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.     

Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis.

Different point mutations in the nucleolar protein fibrillarin (Nop1p in Saccharomyces cerevisiae) can inhibit different steps in ribosome synthesis. A screen for mutations that are synthetically lethal (sl) with the nop1-5 allele, which inhibits pre-rRNA processing, identified NOP56. An independent sl mutation screen with nop1-3, which inhibits pre-rRNA methylation, identified a mutation in NOP58. Strikingly, Nop56p and Nop58p are highly homologous (45% identity). Both proteins were found to be essential and localized to the nucleolus. A temperature-sensitive lethal mutant allele, nop56-2, inhibited many steps in pre-rRNA processing, particularly on the pathway of 25S/5.8S rRNA synthesis, and led to defects in 60S subunit assembly. Epitope-tagged constructs show that both Nop56p and Nop58p are associated with Noplp in complexes, Nop56p and Nop1p exhibiting a stoichiometric association. These physical interactions presumably underlie the observed sl phenotypes. Well-conserved homologs are present in a range of organisms, including humans (52% identity between human hNop56p and yeast Nop56p), suggesting that these complexes have been conserved in evolution.     

Nop53p, an essential nucleolar protein that interacts with Nop17p and Nip7p, is required for pre-rRNA processing in Saccharomyces cerevisiae

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two-hybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.     

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 two proteins Pat1p (Mrt1p) and Spb8p interact in vivo, are required for mRNA decay, and are functionally linked to Pab1p

We report here the characterization of a bypass suppressor of pab1Delta which leads to a fourfold stabilization of the unstable MFA2 mRNA. Cloning of the wild-type gene for that suppressor reveals that it is identical to PAT1 (YCR077c), a gene whose product was reported to interact with Top2p. PAT1 is not an essential gene, but its deletion leads to a thermosensitive phenotype. Further analysis has shown that PAT1 is allelic with mrt1-3, a mutation previously reported to affect decapping and to bypass suppress pab1Delta, as is also the case for dcp1, spb8, and mrt3. Coimmunoprecipitation experiments show that Pat1p is associated with Spb8p. On sucrose gradients, the two proteins cosediment with fractions containing the polysomes. In the absence of Pat1p, however, Spb8p no longer cofractionates with the polysomes, while the removal of Spb8p leads to a sharp decrease in the level of Pat1p. Our results suggest that some of the factors involved in mRNA degradation could be associated with the mRNA that is still being translated, awaiting a specific signal to commit the mRNA to the degradation pathway.     

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.     

Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast.

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.     

Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae

A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15°C and slow growth at 20 and 25°C. Growth of a nop12Δ strain at 15 and 20°C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.     

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.     

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.     

Characterization of Saccharomyces cerevisiae Nop17p, a novel Nop58p-interacting protein that is involved in Pre-rRNA processing

In eukaryotes, pre-rRNA processing depends on cis-acting elements and on a large number of non-ribosomal trans-acting factors, including endonucleases and exonucleases, RNA helicases, rRNA modifying enzymes and components of snoRNPs. The exosome is a conserved eukaryotic protein complex containing multiple 3'-5' exonucleases, which has been implicated in pre-rRNA, snoRNA and snRNA processing, as well as in mRNA degradation. In order to identify new proteins involved in rRNA processing, we have screened a yeast two-hybrid cDNA library, to isolate proteins interacting with the exosome subunit Rrp43p. In this screen, a novel nucleolar protein, Nop17p, was identified which also interacts with the box C/D snoRNP protein Nop58p. The NOP17 gene is not essential for cell viability but its deletion causes a temperature-sensitive phenotype. Pre-rRNA processing analyses revealed that rRNA formation is affected in the Deltanop17 strain subjected to the non-permissive temperature, although it is not blocked completely. In addition, primer extension analyses of RNA isolated from Nop17p-depleted cells subjected to the non-permissive temperature indicates that the pre-rRNA is undergoing different modification or degradation processes in these cells as compared to the parental strain. Nop17p was recently described in the same complex as Nop58p and, interestingly, its depletion leads to mislocalization of Nop1p, Nop56p, Nop58p and Snu13p, which are the core proteins of the box C/D ribonucleoprotein (snoRNP), indicating that Nop17p function is required either for nucleolar retention or for the proper assembly of the box C/D snoRNP.     

Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein

The nucleolar Saccharomyces cerevisiae protein Nep1 was previously shown to bind to a specific site of the 18S rRNA and to be involved in assembly of Rps19p into pre-40S ribosome subunits. Here we report on the identification of tma23 and nop6 mutations as recessive suppressors of a nep1(ts) mutant allele and the nep1 deletion as well. Green fluorescent protein fusions localized Tma23p and Nop6p within the nucleolus, indicating their function in ribosome biogenesis. The high lysine content of both proteins and an RNA binding motif in the Nop6p amino acid sequence suggest RNA-binding functions for both factors. Surprisingly, in contrast to Nep1p, Tma23p and Nop6p seem to be specific for fungi as no homologues could be found in higher eukaryotes. In contrast to most other ribosome biogenesis factors, Tma23p and Nop6p are nonessential in S. cerevisiae. Interestingly, the tma23 mutants showed a considerably increased resistance against the aminoglycoside G418, probably due to a structural change in the 40S ribosomal subunit, which could be the result of incorrectly folded 18S rRNA gene, missing rRNA modifications or the lack of a ribosomal protein.     

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

Eukaryotic nucleoli contain a large family of box C+D small nucleolar RNA (snoRNA) species, all of which are associated with a common protein Nop1p/fibrillarin. Nop58p was identified in a screen for synthetic lethality with Nop1p and shown to be an essential nucleolar protein. Here we report that a Protein A-tagged version of Nop58p coprecipitates all tested box C+D snoRNAs and that genetic depletion of Nop58p leads to the loss of all tested box C+D snoRNAs. The box H+ACA class of snoRNAs are not coprecipitated with Nop58p, and are not codepleted. The yeast box C+D snoRNAs include two species, U3 and U14, that are required for the early cleavages in pre-rRNA processing. Consistent with this, Nop58p depletion leads to a strong inhibition of pre-rRNA processing and 18S rRNA synthesis. Unexpectedly, depletion of Nop58p leads to the accumulation of 3' extended forms of U3 and U24, showing that the protein is also involved in snoRNA synthesis. Nop58p is the second common component of the box C+D snoRNPs to be identified and the first to be shown to be required for the stability and for the synthesis of these snoRNAs.     

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.     

Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs.

The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinity-purified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively. Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus. Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs. Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre-rRNA required for the synthesis of mature 18S rRNA. These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p. Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs.     

Structural and thermodynamic evidence for a stabilizing role of Nop5p in S-adenosyl-L-methionine binding to fibrillarin

In Archaea, fibrillarin and Nop5p form the core complex of box C/D small ribonucleoprotein particles, which are responsible for site-specific 2'-hydroxyl methylation of ribosomal and transfer RNAs. Fibrillarin has a conserved methyltransferase fold and employs S-adenosyl-l-methionine (AdoMet) as the cofactor in methyl transfer reactions. Comparison between recently determined crystal structures of free fibrillarin and fibrillarin-Nop5p-AdoMet tertiary complex revealed large conformational differences at the cofactor-binding site in fibrillarin. To identify the structural elements responsible for these large conformational differences, we refined a crystal structure of Archaeoglobus fulgidus fibrillarin-Nop5p binary complex at 3.5 A. This structure exhibited a pre-formed backbone geometry at the cofactor binding site similar to that when the cofactor is bound, suggesting that binding of Nop5p alone to fibrillarin is sufficient to stabilize the AdoMet-binding pocket. Calorimetry studies of cofactor binding to fibrillarin alone and to fibrillarin-Nop5p binary complex provided further support for this role of Nop5p. Mutagenesis and thermodynamic data showed that a cation-pi bridge formed between Tyr-89 of fibrillarin and Arg-169 of Nop5p, although dispensable for in vitro methylation activity, could partially account for the enhanced binding of cofactor to fibrillarin by Nop5p. Finally, assessment of cofactor-binding thermodynamics and catalytic activities of enzyme mutants identified three additional fibrillarin residues (Thr-70, Glu-88, and Asp-133) to be important for cofactor binding and for catalysis.     

Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease.

An external stem, essential for the release of small nucleolar RNAs (snoRNAs) from their pre-mRNAs, flanks the majority of yeast intron-encoded snoRNAs. Even if this stem is not a canonical Rnt1p substrate, several experiments have indicated that the Rnt1p endonuclease is required for snoRNA processing. To identify the factors necessary for processing of intron-encoded snoRNAs, we have raised in vitro extracts able to reproduce such activity. We found that snoRNP factors are associated with the snoRNA- coding region throughout all the processing steps, and that mutants unable to assemble snoRNPs have a processing-deficient phenotype. Specific depletion of Nop1p completely prevents U18 snoRNA synthesis, but does not affect processing of a dicistronic snoRNA-coding unit that has a canonical Rnt1p site. Correct cleavage of intron-encoded U18 and snR38 snoRNAs can be reproduced in vitro by incubating together purified Nop1p and Rnt1p. Pull-down experiments showed that the two proteins interact physically. These data indicate that cleavage of U18, snR38 and possibly other intron-encoded snoRNAs is a regulated process, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP assembly has occurred.