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.

The synthesis of ribosomes involves many small nucleolar ribonucleoprotein particles (snoRNPs) as transacting factors. Yeast strains lacking the snoRNA, snR10, are viable but are impaired in growth and delayed in the early pre-rRNA cleavages at sites A 0 , A 1 , and A 2 , which lead to the synthesis of 18S rRNA. The same cleavages are inhibited by genetic depletion of the essential snoRNP protein Gar1p. Screens for mutations showing synthetic lethality with deletion of the SNR10 gene or with a temperature-sensitive gar1 allele both identified the ROK1 gene, encoding a putative, ATP-dependent RNA helicase of the DEAD-box family. The ROK1 gene is essential for viability, and depletion of Rok1p inhibits pre-rRNA processing at sites A 0 , A 1 , and A 2 , thereby blocking 18S rRNA synthesis. Indirect immunofluorescence by using a ProtA-Rok1p construct shows the protein to be predominantly nucleolar. These results suggest that Rok1p is required for the function of the snoRNP complex carrying out the early pre-rRNA cleavage reactions.Ribosome biogenesis in eukaryotes takes place largely in a specialized nuclear compartment, the nucleolus (reviewed in reference 43). Here, approximately 80 ribosomal proteins associate with the four mature rRNA molecules to form the large and small ribosomal subunits. Three of the four rRNAs (18S, 5.8S, and 25-28S rRNA) are produced from a single precursor (pre-rRNA), which, in addition to the mature rRNA sequences, contains two external transcribed spacers (ETS), the 5Ј ETS and 3Ј ETS, and two internal transcribed spacers (ITS), ITS1 and ITS2. During maturation of the pre-rRNA, the transcribed spacers are removed in a series of processing steps carried out by endonucleases and exonucleases (see references 13 and 64 for recent reviews).In yeast, the 35S pre-rRNA is cleaved at sites A 0 , A 1 , and A 2 , yielding the 20S pre-rRNA, which is subsequently converted into the mature 18S rRNA (Fig. 1). A large number of transacting factors that are required for the three early cleavages have been identified. The major class comprises the small nucleolar ribonucleoprotein particles (snoRNPs), each consisting of a small RNA molecule (snoRNA) associated with a set of proteins (42). The U3, U14, and snR30 snoRNAs and the snoRNP proteins Nop1p, Sof1p, and Gar1p are all essential for cleavage at sites A 0 , A 1 , and A 2 , while the absence of snR10 delays processing (5,21,28,30,38,44,59,61). Gar1p is associated with a large subset of the snoRNAs, including snR10 and snR30, which all contain common sequence motifs, most notably an ACA sequence located 3 nucleotides from the 3Ј end (4). Phenotypes observed on depletion of Gar1p and the snR30 snoRNA are very similar (21, 44), suggesting that the pre-rRNA processing defect observed on depletion of Gar1p is due mainly to inactivation of the snR30 snoRNP. Loss of snR10 gives a similar but weaker phenotype (59). The functions of U3 and U14 require direct base pairing of the RNA moiety with the pre-rRNA (5-7, 39), but the actual roles that the snoRNPs play i...

It is generally assumed that, in Saccharomyces cerevisiae, immature 40S ribosomal subunits are not competent for translation initiation. Here, we show by different approaches that, in wild-type conditions, a portion of pre-40S particles (pre-SSU) associate with translating ribosomal complexes. When cytoplasmic 20S pre-rRNA processing is impaired, as in Rio1p-or Nob1p-depleted cells, a large part of pre-SSUs is associated with translating ribosomes complexes. Loading of pre-40S particles onto mRNAs presumably uses the canonical pathway as translation-initiation factors interact with 20S pre-rRNA. However, translation initiation is not required for 40S ribosomal subunit maturation. We also provide evidence suggesting that cytoplasmic 20S pre-rRNAs that associate with translating complexes are turned over by the no go decay (NGD) pathway, a process known to degrade mRNAs on which ribosomes are stalled. We propose that the cytoplasmic fate of 20S pre-rRNA is determined by the balance between pre-SSU processing kinetics and sensing of ribosome-like particles loaded onto mRNAs by the NGD machinery, which acts as an ultimate ribosome quality check point.

Recent proteomic analyses are revealing the dynamics of preribosome assembly. Following cleavage at processing site A 2 , which generates the 20S pre-rRNA (the immediate precursor to the 18S rRNA), early RRPs (ribosomal RNA processing factors) are released in bulk from the preribosomes, and the resulting pre-40S subunits are left associated with a limited set of proteins that we refer to as the SSU RRP complex. Dim2p, a core constituent of the SSU RRP complex and conserved KH-domain containing protein, is required for pre-rRNA processing and is associated with early nucleolar and late cytoplasmic pre-rRNA species. Consistently, Dim2p shuttles between the nucle(ol)us and the cytoplasm, a trafficking that is tightly regulated by growth. The association of Dim2p with the 18S rRNA dimethyltransferase Dim1p, as well as its requirement for pre-rRNA processing at cleavage sites A 1 and A 2 and for 18S rRNA dimethylation, suggest that Dim2p may recruit Dim1p to nucleolar pre-rRNAs through its KH domain.

No abstract

Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs). Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudouridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis. To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains. We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases. Rrp8p localizes to the nucleolus. A yeast strain lacking this protein is viable at 30 8C but displays strong growth impairment at lower temperatures. In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed.