Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era,an essential GTPase in Escherichia coli

Era is a small GTP-binding protein and essential for cell growth in Escherichia coli. It consists of two domains: N-terminal GTP-binding and C-terminal RNA-binding KH domains. It has been shown to bind to 16S rRNAs and 30S ribosomal subunits in vitro. Here, we report that a precursor of 16S rRNA accumulates in Era-depleted cells. The accumulation of the precursors is also seen in a cold-sensitive mutant, E200K, in which the mutation site is located in the C-terminal domain. The major precursor molecule accumulated seems to be 17S rRNA, containing extra sequences at both 5' and 3' ends of 16S rRNA. Moreover, the amounts of both 30S and 50S ribosomal subunits relative to the amount of 70S monosomes increase in Era-depleted and E200K mutant cells. The C-terminal KH domain has a high structural similarity to the RbfA protein, a cold shock protein that also specifically associates with 30S ribosomal subunits. RbfA is essential for cell growth at low temperature, and a precursor of 16S rRNA accumulates in an rbfA deletion strain. The 16S rRNA precursor seems to be identical in size to that accumulated in Era mutant cells. Surprisingly, the cold-sensitive cell growth of the rbfA deletion cells was partially suppressed by overproduction of the wild-type Era. The C-terminal domain alone was not able to suppress the cold-sensitive phenotype, whereas Era-dE, which has a 10-residue deletion in a putative effector region of the N-terminal domain, functioned as a more efficient suppressor than the wild-type Era. It was found that Era-dE suppressed defective 16S rRNA maturation, resuming a normal polysome profile to reduce highly accumulated free 30S and 50S subunits in the rbfA deletion cells. These results indicate that Era is involved in 16S rRNA maturation and ribosome assembly.     

Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae.

The genetic depletion of yeast Rrp5p results in a synthesis defect of both 18S and 5.8S ribosomal RNAs (Venema J, Tollervey D. 1996. EMBO J 15:5701-5714). We have isolated the RRP5gene in a genetic approach aimed to select for yeast factors interfering with protein import into mitochondria. We describe here a striking feature of Rrp5p amino acid sequence, namely the presence of twelve putative S1 RNA-binding motifs and seven tetratricopeptide repeats (TPR) motifs. We have constructed two conditional temperature-sensitive alleles of RRP5 gene and analyzed them for associated rRNA-processing defects. First, a functional "bipartite gene" was generated revealing that the S1 and TPR parts of the protein can act independently of each other. We also generated a two amino acid deletion in TPR unit 1 (rrp5delta6 allele). The two mutant forms of Rrp5p were shown to cause a defect in 18S rRNA synthesis with no detectable effects on 5.8S rRNA production. However, the rRNA processing pathway was differently affected in each case. Interestingly, the ROK1 gene which, like RRP5, was previously isolated in a screen for synthetic lethal mutations with snR10 deletion, was here identified as a high copy suppressor of the rrp5delta6 temperature-sensitive allele. ROK1 also acts as a low copy suppressor but cannot bypass the cellular requirement for RRP5. Furthermore, we show that suppression by the Rok1p putative RNA helicase rescues the 18S rRNA synthesis defect caused by the rrp5delta6 mutation.     

A 3' splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in Neurospora crassa

We showed previously that the cyt-21+ gene of Neurospora crassa encodes a mitochondrial ribosomal protein homologous to Escherichia coli ribosomal protein S-16 (Kuiper, M. T. R., Akins, R. A., Holtrop, M., de Vries, H., and Lambowitz, A. M. (1988) J. Biol. Chem. 263, 2840-2847). A mutation in this gene, cyt-21-1, results in deficiency of mitochondrial small ribosomal subunits and small rRNA (Collins, R. A., Bertrand, H., LaPolla, R. J., and Lambowitz, A. M. (1979) Mol. Gen. Genet. 177, 73-84). In the present work, cloning and sequencing of the cyt-21-1 mutant allele show that it contains a single dG to dA transition at the 3' splice site AG of the first intron in the protein coding region. This mutation leads to inactivation of the normal 3' splice site and activation of a cryptic 3' splice site, 15 nucleotides downstream. The use of this cryptic splice site results in an in-frame deletion of 5 amino acids from the cyt-21 protein. Comparison of mutant and wild-type mitochondrial small ribosomal subunit proteins showed one protein, S-24, with an altered electrophoretic mobility, consistent with the predicted deletion. The mutant ribosomal protein is still capable of binding to mitochondrial small ribosomal subunits, but results in abnormal mitochondrial ribosome assembly.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Further characterization of recessive suppression in yeast. Isolation of the low-temperature sensitive mutant of Saccharomyces cerevisiae defective in the assembly of 60 S ribosomal subunit

It has been shown that recessive suppressor mutations in the yeast Saccharomyces cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10 degrees C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P-2156, was studied in detail. After a prolonged period of incubation (70 h) under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.     

Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome''s central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.     

A novel mutation in ribosomal protein S4 that affects the function of a mutated RF1

Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination. RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA. RF1 and RF2 have been shown to bind to several ribosomal proteins. To study this interaction in vivo, prfA1, a mutant form of RF1 has been used. A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 degrees C and shows an increased misreading of UAG and UAA. In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts(+); on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1. The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive. The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16. These three mutations all confer both a Ts (44 degrees C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not. The three alleles were sequenced and shown to be truncations of the S4 protein. None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation. Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants. Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough.     

Yar1 protects the ribosomal protein Rps3 from aggregation

2000 ribosomes have to be synthesized in yeast every minute. Therefore the fast production of ribosomal proteins, their efficient delivery to the nucleus and correct incorporation into ribosomal subunits are prerequisites for optimal growth rates. Here, we report that the ankyrin repeat protein Yar1 directly interacts with the small ribosomal subunit protein Rps3 and accompanies newly synthesized Rps3 from the cytoplasm into the nucleus where Rps3 is assembled into pre-ribosomal subunits. A yar1 deletion strain displays a similar phenotype as an rps3 mutant strain, showing an accumulation of 20S pre-rRNA and a 40S export defect. The combination of an rps3 mutation with a yar1 deletion leads to an enhancement of these phenotypes, while increased expression of RPS3 suppresses the defects of a yar1 deletion strain. We further show that Yar1 protects Rps3 from aggregation in vitro and increases its solubility in vivo. Our data suggest that Yar1 is a specific chaperone for Rps3, which serves to keep Rps3 soluble until its incorporation into the pre-ribosome.     

Hybrid protein between ribosomal protein S16 and RimM of Escherichia coli retains the ribosome maturation function of both proteins

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimM mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion between rpsP, encoding the ribosomal protein S16, and rimM on the chromosome as a result of mutations in the rpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.     

Synergistic defect in 60S ribosomal subunit assembly caused by a mutation of Rrs1p, a ribosomal protein L11-binding protein, and 3'-extension of 5S rRNA in Saccharomyces cerevisiae

Rrs1p, a ribosomal protein L11-binding protein, has an essential role in biogenesis of 60S ribosomal subunits. We obtained conditionally synthetic lethal allele with the rrs1-5 mutation and determined that the mutation is in REX1, which encodes an exonuclease. The highly conserved leucine at 305 was substituted with tryptophan in rex1-1. The rex1-1 allele resulted in 3′-extended 5S rRNA. Polysome analysis revealed that rex1-1 and rrs1-5 caused a synergistic defect in the assembly of 60S ribosomal subunits. In vivo and in vitro binding assays indicate that Rrs1p interacts with the ribosomal protein L5–5S rRNA complex. The rrs1-5 mutation weakens the interaction between Rrs1p with both L5 and L11. These data suggest that the assembly of L5–5S rRNA on 60S ribosomal subunits coordinates with assembly of L11 via Rrs1p.     

Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.     

Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli

A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wild-type Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and overexpressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures.     

Restricting conformational flexibility of the switch II region creates a dominant-inhibitory phenotype in Obg GTPase Nog1

Nog1 is a conserved eukaryotic GTPase of the Obg family involved in the biogenesis of 60S ribosomal subunits. Here we report the unique dominant-inhibitory properties of a point mutation in the switch II region of mouse Nog1; this mutation is predicted to restrict conformational mobility of the GTP-binding domain. We show that although the mutation does not significantly affect GTP binding, ectopic expression of the mutant in mouse cells disrupts productive assembly of pre-60S subunits and arrests cell proliferation. The mutant impairs processing of multiple pre-rRNA intermediates, resulting in the degradation of the newly synthesized 5.8S/28S rRNA precursors. Sedimentation analysis of nucleolar preribosomes indicates that defective Nog1 function inhibits the conversion of 32S pre-rRNA-containing complexes to a smaller form, resulting in a drastic accumulation of enlarged pre-60S particles in the nucleolus. These results suggest that conformational changes in the switch II element of Nog1 have a critical importance for the dissociation of preribosome-bound factors during intranucleolar maturation and thereby strongly influence the overall efficiency of the assembly process.     

Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase

The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other E. coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E. coli genes that were capable of rescuing the rrmJ mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA.     

A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome.

A single base substitution mutation from guanine to cytosine was constructed at position 2661 of Escherichia coli 23S rRNA and cloned into the rrnB operon of the multi-copy plasmid pKK3535. The mutant plasmid was transformed into E.coli to determine the effect of the mutation on cell growth as well as the structural and functional properties of the mutant ribosomes in vivo and in vitro. The results show that the mutant ribosomes have a slower elongation rate and an altered affinity for EF-Tu-tRNA-GTP ternary complex. This supports previous findings which indicated that position 2661 is part of a region of 23S rRNA that forms a recognition site for binding of the ternary complex in the ribosomal A site. Combinations of the 2661 mutation with various mutations in ribosomal protein S12 also demonstrate that elements of both ribosomal subunits work in concert to form this binding site.     

Intersection of the Kap123p-mediated nuclear import and ribosome export pathways

Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.