Late Cytoplasmic Maturation of the Small Ribosomal Subunit Requires RIO Proteins in Saccharomyces cerevisiae

Abstract

Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

ACKNOWLEDGMENTS

We are grateful to J. Warner, Ed Hurt, and A. Tartakoff for providing us with antibodies, plasmids, and S. cerevisiae strains. We thank members of the Ferrer laboratory for helpful discussions and Y. Henry for critical reading of the manuscript. We also thank Y. de Préval for synthesis of oligonucleotides, D. Villa for the photographs, and A. Rivals for technical support.

This work was supported by the CNRS and the Université Paul Sabatier and grants from the Programme de Recherches Fondamentales en Microbiologie et Maladies Infectieuses et Parasitaires du Ministère de Education Nationale (MENRT) to M.C.F. E.V. was supported by grants from the MENRT and the Fondation pour la Recherche Médicale.