The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles

Ribosome biogenesis takes place successively in the nucleolar, nucleoplasmic, and cytoplasmic compartments. Numerous nonribosomal factors transiently associate with the nascent ribosomes, but the mechanisms driving ribosome formation are mostly unknown. Here, we show that an energy-consuming enzyme, the AAA-type (ATPases associated with various cellular activities) ATPase Rix7, restructures a novel pre-60S particle at the transition from the nucleolus to nucleoplasm. Rix7 interacts genetically with Nsa1 and is targeted to the Nsa1-defined preribosomal particle. In vivo, Nsa1 cannot dissociate from pre-60S particles in rix7 mutants, causing nucleolar Nsa1 to escape to the cytoplasm, where it remains associated with aberrant 60S subunits. Altogether, our data suggest that Rix7 is required for the release of Nsa1 from a discrete preribosomal particle, thereby triggering the progression of 60S ribosome biogenesis.     

The p21-activated protein kinase inhibitor Skb15 and its budding yeast homologue are 60S ribosome assembly factors

Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G(1), and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.     

Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNA processing and nuclear export of 60 S subunits

Rea1, the largest predicted protein in the yeast genome, is a member of the AAA(+) family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.     

Cic1p/Nsa3p is required for synthesis and nuclear export of 60S ribosomal subunits

Cic1p/Nsa3p was previously reported to be associated with the 26S proteasome and required for the degradation of specific substrates, but was also shown to be associated with early pre-60S particles and to be localized to the nucleolus. Here we report that Cic1p/Nsa3p is required for the synthesis of 60S ribosome subunits. A temperature-sensitive lethal cic1-2 point mutation inhibits synthesis of the mature 5.8S and 25S rRNAs. Release of the pre-60S particles from the nucleolus to the nucleoplasm was also inhibited as judged by the nuclear accumulation of an Rpl11b-GFP reporter construct. We suggest that Cic1p/Nsa3p associates early with nascent preribosomal particles and is required for correct processing and nuclear release of large ribosomal subunit precursors.     

Yeast Pescadillo is required for multiple activities during 60S ribosomal subunit synthesis

The Pescadillo protein was identified via a developmental defect and implicated in cell cycle progression. Here we report that human Pescadillo and its yeast homolog (Yph1p or Nop7p) are localized to the nucleolus. Depletion of Nop7p leads to nuclear accumulation of pre-60S particles, indicating a defect in subunit export, and it interacts genetically with a tagged form of the ribosomal protein Rpl25p, consistent with a role in subunit assembly. Two pre-rRNA processing pathways generate alternative forms of the 5.8S rRNA, designated 5.8S(L) and 5.8Ss. In cells depleted for Nop7p, the 27SA3 pre-rRNA accumulated, whereas later processing intermediates and the mature 5.8Ss rRNA were depleted. Less depletion was seen for the 5.8S(L) pathway. TAP-tagged Nop7p coprecipitated precursors to both 5.8S(L) and 5.8Ss but not the mature rRNAs. We conclude that Nop7p is required for efficient exonucleolytic processing of the 27SA3 pre-rRNA and has additional functions in 60S subunit assembly and transport. Nop7p is a component of at least three different pre-60S particles, and we propose that it carries out distinct functions in each of these complexes.     

Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C₁ and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7^Lb within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.     

The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus.     

The ribosome assembly factor Nop53 has a structural role in the formation of nuclear pre-60S intermediates,affecting late maturation events

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S ‘foot’. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.     

Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.     

Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis

Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied trans-acting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA₃, 27SB_S and 7S_L/S precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.     

The Cytosolic J-protein, Jjj1, and Rei1 Function in the Removal of the Pre-60 S Subunit Factor Arx1

Although the biogenesis of ribosomal subunits occurs predominantly in the nucleus, final remodeling steps take place in the cytosol. One cytosolic step has two components: 1) the removal of the maturation factor Arx1, which transits from the nucleus to the cytosol with the pre-60 S subunit, and 2) its subsequent transport back into the nucleus. Two cytosolic proteins, Rei1 and Jjj1, are required, but their individual contributions to this step are not understood. Here we report that Rei1 and Jjj1 directly interact. This interaction is mediated by a C-terminal segment of Jjj1 encompassing a region rich in charged residues, flanked by C₂H₂-type zinc fingers. Deletion of the charged region results in defects in 60 S subunit biogenesis in vivo. In addition, we report resolution of an apparent contradiction in the literature regarding the association of Arx1 with the pre-60 S subunit in the absence of Rei1. The association of Arx1 with ribosomes is sensitive to the concentration of magnesium ions when Rei1 is absent. At near physiological concentrations, Arx1 remains associated with the pre-60 S particle, as it does in the absence of Jjj1; at higher concentrations, Arx1 dissociates in the absence of Rei1 but not in the absence of Jjj1. As both Rei1 and Jjj1 are required for dissociation of Arx1 from the pre-60 S subunit, and the region of Jjj1 that mediates interaction with Rei1 is required in vivo for 60 S subunit biogenesis, our data support the idea that the primary role of both Rei1 and Jjj1 is the first step of the Arx1 removal/recycling process.     

Mak5p,which is required for the maintenance of the M1 dsRNA virus,is encoded by the yeast ORF<Emphasis Type="Italic"> YBR142w</Emphasis> and is involved in the biogenesis of the 60S subunit of the ribosome

In this study, we show that the Saccharomyces cerevisiae ORF YBR142w, which encodes a putative DEAD-box RNA helicase, corresponds to MAK5. The mak5-1 allele is deficient in the maintenance of the M1 dsRNA virus, resulting in a killer minus phenotype. This allele carries two mutations, G218D in the conserved ATPase A-motif and P618S in a non-conserved region. We have separated these mutations and shown that it is the G218D mutation that is responsible for the killer minus phenotype. Mak5p is an essential nucleolar protein; depletion of the protein leads to a reduction in the level of 60S ribosomal subunits, the appearance of half-mer polysomes, and a delay in production of the mature 25S and 5.8S rRNAs. Thus, Mak5p is involved in the biogenesis of 60S ribosomal subunits.Communicated by F. Messenguy     

Nop53p is required for late 60S ribosome subunit maturation and nuclear export in yeast

We report that Ypl146cp/Nop53p is associated with pre-60S ribosomal complexes and localized to the nucleolus and nucleoplasm. In cells depleted of Nop53p synthesis of the rRNA components of the 60S ribosomal subunit is severely inhibited, with strikingly strong accumulation of the 7S pre-rRNA and a 5' extended form of the 25S rRNA. In cells depleted of Nop53p pre-60S subunits accumulate in the nucleus. However, a heterokaryon assay demonstrated that Nop53p is not transferred between nuclei, indicating that it is not released into the cytoplasm. We conclude that Nop53p is a late-acting factor in the nuclear maturation of 60S ribosomal subunits, which is required for normal acquisition of export competence. The strong accumulation of preribosomes in the Nop53p-depleted strain further suggests that it may participate in targeting aberrant preribosomes to surveillance and degradation pathways.     

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.     

Rbp95 binds to 25S rRNA helix H95 and cooperates with the Npa1 complex during early pre-60S particle maturation

Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3′ region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.     

Characterization of Saccharomyces cerevisiae Npa2p (Urb2p) reveals a low-molecular-mass complex containing Dbp6p, Npa1p (Urb1p), Nop8p, and Rsa3p involved in early steps of 60S ribosomal subunit biogenesis

We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with the rsa3 null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA(2) pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis.     

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.     

A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits

Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm. Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm. A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle. Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure. Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits. Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.     

Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps.

Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP-binding site, which is essential in vivo. Kinetic and steady-state measurements of the levels of pre-rRNAs in Nog2p-depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SB(S) and 7S(S) precursors. We found Nog2p physically associated with large pre-60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP-binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre-60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP-dependent association of Nog2p with the pre-ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.     

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.