Bms1p, a G-domain-containing protein, associates with Rcl1p and is required for 18S rRNA biogenesis in yeast

Maturation of 18S rRNA and biogenesis of the 40S ribosomes in yeast requires a large number of trans-acting factors, including the U3 small nucleolar ribonucleoprotein (U3 snoRNP), and the recently characterized cyclase-like protein Rcl1p. U3 snoRNP is a key particle orchestrating early 35S rRNA cleavage events. A unique property of Rcl1p is that it specifically associates with U3 snoRNP, but this association appears to occur only at the level of nascent ribosomes and not with the U3 monoparticle. Here we report the characterization of Bms1p, a protein that associates with Rcl1p in multiple structures, including a specific complex sedimenting at around 10S. Like Rcl1p, Bms1p is an essential, evolutionarily conserved, nucleolar protein, and its depletion interferes with processing of the 35S pre-rRNA at sites A0, A1, and A2, and the formation of 40S subunits. The N-terminal domain of Bms1p has structural features found in regulatory GTPases and we demonstrate that mutations of amino acids implicated in GTP/GDP binding affect Bms1p activity in vivo. The results indicate that Bms1p may act as a molecular switch during maturation of the 40S ribosomal subunit in the nucleolus.     

The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre-40S particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit. Upon depletion of Rps15p, 20S pre-rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre-rRNA early cleavages. In contrast, Rps18p, which contacts Rps15p in the small subunit, is required upstream for pre-rRNA processing at site A2. Most pre-40S specific factors are correctly associated with the intermediate particles accumulating in the nucleus upon Rps15p depletion, except the late-binding proteins Tsr1p and Rio2p. Here we show that these two proteins are dispensable for nuclear exit; instead, they participate in 20S pre-rRNA processing in the cytoplasm. We conclude that, during the final maturation steps in the nucleus, incorporation of the ribosomal protein Rps15p is specifically required to render the pre-40S particles competent for translocation to the cytoplasm.     

The ATPase and helicase activities of Prp43p are stimulated by the G‐patch protein Pfa1p during yeast ribosome biogenesis

Prp43p is a RNA helicase required for pre‐mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G‐patch protein Pfa1p, a component of pre‐40S pre‐ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G‐patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre‐40S pre‐ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre‐rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre‐rRNA processing defect of Δpfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre‐rRNA‐to‐18S rRNA conversion.     

90S pre-ribosomes include the 35S pre-rRNA,the U3 snoRNP,and 40S subunit processing factors but predominantly lack 60S synthesis factors

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.     

Nob1p is required for cleavage of the 3' end of 18S rRNA

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.     

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.     

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.     

Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.     

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.     

Yeast Krr1p physically and functionally interacts with a novel essential Kri1p, and both proteins are required for 40S ribosome biogenesis in the nucleolus

Using a two-hybrid screening with TOM1, a putative ubiquitin-ligase gene of Saccharomyces cerevisiae, we isolated KRR1, a homologue of human HRB2 (for human immunodeficiency virus type 1 Rev-binding protein 2). To characterize the gene function, we constructed temperature-sensitive krr1 mutants and isolated two multicopy suppressors. One suppressor is RPS14A, encoding a 40S ribosomal protein. The C-terminal-truncated rpS14p, which was reported to have diminished binding activity to 18S rRNA, failed to suppress the krr1 mutant. The other suppressor is a novel gene, KRI1 (for KRR1 interacting protein; YNL308c). KRI1 is essential for viability, and Kri1p is localized to the nucleolus. We constructed a galactose-dependent kri1 strain by placing KRI1 under control of the GAL1 promoter, so that expression of KRI1 was shut off when transferring the culture to glucose medium. Polysome and 40S ribosome fractions were severely decreased in the krr1 mutant and Kri1p-depleted cells. Pulse-chase analysis of newly synthesized rRNAs demonstrated that 18S rRNA is not produced in either mutant. However, wild-type levels of 25S rRNA are made in either mutant. Northern analysis revealed that the steady-state levels of 18S rRNA and 20S pre-rRNAs were reduced in both mutants. Precursors for 18S rRNA were detected but probably very unstable in both mutants. A myc-tagged Kri1p coimmunoprecipitated with a hemagglutinin-tagged Krr1p. Furthermore, the krr1 mutant protein was defective in its interaction with Kri1p. These data lead us to conclude that Krr1p physically and functionally interacts with Kri1p to form a complex which is required for 40S ribosome biogenesis in the nucleolus.     

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.     

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.     

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.     

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.     

Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast.

We have isolated and characterized Mpp10p, a novel protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) from the yeast Saccharomyces cerevisiae. The MPP10 protein was first identified in human cells by its reactivity with an antibody that recognizes specific sites of mitotic phosphorylation. To study the functional role of MPP10 in pre-rRNA processing, we identified the yeast protein by performing a GenBank search. The yeast Mpp10p homolog is 30% identical to the human protein over its length. Antibodies to the purified yeast protein recognize a 110-kDa polypeptide in yeast extracts and immunoprecipitate the U3 snoRNA, indicating that Mpp10p is a specific protein component of the U3 snoRNP in yeast. As a first step in the genetic analysis of Mpp10p function, diploid S. cerevisiae cells were transformed with a null allele. Sporulation and tetrad analysis indicate that MPP10 is an essential gene. A strain was constructed where Mpp10p is expressed from a galactose-inducible, glucose- repressible promoter. After depletion of Mpp10p by growth in glucose, cell growth is arrested and levels of 18S and its 20S precursor are reduced or absent while the 23S and 35S precursors accumulate. This pattern of accumulation of rRNA precursors suggests that Mpp10p is required for cleavage at sites A0, A1, and A2. Pulse-chase analysis of newly synthesized pre-rRNAs in Mpp10p-depleted yeast confirms that little mature 18S rRNA formed. These results reveal a novel protein essential for ribosome biogenesis and further elucidate the composition of the U3 snoRNP.     

Crucial role of the Rcl1p–Bms1p interaction for yeast pre-ribosomal RNA processing

The essential Rcl1p and Bms1p proteins form a complex required for 40S ribosomal subunit maturation. Bms1p is a GTPase and Rcl1p has been proposed to catalyse the endonucleolytic cleavage at site A₂ separating the pre-40S and pre-60S maturation pathways. We determined the 2.0 Å crystal structure of Bms1p associated with Rcl1p. We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A₂. Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing. We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.     

Cracking pre‐40S ribosomal subunit structure by systematic analyses of RNA–protein cross‐linking

Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre‐ribosomal particles. We report rRNA‐binding sites for six late‐acting 40S ribosome synthesis factors, three of which cluster around the 3′ end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in ‘beak’ structure formation during 40S maturation—and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1‐ and Tsr1‐binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3′‐endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre‐rRNA. Nob1 binds before pre‐rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.     

Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3'-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis.