepted 19 January 2005

H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) are essential for the maturation and pseudouridylation of the precursor of rRNAs and other stable RNAs. Although the RNA and protein components of these RNPs have been identified, the mechanisms by which they are assembled in vivo are poorly understood. Here we show that the RNA binding protein Naf1p, which is required for H/ACA snoRNPs stability, associates with RNA polymerase II-associated proteins Spt16p, Tfg1p, and Sub1p and with H/ACA snoRNP proteins. Chromatin immunoprecipitation experiments show that Naf1p and the pseudouridylsynthetase Cbf5p cross-link specifically with the chromatin of H/ACA small nucleolar RNA (snoRNA) genes. Naf1p and Cbf5p cross-link predominantly with the 3' end of these genes, in a pattern similar to that observed for transcription elongation factor Spt16p. Cross-linking of Naf1p to H/ACA snoRNA genes requires active transcription and intact H/ACA snoRNA sequences but does not require the RNA polymerase II CTD kinase Ctk1p. These results suggest that Naf1p and Cbf5p are recruited in a cotranscriptional manner during H/ACA snoRNP assembly, possibly by binding to the nascent H/ACA snoRNA transcript during elongation or termination of transcription of H/ACA snoRNA genes.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry and Molecular Biology Institute, UCLA, Box 951569, Los Angeles, CA 90095-1569. Phone: (310) 825-4399. Fax: (310) 206-4038. E-mail: guillom{at}chem.ucla.edu.

Molecular and Cellular Biology, April 2005, p. 3295-3304, Vol. 25, No. 8
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.8.3295-3304.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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