Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein

Numerous non-ribosomal trans-acting factors involved in pre-ribosomal RNA processing have been characterized, but none of them is specifically required for the last cytoplasmic steps of 18S rRNA maturation. Here we demonstrate that Rio1p/Rrp10p is such a factor. Previous studies showed that the RIO1 gene is essential for cell viability and conserved from archaebacteria to man. We isolated a RIO1 mutant in a screen for mutations synthetically lethal with a mutant allele of GAR1, an essential gene required for 18S rRNA production and rRNA pseudouridylation. We show that RIO1 encodes a cytoplasmic non-ribosomal protein, and that depletion of Rio1p blocks 18S rRNA production leading to 20S pre-rRNA accumulation. In situ hybridization reveals that, in Rio1p depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. This strongly suggests that Rio1p is involved in the cytoplasmic cleavage of 20S pre-rRNA at site D, producing mature 18S rRNA. Thus, Rio1p has been renamed Rrp10p (ribosomal RNA processing #10). Rio1p/Rrp10p is the first non-ribosomal factor characterized specifically required for 20S pre-rRNA processing.     

Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae

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.     

Bypassing the rRNA processing endonucleolytic cleavage at site A2 in Saccharomyces cerevisiae

Rrp5p is the only ribosomal RNA processing trans-acting factor that is required for the synthesis of both 18S and 5.8S rRNAs in Saccharomyces cerevisiae. Mutational analyses have characterized modified forms of Rrp5p that either affect formation of 18S rRNA by inhibiting cleavage at sites A0/A1/A2, or synthesis of 5.8S rRNA by inhibiting cleavage at site A3. Here, we examine the rRNA maturation process associated with a RRP5 bipartite allele that codes for two noncontiguous parts of the protein. This slow-growing bipartite mutant has a unique rRNA-processing phenotype that proceeds without endonucleolytic cleavage at site A2. In wild-type cells, the A2 cleavage takes place on the 32S pre-rRNA and is responsible for the formation of 20S and 27SA2 species, the precursors of mature 18S and 5.8S/25S rRNAs, respectively. In the bipartite strain, such precursors were not detectable as judged by Northern analysis or in vivo labeling. They were replaced by the aberrant 21S species and the bypassing 27SA3 precursor, both descended from direct cleavage of 32S pre-rRNA at site A3, which provides an alternative rRNA maturation pathway in this strain. The 21S pre-rRNA is the sole detectable and most likely available precursor of 18S rRNA in this particular strain, indicating that 18S rRNA can be directly produced from 21S. Furthermore, 21S species were found associated with 43S preribosomal particles as similarly observed for the 20S pre-rRNA in the wild-type cells.     

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.     

Deletions in the S1 domain of Rrp5p cause processing at a novel site in ITS1 of yeast pre-rRNA that depends on Rex4p

Rrp5p is the only protein so far known to be required for the processing of yeast pre-rRNA at both the early sites A0, A1 and A2 leading to 18S rRNA and at site A3, the first step specific for the pathway leading to 5.8S/25S rRNA. Previous in vivo mutational analysis of Rrp5p demonstrated that the first 8 of its 12 S1 RNA-binding motifs are involved in the formation of the 'short' form of 5.8S rRNA (5.8S(S)), which is the predominant species under normal conditions. We have constructed two strains in which the genomic RRP5 gene has been replaced by an rrp5 deletion mutant lacking either S1 motifs 3-5 (rrp5-Delta3) or 5-8 (rrp5-Delta4). The first mutant synthesizes almost exclusively 5.8S(L) rRNA, whereas the second one still produces a considerable amount of the 5.8S(S) species. Nevertheless, both mutations were found to block cleavage at site A3 completely. Instead, a novel processing event occurs at a site in a conserved stem-loop structure located between sites A2 and A3, which we have named A4. A synthetic lethality screen using the rrp5-Delta3 and rrp-Delta4 mutations identified the REX4 gene, which encodes a non-essential protein belonging to a class of related yeast proteins that includes several known 3'-->5' exonucleases. Inactivation of the REX4 gene in rrp5-Delta3 or rrp-Delta4 cells abolished cleavage at A4, restored cleavage at A3 and returned the 5.8S(S):5.8S(L) ratio to the wild-type value. The sl phenotype of the rrp5Delta/rex4(-) double mutants appears to be due to a severe disturbance in ribosomal subunit assembly, rather than pre-rRNA processing. The data provide direct evidence for a crucial role of the multiple S1 motifs of Rrp5p in ensuring the correct assembly and action of the processing complex responsible for cleavage at site A3. Furthermore, they clearly implicate Rex4p in both pre-rRNA processing and ribosome assembly, even though this protein is not essential for yeast.     

RRP5 is required for formation of both 18S and 5.8S rRNA in yeast.

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S) are synthesized as a single precursor which is subsequently processed into the mature rRNAs by a complex series of cleavage and modification reactions. In the yeast Saccharomyces cerevisiae, the early pre-rRNA cleavages at sites A0, A1 and A2, required for the synthesis of 18S rRNA, are inhibited in strains lacking RNA or protein components of the U3, U14, snR10 and snR30 small nucleolar ribonucleoproteins (snoRNPs). The subsequent cleavage at site A3, required for formation of the major, short form of 5.8S rRNA, is carried out by another ribonucleoprotein, RNase MRP. A screen for mutations showing synthetic lethality with deletion of the non-essential snoRNA, snR10, identified a novel gene, RRP5, which is essential for viability and encodes a 193 kDa nucleolar protein. Genetic depletion of Rrp5p inhibits the synthesis of 18S rRNA and, unexpectedly, also of the major short form of 5.8S rRNA. Pre-rRNA processing is concomitantly impaired at sites A0, A1, A2 and A3. This distinctive phenotype makes Rrp5p the first cellular component simultaneously required for the snoRNP-dependent cleavage at sites A0, A1 and A2 and the RNase MRP-dependent cleavage at A3 and provides evidence for a close interconnection between these processing events. Putative RRP5 homologues from Caenorhabditis elegans and humans were also identified, suggesting that the critical function of Rrp5p is evolutionarily conserved.     

Deletion of the three distal S1 motifs of Saccharomyces cerevisiae Rrp5p abolishes pre-rRNA processing at site A(2) without reducing the production of functional 40S subunits

Yeast Rrp5p, one of the few trans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure. Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A(0) to A(2) and thus for the formation of 18S rRNA. The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A(3) and thus for the production of the short form of 5.8S rRNA. Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutant rrp5Delta6) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis. Here we show that, nevertheless, in the rrp5Delta6 mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A(2). Instead, the 32S precursor is processed directly at site A(3), producing exclusively 21S rather than 20S pre-rRNA. This is the first evidence that the 21S precursor, which was observed previously only in cells showing a substantial growth defect or as a minor species in addition to the normal 20S precursor, is an efficient substrate for 18S rRNA synthesis. Maturation of the 21S precursor occurs via the same endonucleolytic cleavage at site D as that used for 20S pre-rRNA maturation. The resulting D-A(3) fragment, however, is degraded by both 5'-->3' and 3'-->5' exonuclease digestions, the latter involving the exosome, in contrast to the exclusively 5'-->3' exonucleolytic digestion of the D-A(2) fragment. We also show that rrp5Delta6 cells are hypersensitive to both hygromycin B and cycloheximide, suggesting that, despite their wild-type growth rate, their preribosomes or ribosomes may be structurally abnormal.     

Dim2p, a KH-domain protein required for small ribosomal subunit synthesis

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.     

Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2

During their biogenesis, 40S ribosomal subunit precursors are exported from the nucleus to the cytoplasm, where final maturation occurs. In this study, we show that the protein kinase human Rio2 (hRio2) is part of a late 40S preribosomal particle in human cells. Using a novel 40S biogenesis and export assay, we analyzed the contribution of hRio2 to late 40S maturation. Although hRio2 is not absolutely required for pre-40S export, deletion of its binding site for the export receptor CRM1 decelerated the kinetics of this process. Moreover, in the absence of hRio2, final cytoplasmic 40S maturation is blocked because the recycling of several trans-acting factors and cytoplasmic 18S-E precursor ribosomal RNA (rRNA [pre-rRNA]) processing are defective. Intriguingly, the physical presence of hRio2 but not its kinase activity is necessary for the release of hEnp1 from cytoplasmic 40S precursors. In contrast, hRio2 kinase activity is essential for the recycling of hDim2, hLtv1, and hNob1 as well as for 18S-E pre-rRNA processing. Thus, hRio2 is involved in late 40S maturation at several distinct steps.     

The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA.

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.     

The roles of S1 RNA-binding domains in Rrp5's interactions with pre-rRNA

RNA-binding proteins mediate the function of all RNAs. Since few distinct RNA-binding domains (RBDs) exist, with most RBDs contacting only a few nucleotides, RNA-binding proteins often combine multiple RNA-binding motifs to achieve a higher affinity and selectivity for their targets. Rrp5, a ribosome assembly factor essential for both 40S and 60S ribosome maturation, is an extreme example as it contains 12 tandem S1 RNA-binding domains. In this study, we use a combination of RNA binding and DMS probing experiments to probe interactions of Rrp5 with pre-rRNA mimics. Our data localize Rrp5's binding site to three distinct regions within internal transcribed spacer 1 (ITS1), the sequence between 18S and 5.8S rRNAs. One of these regions is directly adjacent to a recently uncovered helical structure, which prevents premature cleavage at the 3'-end of 18S rRNA. This finding, together with previous results, suggests a role for Rrp5 in regulating the above-mentioned helical element. Furthermore, we have produced two truncated forms of the protein, Rrp5N and Rrp5C, which together encompass the entire protein and fully restore growth. Quantitative analysis of the RNA affinity of these Rrp5 fragments indicates that the first nine S1 motifs contribute much of Rrp5's RNA affinity, while the last three domains alone provide its specificity for the pre-rRNA. This surprising division of labor is unique, as it suggests that S1 domains can bind RNA both specifically as well as nonspecifically with high affinity; this has important implications for the molecular details of the Rrp5?pre-rRNA complex.     

Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p

NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001–5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3′-to-5′ exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.     

Functional analysis of Rrp7p, an essential yeast protein involved in pre-rRNA processing and ribosome assembly.

During the functional analysis of open reading frames (ORFs) identified during the sequencing of chromosome III of Saccharomyces cerevisiae, the previously uncharacterized ORF YCL031C (now designated RRP7) was deleted. RRP7 is essential for cell viability, and a conditional null allele was therefore constructed, by placing its expression under the control of a regulated GAL promoter. Genetic depletion of Rrp7p inhibited the pre-rRNA processing steps that lead to the production of the 20S pre-rRNA, resulting in reduced synthesis of the 18S rRNA and a reduced ratio of 40S to 60S ribosomal subunits. A screen for multicopy suppressors of the lethality of the GAL::rrp7 allele isolated the two genes encoding a previously unidentified ribosomal protein (r-protein) that is highly homologous to the rat r-protein S27. When present in multiple copies, either gene can suppress the lethality of an RRP7 deletion mutation and can partially restore the ribosomal subunit ratio in Rrp7p-depleted cells. Deletion of both r-protein genes is lethal; deletion of either single gene has an effect on pre-rRNA processing similar to that of Rrp7p depletion. We believe that Rrp7p is required for correct assembly of rpS27 into the preribosomal particle, with the inhibition of pre-rRNA processing appearing as a consequence of this defect.     

Rok1p is a putative RNA helicase required for rRNA processing.

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 A0, A1, and A2, 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 A0, A1, and A2, 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.     

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

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 pseudo-uridines 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 degrees C 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.     

The putative NTPase Fap7 mediates cytoplasmic 20S pre-rRNA processing through a direct interaction with Rps14

One of the proteins identified as being involved in ribosome biogenesis by high-throughput studies, a putative P-loop-type kinase termed Fap7 (YDL166c), was shown to be required for the conversion of 20S pre-rRNA to 18S rRNA. However, the mechanism underlying this function has remained unclear. Here we demonstrate that Fap7 is strictly required for cleavage of the 20S pre-rRNA at site D in the cytoplasm. Genetic depletion of Fap7 causes accumulation of only the 20S pre-rRNA, which could be detected not only in 43S preribosomes but also in 80S-sized complexes. Fap7 is not a structural component of 43S preribosomes but likely transiently interacts with them by directly binding to Rps14, a ribosomal protein that is found near the 3' end of the 18S rRNA. Consistent with an NTPase activity, conserved residues predicted to be required for nucleoside triphosphate (NTP) hydrolysis are essential for Fap7 function in vivo. We propose that Fap7 mediates cleavage of the 20S pre-rRNA at site D by directly interacting with Rps14 and speculate that it is an enzyme that functions as an NTP-dependent molecular switch in 18S rRNA maturation.     

The roles of Rrp5p in the synthesis of yeast 18S and 5.8S rRNA can be functionally and physically separated.

The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8Ss) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8Ss rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.     

Slx9p facilitates efficient ITS1 processing of pre-rRNA in Saccharomyces cerevisiae

Slx9p (Ygr081cp) is a nonessential yeast protein previously linked genetically with the DNA helicase Sgs1p. Here we report that Slx9p is involved in ribosome biogenesis in the yeast Saccharomyces cerevisiae. Deletion of SLX9 results in a mild growth defect and a reduction in the level of 18S rRNA. Co-immunoprecipitation experiments showed that Slx9p is associated with 35S, 23S, and 20S pre-rRNA, as well as U3 snoRNA and, thus, is a bona fide component of pre-ribosomes. The most striking effects on pre-rRNA processing resulting from deletion of SLX9 is the accumulation of the mutually exclusive 21S and 27SA2 pre-rRNA. Furthermore, deletion of SLX9 is synthetically lethal with mutations in Rrp5p that block cleavage at either site A2 or A3. We conclude that Slx9p has a unique role in the processing events responsible for separating the 66S and 43S pre-ribosomal particles. Interestingly, homologs of Slx9p were found only in other yeast species, indicating that the protein has been considerably less well conserved during evolution than the majority of trans-acting processing factors.     

Yeast Rrp14p is required for ribosomal subunit synthesis and for correct positioning of the mitotic spindle during mitosis

Here we report that Rrp14p/Ykl082p is associated with pre-60S particles and to a lesser extent with earlier 90S pre-ribosomes. Depletion of Rrp14p inhibited pre-rRNA synthesis on both the 40S and 60S synthesis pathways. Synthesis of the 20S precursor to the 18S rRNA was largely blocked, as was maturation of the 27SB pre-rRNA to the 5.8S and 25S rRNAs. Unexpectedly, Rrp14p-depleted cells also showed apparently specific cell-cycle defects. Following release from synchronization in S phase, Rrp14p-depleted cells uniformly arrested in metaphase with short mitotic spindles that were frequently incorrectly aligned with the site of bud formation. In the absence of Bub2p, which is required for the spindle orientation checkpoint, this metaphase arrest was not seen in Rrp14p-depleted cells, which then arrested with multiple buds, several SPBs and binucleate mother cells. These data suggest that Rrp14p may play some role in cell polarity and/or spindle positioning, in addition to its function in ribosome synthesis.