Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5. 8S RRNA processing and 60S ribosome biogenesis

We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G(1) in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Delta, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Delta leads to cell growth arrest in the G(1) phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Delta expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Delta in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.     

Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis

Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored. Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control. Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells. We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps. Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes. Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle. These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits.     

Functional inactivation of the mouse nucleolar protein Bop1 inhibits multiple steps in pre-rRNA processing and blocks cell cycle progression

Bop1 is a conserved nucleolar protein involved in rRNA processing and ribosome assembly in eukaryotes. Expression of its dominant-negative mutant Bop1 Delta in mouse cells blocks rRNA maturation and synthesis of large ribosomal subunits and induces a reversible, p53-dependent cell cycle arrest. In this study, we have conducted a deletion analysis of Bop1 and identified a new mutant, Bop1N2, that also acts as a potent inhibitor of cell cycle progression. Bop1N2 and Bop1 Delta are C-terminal and N-terminal deletion mutants, respectively, and share only 72 amino acid residues. Both mutant proteins are localized to the nucleolus and strongly inhibit rRNA processing, suggesting that activation of a cell cycle checkpoint by Bop1 mutants is linked to their inhibitory effects on rRNA and ribosome synthesis. By using these dominant-negative mutants as well as antisense oligonucleotides to interfere with endogenous Bop1, we identified specific rRNA processing steps that require Bop1 function in mammalian cells. Our data demonstrate that Bop1 is required for proper processing at four distinct sites located within the internal transcribed spacers ITS1 and ITS2 and the 3' external spacer. We propose a model in which Bop1 serves as an essential factor in ribosome formation that coordinates processing of the spacer regions in pre-rRNA.     

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.     

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.     

Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition

Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase. Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.     

Ytm1, Nop7, and Erb1 form a complex necessary for maturation of yeast 66S preribosomes

The essential, conserved yeast nucleolar protein Ytm1 is one of 17 proteins in ribosome assembly intermediates that contain WD40 protein-protein interaction motifs. Such proteins may play key roles in organizing other molecules necessary for ribosome biogenesis. Ytm1 is present in four consecutive 66S preribosomes containing 27SA2, 27SA3, 27SB, and 25.5S plus 7S pre-rRNAs plus ribosome assembly factors and ribosomal proteins. Ytm1 binds directly to Erb1 and is present in a heterotrimeric subcomplex together with Erb1 and Nop7, both within preribosomes and independently of preribosomes. However, Nop7 and Erb1 assemble into preribosomes prior to Ytm1. Mutations in the WD40 motifs of Ytm1 disrupt binding to Erb1, destabilize the heterotrimer, and delay pre-rRNA processing and nuclear export of preribosomes. Nevertheless, 66S preribosomes lacking Ytm1 remain otherwise intact.     

Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit

Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3'-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of diazaborine on rRNA processing.     

Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.     

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.     

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.     

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.     

Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3′ end of 5.8S rRNA and the 5′ end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing.     

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.     

Depletion of U3 small nucleolar RNA inhibits cleavage in the 5'' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA.

Multiple processing events are required to convert a single eukaryotic pre-ribosomal RNA (pre-rRNA) into mature 18S (small subunit), 5.8S and 25-28S (large subunit) rRNAs. We have asked whether U3 small nucleolar RNA is required for pre-rRNA processing in vivo by depleting Saccharomyces cerevisiae of U3 by conditional repression of U3 synthesis. The resulting pattern of accumulation and depletion of specific pre-rRNAs indicates that U3 is required for multiple events leading to the maturation of 18S rRNA. These include an initial cleavage within the 5' external transcribed spacer, resembling the U3 dependent initial processing event of mammalian pre-rRNA. Formation of large subunit rRNAs is unaffected by U3 depletion. The similarity between the effects of U3 depletion and depletion of U14 small nucleolar RNA and the nucleolar protein fibrillarin (NOP1) suggests that these could be components of a single highly conserved processing complex.     

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.     

Coevolution of ribosomal RNA expansion segment 7L and assembly factor Noc2p specializes the ribosome biogenesis pathway between Saccharomyces cerevisiae and Candida albicans

Ribosomes of different species share an evolutionarily conserved core, exhibiting flexible shells formed partially by the addition of species-specific ribosomal RNAs (rRNAs) with largely unexplored functions. In this study, we showed that by swapping the Saccharomyces cerevisiae 25S rRNA genes with non-S. cerevisiae homologs, species-specific rRNA variations caused moderate to severe pre-rRNA processing defects. Specifically, rRNA substitution by the Candida albicans caused severe growth defects and deficient pre-rRNA processing. We observed that such defects could be attributed primarily to variations in expansion segment 7L (ES7^L) and could be restored by an assembly factor Noc2p mutant (Noc2p-K384R). We showed that swapping ES7^L attenuated the incorporation of Noc2p and other proteins (Erb1p, Rrp1p, Rpl6p and Rpl7p) into pre-ribosomes, and this effect could be compensated for by Noc2p-K384R. Furthermore, replacement of Noc2p with ortholog from C. albicans could also enhance the incorporation of Noc2p and the above proteins into pre-ribosomes and consequently restore normal growth. Taken together, our findings help to elucidate the roles played by the species-specific rRNA variations in ribosomal biogenesis and further provide evidence that coevolution of rRNA expansion segments and cognate assembly factors specialized the ribosome biogenesis pathway, providing further insights into the function and evolution of ribosome.     

Npa1p, a component of very early pre-60S ribosomal particles, associates with a subset of small nucleolar RNPs required for peptidyl transferase center modification

We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.