Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3'' end formation of 5.8S rRNA in Saccharomyces cerevisiae.

The temperature-sensitive mutation, dob1-1, was identified in a screen for dependence on overexpression of the yeast translation initiation factor eIF4B (Tif3p). Dob1p is an essential putative ATP-dependent RNA helicase. Polysome analyses revealed an under accumulation of 60S ribosomal subunits in the dob1-1 mutant. Pulse-chase labelling of pre-rRNA showed that this was due to a defect in the synthesis of the 5.8S and 25S rRNAs. Northern and primer extension analyses in the dob1-1 mutant, or in a strain genetically depleted of Dob1p, revealed a specific inhibition of the 3' processing of the 5.8S rRNA from its 7S precursor. This processing recently has been attributed to the activity of the exosome, a complex of 3'-->5' exonucleases that includes Rrp4p. In vivo depletion of Dob1p also inhibits degradation of the 5' external transcribed spacer region of the pre-rRNA. A similar phenotype was observed in rrp4 mutant strains and, moreover, the dob1-1 and rrp4-1 mutations show a strong synergistic growth inhibition. We propose that Dob1p functions as a cofactor for the exosome complex that unwinds secondary structures in the pre-rRNA that otherwise block the progression of the 3'-->5' exonucleases.     

Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.     

Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae

The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. Recent work has identified Ccr4p and Pop2p as components of the major cytoplasmic deadenylase in yeast. We now demonstrate that CCR4 encodes the catalytic subunit of the deadenylase and that Pop2p is dispensable for catalysis. In addition, we demonstrate that at least some of the Ccr4p/Pop2p-associated Not proteins are cytoplasmic, and lesions in some of the NOT genes can lead to defects in mRNA deadenylation rates. The Ccr4p deadenylase is inhibited in vitro by addition of the poly(A) binding protein (Pab1p), suggesting that dissociation of Pab1p from the poly(A) tail may be rate limiting for deadenylation in vivo. In addition, the rapid deadenylation of the COX17 mRNA, which is controlled by a member of the Pumilio family of deadenylation activators Puf3p, requires an active Ccr4p/Pop2p/Not deadenylase. These results define the Ccr4p/Pop2p/Not complex as the cytoplasmic deadenylase in yeast and identify positive and negative regulators of this enzyme complex.     

The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases

The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors. This removal is initiated by endonucleolytic cleavage approximately midway in ITS2. The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3'-->5' exonucleases, many of which are part of the exosome. So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown. Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5'-->3' exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5' end is located closely downstream of the previously mapped 3' end of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p. In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.     

Location of the 5.8S rRNA gene of Saccharomyces cerevisiae.

Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.     

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.     

Identification of cis-acting elements involved in 3'-end formation of Saccharomyces cerevisiae 18S rRNA.

In yeast, the 3' end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D. Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site. To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo. Using clustered point mutations, we found that alterations in the sequence spanning site D from position -5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units. More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position -2), whereas sequence changes at position -1, +1, and +2 relative to site D had no effect. The data further demonstrate that the proposed base pairing between the 3' end of 18S rRNA and the 5' end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits. These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation. The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.     

Saccharomyces cerevisiae Ngl3p is an active 3'-5' exonuclease with a specificity towards poly-A RNA reminiscent of cellular deadenylases

Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5' exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has been identified as a new member of the EEP family of exonucleases based on sequence homology, but its activity and biological roles are presently unknown. Here, we show using in vitro deadenylation assays on defined RNA species mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5' exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p is naturally involved in processing of poly-adenylated RNA and provide insights into the mechanistic variations observed among the redundant set of EEP enzymes found in yeast and higher eukaryotes.     

Binding of proteins from the large ribosomal subunits to 5.8 S rRNA of Saccharomyces cerevisiae

Specific binding of purified proteins from the large ribosomal subunits of Saccharomyces cerevisiae to 5.8 S rRNA was examined by three different methods: nitrocellulose membrane filtration, sucrose density gradient centrifugation, and RNA-Sepharose column chromatography. RNA-protein complex formation was proportional to the amount of proteins added to the reaction mixture. The binding of proteins to the RNA could be saturated. Such RNA-protein complexes were isolated on sucrose density gradients. Protein species present in these complexes were isolated, iodinated, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Eleven proteins, L13, L14, L17, L19, L21, L24, L25, L29, L30, L33, and L39, were identified. By comparison, only six proteins interacted with the 5.8 S rRNA-Sepharose under similar ionic conditions. They were proteins L14, L21, L24, L27, L29, and L30. To better characterize these binding proteins, the interaction of individual proteins with 5.8 S rRNA was studied by nitrocellulose membrane filtration. Proteins L14, L19, L21, L29, L33, and L39 were observed to bind individually with 5.8 S rRNA. Binding of each protein to the RNA could be saturated. The apparent association constants (K'a), measured at 4 degrees C and in 30 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 330 mM KCl, and 6 mM beta-mercaptoethanol, ranged from 1.05 to 3.70 X 10(6) M-1.     

RRP20, a component of the 90S preribosome,is required for pre-18S rRNA processing in Saccharomyces cerevisiae

A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A₁ and A₂, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20.     

Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA.

The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure. In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another. We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA. We found that approximately half of variable region V13 can be deleted without any phenotypic effect. The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality. Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2. Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal. Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S). These results demonstrate that both variable regions play an important role in 60S subunit formation.     

ESF1 is required for 18S rRNA synthesis in Saccharomyces cerevisiae

We report that Esf1p (Ydr365cp), an essential, evolutionarily conserved nucleolar protein, is required for the biogenesis of 18S rRNA in Saccharomyces cerevisiae. Depletion of Esf1p resulted in delayed processing of 35S precursor and a striking loss of 18S rRNA. Esf1p physically associated with ribosomal proteins and proteins involved in 18S rRNA biogenesis. Consistent with its role in 18S rRNA biogenesis, Esf1p also physically associated with U3 and U14 snoRNAs, but did not appear to be a core component of the SSU processome. These data indicate that Esf1p plays a direct role in early pre-rRNA processing.     

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.     

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.     

Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3.

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.     

Rok1p is a putative RNA helicase required for rRNA processing.

The synthesis of ribosomes involves many small nucleolar ribonucleoprotein particles (snoRNPs) as transacting factors. Yeast strains lacking the snoRNA, snR10, are viable but are impaired in growth and delayed in the early pre-rRNA cleavages at sites A0, A1, and A2, which lead to the synthesis of 18S rRNA. The same cleavages are inhibited by genetic depletion of the essential snoRNP protein Gar1p. Screens for mutations showing synthetic lethality with deletion of the SNR10 gene or with a temperature-sensitive gar1 allele both identified the ROK1 gene, encoding a putative, ATP-dependent RNA helicase of the DEAD-box family. The ROK1 gene is essential for viability, and depletion of Rok1p inhibits pre-rRNA processing at sites A0, A1, and A2, thereby blocking 18S rRNA synthesis. Indirect immunofluorescence by using a ProtA-Rok1p construct shows the protein to be predominantly nucleolar. These results suggest that Rok1p is required for the function of the snoRNP complex carrying out the early pre-rRNA cleavage reactions.     

Rio2p,an evolutionarily conserved,low abundant protein kinase essential for processing of 20 S Pre-rRNA in Saccharomyces cerevisiae

Saccharomyces cerevisiae Rio2p (encoded by open reading frame Ynl207w) is an essential protein of unknown function that displays significant sequence similarity to Rio1p/Rrp10p. The latter was recently shown to be an evolutionarily conserved, predominantly cytoplasmic serine/threonine kinase whose presence is required for the final cleavage at site D that converts 20 S pre-rRNA into mature 18 S rRNA. A data base search identified homologs of Rio2p in a wide variety of eukaryotes and Archaea. Detailed sequence comparison and in vitro kinase assays using recombinant protein demonstrated that Rio2p defines a subfamily of protein kinases related to, but both structurally and functionally distinct from, the one defined by Rio1p. Failure to deplete Rio2p in cells containing a GAL-rio2 gene and direct analysis of Rio2p levels by Western blotting indicated the protein to be low abundant. Using a GAL-rio2 gene carrying a point mutation that reduces the kinase activity, we found that depletion of this mutant protein blocked production of 18 S rRNA due to inhibition of the cleavage of cytoplasmic 20 S pre-rRNA at site D. Production of the large subunit rRNAs was not affected. Thus, Rio2p is the second protein kinase that is essential for cleavage at site D and the first in which the processing defect can be linked to its enzymatic activity. Contrary to Rio1p/Rrp10p, however, Rio2p appears to be localized predominantly in the nucleus.