Nucleic Acids Research, 2001, Vol. 29, No. 5 1144-1155
© 2001 Oxford University Press
Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis
Département de Biochimie Médicale, CMU, 1 Rue Michel Servet, CH-1211 Genève 4, Switzerland
RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse–chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A1 and A2 are inhibited and delayed at site A0. Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.
* To whom correspondence should be addressed at present address: Equipe Épissage et Dégradation des ARNs Messagers Eucaryotes, Centre de Génétique Moléculaire, CNRS, Avenue de la Terrasse, F-91198 Gif sur Yvette Cedex, France. Tel: +33 1 69 82 38 83; Fax: +33 1 69 82 38 77; Email: daugeron{at}cgm.cnrs-gif.fr
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