The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinitypurified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively.

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.Seen from a home economics point of view, making ribosomes is the main task of a living cell (36, 37). Synthesis of the ribosome constituents, their processing and assembly into mature particles, and the regulation of the entire process require the participation of numerous factors. Surprisingly, until relatively recently, few players involved in these processes had been identified. However, in the past decade, by means of genetic and biochemical tricks, and thanks to the tractability of the yeast Saccharomyces cerevisiae and, more recently, with in silico searches, dozens of factors (snoRNAs or protein factors) implicated in ribosome biogenesis have been characterized. For most of them, however, their molecular function in this process is not known.During the past year, systematic analyses of protein complexes in S. cerevisiae by tandem affinity chromatography purification (3,7,11,14,16) as well as proteomic analysis of the nucleolus of human cells (1) and definition of putative modular transcriptional networks by computer assisted analysis of the transcriptional expression patterns (18,20) led to a burst of new putative players. Through these approaches, new factors associated with the pre-60S particle, precursor of the large ribosomal subunit (LSU) and thus putatively involved in the maturation-assembly pathway of the LSU, have been identified. Likewise, factors associated with the early processing complexes containing the large 35S pre-rRNA have been characterized (11) and shown to include mostly proteins specifically required for small ribosomal subunit (SSU) production. In contrast, the nonribosomal proteins found in late nucleoprotein complex precursors of the SSU have not been ide...

Eukaryotic rRNAs possess numerous post-transcriptionally modified nucleotides. The most abundant modifications, 2'-O-ribose methylation and pseudouridylation, occur in the nucleolus during rRNA processing. The nucleolus contains a large number of small nucleolar RNAs (snoRNAs) most of which can be classified into two distinct families defined by conserved sequence boxes and common associated proteins. The C and D box-containing snoRNAs are associated with fibrillarin, and most of them function as guide RNAs in site-specific ribose methylation of rRNAs. The nucleolar function of the other class of snoRNAs, which share box H and ACA elements and are associated with a glycine- and arginine-rich nucleolar protein, Gar1p, remains elusive. Here we demonstrate that the yeast Saccharomyces cerevisiae Gar1 snoRNP protein plays an essential and specific role in the overall pseudouridylation of yeast rRNAs. These results establish a novel function for Gar1 protein and indicate that the box H/ACA snoRNAs, or at least a subset of these snoRNAs, function in the site-specific pseudouridylation of rRNAs.