Dynamics of the putative RNA helicase Spb4 during ribosome assembly in Saccharomyces cerevisiae

Spb4 is a putative ATP-dependent RNA helicase that is required for proper processing of 27SB pre-rRNAs and therefore for 60S ribosomal subunit biogenesis. To define the timing of association of this protein with preribosomal particles, we have studied the composition of complexes that copurify with Spb4 tagged by tandem affinity purification (TAP-tagged Spb4). These complexes contain mainly the 27SB pre-rRNAs and about 50 ribosome biogenesis proteins, primarily components of early pre-60S ribosomal particles. To a lesser extent, some protein factors of 90S preribosomal particles and the 35S and 27SA pre-rRNAs also copurify with TAP-tagged Spb4. Moreover, we have obtained by site-directed mutagenesis an allele that results in the R360A substitution in the conserved motif VI of the Spb4 helicase domain. This allele causes a dominant-negative phenotype when overexpressed in the wild-type strain. Cells expressing Spb4(R360A) display an accumulation of 35S and 27SB pre-rRNAs and a net 40S ribosomal subunit defect. TAP-tagged Spb4(R360A) displays a greater steady-state association with 90S preribosomal particles than TAP-tagged wild-type Spb4. Together, our data indicate that Spb4 is a component of early nucle(ol)ar pre-60S ribosomal particles containing 27SB pre-rRNA. Apparently, Spb4 binds 90S preribosomal particles and dissociates from pre-60S ribosomal particles after processing of 27SB pre-rRNA.     

Spb1p is a putative methyltransferase required for 60S ribosomal subunit biogenesis in Saccharomyces cerevisiae.

Several mutants ( spb1 - spb7 ) have been previously identified as cold-sensitive extragenic suppressors of loss-of-function mutations in the poly(A)(+)-binding protein 1 of Saccharomyces cerevisiae. Cloning, sequence and disruption analyses revealed that SPB1 (YCL054W) encodes an essential putative S -adenosylmethionine-dependent methyltransferase. Polysome analyses showed an under-accumulation of 60S ribosomal subunits in the spb1-1 mutant and in a strain genetically depleted of Spb1p. Northern and primer extension analyses indicated that this was due to inhibition of processing of the 27SB precursors, which results in depletion of the mature 25S and 5.8S rRNAs. At later time points of Spb1p depletion, the stability of 40S ribosomal subunits is also affected. These results suggest that Spb1p is involved in 60S ribosomal subunit biogenesis and associates early with the pre-ribosomes. Consistent with this, hemagglutinin epitope-tagged Spb1p localizes to the nucleus with nucleolar enrichment. Despite the expected methyltransferase activity of Spb1p, global methylation of pre-rRNA is not affected upon Spb1p depletion. We propose that Spb1p is required for proper assembly of pre-ribosomal particles during the biogenesis of 60S ribosomal subunits.     

Has1p, a member of the DEAD-box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae

The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre-60S ribosomal complexes. Here, we show that Has1p is an essential trans-acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature-sensitive has1-1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre-rRNA processing by pulse-chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre-rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.     

Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation

The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA(3) to 27SB(S) pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA(3) and 27SB(L) pre-ribosomal RNAs.     

The essential WD-repeat protein Rsa4p is required for rRNA processing and intra-nuclear transport of 60S ribosomal subunits

We report the characterization of a novel factor, Rsa4p (Ycr072cp), which is essential for the synthesis of 60S ribosomal subunits. Rsa4p is a conserved WD-repeat protein that seems to localize in the nucleolus. In vivo depletion of Rsa4p results in a deficit of 60S ribosomal subunits and the appearance of half-mer polysomes. Northern hybridization and primer extension analyses of pre-rRNA and mature rRNAs show that depletion of Rsa4p leads to the accumulation of the 27S, 25.5S and 7S pre-rRNAs, resulting in a reduction of the mature 25S and 5.8S rRNAs. Pulse–chase analyses of pre-rRNA processing reveal that, at least, this is due to a strong delay in the maturation of 27S pre-rRNA intermediates to mature 25S rRNA. Furthermore, depletion of Rsa4p inhibited the release of the pre-60S ribosomal particles from the nucleolus to the nucleoplasm, as judged by the predominantly nucleolar accumulation of the large subunit Rpl25-eGFP reporter construct. We propose that Rsa4p associates early with pre-60S ribosomal particles and provides a platform of interaction for correct processing of rRNA precursors and nucleolar release of 60S ribosomal subunits.     

Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro

We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis. A cold-sensitive allele for that gene (referred to here as spb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here as spb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae. The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content. In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated. Spb1p is a 96.5-kDa protein that is localized to the nucleolus. Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p. Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-L-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases. We show here that Spb1p is able to bind [(3)H]AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.     

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.     

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.     

Ssf1p prevents premature processing of an early pre-60S ribosomal particle

Ssf1p and Ssf2p are two nearly identical and functionally redundant nucleolar proteins. In the absence of Ssf1p and Ssf2p, the 27SA(2) pre-rRNA was prematurely cleaved, inhibiting synthesis of the 27SB and 7S pre-rRNAs and the 5.8S and 25S rRNA components of the large ribosomal subunit. On sucrose gradients, Ssf1p sedimented with pre-60S ribosomal particles. The 27SA(2), 27SA(3), and 27SB pre-rRNAs were copurified with tagged Ssf1p, as were 23 large subunit ribosomal proteins and 21 other proteins implicated in ribosome biogenesis. These included four Brix family proteins, Ssf1p, Rpf1p, Rpf2p, and Brx1p, indicating that the entire family functions in ribosome synthesis. This complex is distinct from recently reported pre-60S complexes in RNA and protein composition. We describe a multistep pathway of 60S preribosome maturation.     

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA(3) to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA(3) pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A(3) assembly factors and for proper assembly of the neighborhoods containing domains I and II.     

Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis

Ribosome biogenesis requires, in addition to rRNA molecules and ribosomal proteins, a multitude of trans-acting factors. Recently it has become clear that in the yeast Saccharomyces cerevisiae many RNA helicases of the DEAD-box and related families are involved in ribosome biogenesis. Here we show that the previously uncharacterised open reading frame YDL031w (renamed DBP10 for DEAD-box protein 10) encodes an essential putative RNA helicase that is required for accurate ribosome biogenesis. Genetic depletion of Dbp10p results in a deficit in 60S ribosomal subunits and an accumulation of half-mer polysomes. Furthermore, pulse-chase analyses of pre-rRNA processing reveal a strong delay in the maturation of 27SB pre-rRNA intermediates into 25S rRNA and 7S pre-rRNA. Northern blot analyses indicate that this delay leads to higher steady-state levels of 27SB species and reduced steady-state levels of 7S pre-rRNA and 25S/5.8S mature rRNAs, thus explaining the final deficit in 60S subunit and the formation of half-mer polysomes. Consistent with a direct role in ribosome biogenesis, Dbp10p was found to be located predominantly in the nucleolus.     

Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C₂.     

Rpf2p,an evolutionarily conserved protein,interacts with ribosomal protein L11 and is essential for the processing of 27 SB Pre-rRNA to 25 S rRNA and the 60 S ribosomal subunit assembly in Saccharomyces cerevisiae

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p is essential for growth and is mainly localized in the nucleolus. The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly. Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA.     

Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway.

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.     

Saccharomyces cerevisiae nucleolar protein Nop7p is necessary for biogenesis of 60S ribosomal subunits

To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A.     

The splicing factor Prp43p, a DEAH box ATPase, functions in ribosome biogenesis

Biogenesis of the small and large ribosomal subunits requires modification, processing, and folding of pre-rRNA to yield mature rRNA. Here, we report that efficient biogenesis of both small- and large-subunit rRNAs requires the DEAH box ATPase Prp43p, a pre-mRNA splicing factor. By steady-state analysis, a cold-sensitive prp43 mutant accumulates 35S pre-rRNA and depletes 20S, 27S, and 7S pre-rRNAs, precursors to the small- and large-subunit rRNAs. By pulse-chase analysis, the prp43 mutant is defective in the formation of 20S and 27S pre-rRNAs and in the accumulation of 18S and 25S mature rRNAs. Wild-type Prp43p immunoprecipitates pre-rRNAs and mature rRNAs, indicating a direct role in ribosome biogenesis. The Prp43p-Q423N mutant immunoprecipitates 27SA2 pre-rRNA threefold more efficiently than the wild type, suggesting a critical role for Prp43p at the earliest stages of large-subunit biogenesis. Consistent with an early role for Prp43p in ribosome biogenesis, Prp43p immunoprecipitates the majority of snoRNAs; further, compared to the wild type, the prp43 mutant generally immunoprecipitates the snoRNAs more efficiently. In the prp43 mutant, the snoRNA snR64 fails to methylate residue C2337 in 27S pre-rRNA, suggesting a role in snoRNA function. We propose that Prp43p promotes recycling of snoRNAs and biogenesis factors during pre-rRNA processing, similar to its recycling role in pre-mRNA splicing. The dual function for Prp43p in the cell raises the possibility that ribosome biogenesis and pre-mRNA splicing may be coordinately regulated.     

ERB1, the yeast homolog of mammalian Bop1, is an essential gene required for maturation of the 25S and 5.8S ribosomal RNAs

We have recently shown that the mammalian nucleolar protein Bop1 is involved in synthesis of the 28S and 5.8S ribosomal RNAs (rRNAs) and large ribosome subunits in mouse cells. Here we have investigated the functions of the Saccharomyces cerevisiae homolog of Bop1, Erb1p, encoded by the previously uncharacterized open reading frame YMR049C. Gene disruption showed that ERB1 is essential for viability. Depletion of Erb1p resulted in a loss of 25S and 5.8S rRNAs synthesis, while causing only a moderate reduction and not a complete block in 18S rRNA formation. Processing analysis showed that Erb1p is required for synthesis of 7S pre-rRNA and mature 25S rRNA from 27SB pre-rRNA. In Erb1p-depleted cells these products of 27SB processing are largely absent and 27SB pre-rRNA is under-accumulated, apparently due to degradation. In addition, depletion of Erb1p caused delayed processing of the 35S pre-rRNA. These findings demonstrate that Erb1p, like its mammalian counterpart Bop1, is required for formation of rRNA components of the large ribosome particles. The similarities in processing defects caused by functional disruption of Erb1p and Bop1 suggest that late steps in maturation of the large ribosome subunit rRNAs employ mechanisms that are evolutionarily conserved throughout eukaryotes.     

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.     

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

Ribosome biogenesis requires >100 nonribosomal proteins, which are associated with different preribosomal particles. The substrates, the interacting partners, and the timing of action of most of these proteins are largely unknown. To elucidate the functional environment of the putative ATP-dependent RNA helicase Dbp6p from Saccharomyces cerevisiae, which is required for 60S ribosomal subunit assembly, we have previously performed a synthetic lethal screen and thereby revealed a genetic interaction network between Dbp6p, Rpl3p, Nop8p, and the novel Rsa3p. In this report, we extended the characterization of this functional network by performing a synthetic lethal screen with the rsa3 null allele. This screen identified the so far uncharacterized Npa1p (YKL014C). Polysome profile analysis indicates that there is a deficit of 60S ribosomal subunits and an accumulation of halfmer polysomes in the slowly growing npa1-1 mutant. Northern blotting and primer extension analysis shows that the npa1-1 mutation negatively affects processing of all 27S pre-rRNAs and the normal accumulation of both mature 25S and 5.8S rRNAs. In addition, 27SA(2) pre-rRNA is prematurely cleaved at site C(2). Moreover, GFP-tagged Npa1p localizes predominantly to the nucleolus and sediments with large complexes in sucrose gradients, which most likely correspond to pre-60S ribosomal particles. We conclude that Npa1p is required for ribosome biogenesis and operates in the same functional environment of Rsa3p and Dbp6p during early maturation of 60S ribosomal subunits.     

Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly.

Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit.