Ribosomal protein L33 is required for ribosome biogenesis, subunit joining, and repression of GCN4 translation - PubMed (original) (raw)
Ribosomal protein L33 is required for ribosome biogenesis, subunit joining, and repression of GCN4 translation
Pilar Martín-Marcos et al. Mol Cell Biol. 2007 Sep.
Abstract
We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd- phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37 degrees C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (DeltaA). At 28 degrees C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in DeltaA cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by DeltaA. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomal-subunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNAiMet suppresses the Gcd- phenotype of rpl33a-G76R cells, dissociation of tRNAiMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.
Figures
FIG. 1.
Gcd− (3ATR), Slg−, cell morphology, and antibiotic-sensitivity phenotypes of the H275 mutant (gcd17-1/rpl33a-G76R). (A) 3ATR phenotype of H275 relative to the 3ATS phenotype of the isogenic WT GCD17 strain H117 (gcn2-101 gcn3-101). Isolated colonies of each strain were replica printed to plates containing 10 mM 3AT and to minimally supplemented SD medium plates and incubated for 3 days at 28°C. (B) Slg− phenotype of H275 at 18°C, 28°C, and 37°C. Cells were streaked for single colonies on SD plates that were incubated at 18°C (10 days), 28°C (3 days), or 37°C (4 days). (C) Aberrant cell morphologies of the H275 mutant. Cells of H117 and of the H275 mutant were grown for 24 h in liquid YPD at 28°C or 37°C, and pictures were taken under a phase-contrast microscope (magnification, ×40). (D) Increased sensitivity of the H275 mutant to the protein synthesis inhibitors paromomycin (500 μg/ml), cycloheximide (0.025 μg/ml), and sparsomycin (5 μg/ml) relative to that of H117. Serial dilutions of cells (104 to 10 cells) grown for 12 h in YPD were plotted on YPD plates and on YPD plates containing the indicated concentrations of antibiotics and incubated for 3 or 4 days at 28°C. (E) The gcd17-1/rpl33a-G76R mutation increases the transcription of the HIS4 gene. Cells of isogenic strains H117 (gcn GCD17) and H275 (gcn gcd17-1) and of the WT strain F35 (GCN GCD) were grown under repressing (R) (SD medium) and derepressing (DR) conditions of tryptophan starvation induced by 0.5 mM 5MT. Total RNA was extracted and 10 μg analyzed by Northern blot analysis in three independent blots, using radiolabeled probes specific to visualize HIS4, GCN4, and PYK1 mRNAs (Materials and Methods). The hybridization signals were quantified by PhosphorImaging analysis, and values normalized relative to PYK1 mRNA are given in percentages below each panel relative to the corresponding values in F35, which were set to 100%.
FIG. 2.
Theoretical 3D model for rpL33. (A) Alignment of rpL33A's primary sequence with that of Pyrococcus furiosus 1SQR and secondary-structure predictions for rpL33. h, predicted alpha helix, s, predicted beta sheet. (B) Overlap of the main-chain backbones of 1SQR (blue) and rpL33A (red), showing a loop of rpL33A absent in 1SQR (green). (C) Predicted β-barrel 3D structure of rpL33 (yellow), with the position of an α-helix (dark pink) and of the G76R mutation in rpl33a-G76R (green). The diagram shown in panel B and the diagram in the upper portion of panel C are in the same orientation, and that in the bottom of panel C is rotated 90° to the right. (D) Glycine 76 (red) is predicted to form hydrogen bonds inside a rigid loop; its primary sequence is part of the 22-amino-acid carboxy-terminal domain conserved in the 99 members of the r-protein L35Ae family.
FIG. 3.
The rpl33a-G76R mutation leads to a shortage in 60S ribosomal subunits and to the accumulation of half-mer polysomes. (A) The rpl33a-G76R strain H275 (bottom panels) and the isogenic RPL33A WT strain H117 (top panels) were grown in YPD to mid-logarithmic phase at 28°C (OD600, ∼0.8) and then shifted for 2 or 4 h at 37°C. Cycloheximide was added at 100 μg/ml before harvesting of cells, and WCE containing ribosomes and polyribosomes were prepared in the presence of 30 mM Mg2+ and separated by velocity sedimentation on 7% to 50% sucrose gradients. In the top panels, peaks representing free-ribosomal 40S and 60S subunits and 80S monosomes are indicated, and half-mer polysomes appearing in the H275 mutant profiles are marked by vertical arrows. The P/M ratios were calculated for profiles obtained from cells grown at 28°C (at time zero [graphs marked t = 0]) and after incubation at 37°C (graphs marked 2h and 4h), and the averages of values obtained in three independent experiments are indicated on the A_254 tracings. (B) Strains H117 (RPL33A), H275 (rpl33a-G76R), Hm506 (Δ_A), Hm502 (Δ_B_), and Hm505 (rpl33a-G76R ΔB) were cultured as described for panel A, but WCE were prepared in the absence of cycloheximide and Mg2+ and resolved by velocity sedimentation through 7% to 50% sucrose gradients. The mean ratios of total 60S/40S subunits determined from three replicate experiments are indicated below the _A_254 tracings, and the 25S/18S rRNA ratios estimated with an Agilent Technologies model 2100 bioanalyzer are indicated inside parentheses below the 60S/40S rRNA ratios.
FIG. 4.
Deficient and aberrant processing of pre-rRNAs in rpl33a-G76R cells shown by Northern analysis. (A) Scheme of the pre-rRNA processing pathway. The 35S pre-rRNA contains the sequences for mature rRNAs (18S, 5.8S, and 25S) separated by two internal transcribed spaces (ITS1 and ITS2) and flanked by two external transcribed spaces (5′ETS and 3′ETS). The rRNAs are shown as filled bars and the transcribed spaces represented as lines. The processing sites are indicated above the diagram by the uppercase letters A to E. The annealing positions of oligonucleotides 1 to 7 used as hybridization probes are indicated beneath all of the rRNA species that they detect. (B to D) Strains H117 (RPL33A) and mutant H275 (rpl33a-G76R) were cultured in liquid YPD medium at 28°C to mid-logarithmic phase (OD600, ∼0.8) and shifted to 37°C for 2, 4, or 6 h. Total RNA was extracted, and samples containing 10 μg were resolved on a 1.2% agarose-4% formaldehyde gel and subjected to Northern analysis. (B) The blot was first dyed with methylene blue to test the integrity of the 25S and the 18S rRNAs. (C) The blot was consecutively hybridized with probes whose positions are depicted in panel A. The RNA species, visualized with the probes indicated at the left, are labeled on the right. The probe specific for 5S rRNA is indicated as (8) (Materials and Methods). (D) Hybridization with a probe specific for U3 (9) (Materials and Methods), which was used as internal control for loading. (E) Schematic diagram showing the potential origin of aberrant and less common pre-rRNA species indicated in panel C.
FIG. 5.
Comparison of the Gcd− and Slg− phenotypes of several rpl33 mutants. (A) Steady-state amounts of RPL33 mRNAs. Cells of Hm506 (Δ_A_) and Hm502 (Δ_B_) were grown in YPD to mid-logarithmic phase at 28°C (OD600, ∼0.8) and transferred for 30 min or 2 h at 37°C. Total RNA was extracted and 10 μg analyzed by Northern blotting using a probe that hybridizes to both RPL33A and RPL33B mRNAs and a probe for SCR1, used as the loading control (10) (Materials and Methods). (B) The Slg− phenotypes of Hm502 (Δ_B_), Hm506 (Δ_A_), and Hm505 (rpl33a-G76R ΔB) mutants are shown relative to that of the original mutant H275 (rpl33a-G76R) and to the WT phenotype (Slg+) of the parental strain H117 (gcn2 gcn3 RPL33A RPL33B). The Slg+ phenotype of the WT strain S288C (GCN2 GCN3 RPL33A RPL33B) is shown as a reference. The growth phenotypes were analyzed by streaking single colonies on YPD medium and incubating the plates for 8 days at 18°C and for 3 days at 28°C or at 37°C. (C) The Gcd− phenotype (3ATR) of the same mutants as those shown in panel B is shown relative to that of the original mutant H275 and to the 3ATS (Gcn−) phenotype of the parental strain H117. The 3ATR phenotype of the WT strain S288C is shown as a reference. Isolated colonies of each strain were replica printed to 10 mM 3AT and to SD plates and incubated for 3 days at 28°C. (D) The rpl33a-G76R and Δ_A_ mutations lead to constitutive derepression of GCN4-lacZ independently of the positive GCN regulators. GCN4-lacZ fusions were introduced into yeast strains on low-copy-number plasmids p180, p226, p227, and pM226 (labeled boxes with a triangle at the right end). The relevant genotypes of the strains are indicated on the left below these diagrams. The gcn2-101 gcn3-101 strains H466 (RPL33A), Hm526 (Δ_A_), and Hm527 (rpl33a-G76R) are isogenic. The four uORFs in the leader sequence of p180 are shown as open boxes, and point mutations that remove the AUG codons of uORF1 to -3 (p226) or uORF1 to -4 (p227) are shown as ×s. uORF1 in pM226, located at the position of uORF4 and elongated to overlap the beginning of GCN4, is indicated by a rectangle. β-Galactosidase activity was measured in cells grown to mid-logarithmic phase under nonstarvation, repressing (R), or derepressing (DR) conditions of histidine starvation induced by 3AT. Values are averages of results obtained in three independent experiments with two independent transformants. Units of β-galactosidase activity were calculated as indicated in Materials and Methods.
FIG. 6.
Genetic interactions of rpl33a-G76R. (A) Weak suppression of rpl33a-G76R by hc_NOP7_ and hc_DRS1_ plasmids. Transformants of the Hm337 mutant (rpl33a-G76R) carrying an empty vector (pRS426) or high-copy-number plasmids bearing RPL33A (pPM13), NOP7 (pJW6039), or DRS1 (pJW3015) were grown in SD-Ura medium at 28°C and serial dilutions (103 to 10 cells) plotted on plates of the same medium, which were incubated for 3 days at 37°C (left panel). Isolated colonies of each transformant were replica printed to plates containing 10 mM 3AT and to minimally supplemented SD plates and incubated for 3 days at 28°C (right panels). (B) Allele-specific suppression of the nop7 and drs1 mutations by the hc_RPL33A_ plasmid. Mutant drs1-1 and nop7-A strains bearing an hc_RPL33A_ plasmid (pPM13), empty vector (pR8426), or high-copy-number plasmids bearing DRS1 (pJW3015) or NOP7 (pJW6039) were grown as described for panel A and plotted on SC-Ura plates, which were incubated for 2 days at 28°C or for 5 days at 18°C. (C) Dosage suppressors of the Slg− phenotype of the rpl33a-G76R mutant. Transformants of Hm337 (rpl33a-G76R) carrying an empty vector (pRS426), a single-copy plasmid bearing RPL33A (pPM2), or high-copy-number plasmids bearing RPL33A (pPM13), PAB1 (pAS425), IMT4 (p2635), or the three subunits of eIF2 plus IMT4 (hc-TC; p3000) were streaked for single colonies on minimally supplemented SD medium and incubated at 28°C (3 days) or 37°C (4 days). (D) The Gcd− phenotype of rpl33a-G76R is partially suppressed by the hc_IMT4_ plasmid and the hc-TC, but not by the hc_PAB1_ plasmid. Isolated colonies of the same transformants as those described in panel C were replica printed to plates containing 10 mM 3AT and to SD plates and incubated for 3 days at 28°C.
References
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