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

Abstract

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.

ACKNOWLEDGMENTS

We thank A. Henras, D. Robyr, and S. Buratowski for advice on ChIP experiments; K. Sakurai for help and support; and A. Henras for critical reading of the manuscript. We thank Michèle Caizergues-Ferrer for fruitful discussion and Christophe Dez for assistance with TAP tag purification.

P.K.Y. was supported by a UCLA dissertation year fellowship, C.H. was supported by a Ph.D. fellowship from the Ministère Délégué à la Recherche et aux Nouvelles Technologies. This research was supported by NIH grant GM61518 (G.C.); the CNRS, Université Paul Sabatier, and La Ligue Nationale contre le Cancer (Equipe Labelisée) (Y.H.); and the Région Midi-Pyrénées and the Génopole Toulouse Midi-Pyrénées (B.M.).