Abstract
During rRNA biogenesis, multiple RNA and protein substrates are modified and assembled through the coordinated activity of many factors. In Saccharomyces cerevisiae, the double-stranded RNA nuclease Rnt1p and the H/ACA snoRNA pseudouridylase complex participate in the transformation of the nascent pre-rRNA transcript into 35S pre-rRNA. Here we demonstrate the binding of a component of the H/ACA complex (Gar1p) to Rnt1p in vivo and in vitro in the absence of other factors. In vitro, Rnt1p binding to Gar1p is mutually exclusive of its RNA binding and cleavage activities. Mutations in Rnt1p that disrupt Gar1p binding do not inhibit RNA cleavage in vitro but slow RNA processing, prevent nucleolar localization of H/ACA snoRNA-associated proteins, and reduce pre-rRNA pseudouridylation in vivo. These results demonstrate colocalization of various components of the rRNA maturation complex and suggest a mechanism that links rRNA pseudouridylation and cleavage factors.
We thank J. Carbon, S. Fournier, J. Warner, J. Aris, J. P. Gélugne, P. James, S. Labbé, D. Thiele, and S. J. Elledge for sending plasmids, strains, and antibodies. We are indebted to B. Chabot, R. Wellinger, and all the members of the laboratory of Sherif Abou Elela for critical reading of the manuscript.
This work was supported by grant 216854 from the Natural Sciences and Engineering Research Council of Canada (NSERC) and grant MOP-14305 from the Canadian Institute for Health Research (CIHR). Support for the RNA group core was provided by CIHR. S.A.E. is Chercheur-Boursier Junior II of the Fonds de la Recherche en Santé du Québec.