Figure 1.
The rpl3[W255C] mutation results in a deficit in 40S ribosomal subunits.
Strain JDY319 (rpl3::HIS3MX6) expressing either wild-type RPL3 or the indicated rpl3 alleles, harboured on the plasmid YCplac111, were grown in YPD at 23°C to exponential phase. Cell extracts were prepared and 10 A260 of each extract were resolved on 7–50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of 40S, 60S, 80S and polysomes are indicated. Half-mers are labelled by arrows.
Figure 2.
The rpl3[W255C] mutant accumulates 20S pre-rRNA.
The wild-type and the rpl3 mutants described in the Figure 1 and the rps14A[R136A] strain were exponentially grown in YPD medium at 23°C. Total RNA was prepared and equal amounts of RNA (5 µg) were subjected to Northern blot hydridisation. A. Northern blot analysis of high-molecular weight pre- and mature rRNAs. Note that two exposure times are shown to better visualize differences in 20S pre-rRNA levels. B. Northern blot analysis of low-molecular weight pre- and mature rRNAs. Signal intensities were measured by phosphorimager scanning; values (indicated below each panel) were normalized to those obtained for the wild-type control, arbitrarily set at 1.0. Probes, between parentheses, are described in Figure S1A and Table S3.
Figure 3.
The 20S pre-rRNA accumulates in the cytoplasm of rpl3[W255C] cells.
A. Wild-type and rpl3[W255C] cells expressing either L25-eGFP or S2-eGFP were exponentially grown in SD-Ura at 23°C. The GFP signal was analysed by fluorescence microscopy. B. Wild-type and rpl3[W255C] cells were grown in YPD at 23°C. Cells were fixed with formaldehyde, spheroblasted, and subjected to FISH using a Cy3-labelled probe complementary to the D/A2 segment of ITS1 (Table S3). DAPI staining visualises the nucleoplasm. C. Levels of dimethylated 20S pre-rRNA in the wild-type strain and the rpl3[W255C] and rpl3[Q371H] mutants. RNA was extracted from cells of these strains following exponential growth in YPD at 23°C and analysed by primer extension with probe c (Figure S1 and Table S3). The position of the primer extension stops due to the presence of the modifications is indicated.
Figure 4.
20S pre-rRNA containing 40S subunits get incorporated into polysomes in rpl3[W255C] cells.
The wild-type strain and the rpl3[W255C], rpl3[Q371H] and rps14A[R136A] mutants were grown in YPD at 23°C. Cell extracts were prepared and 8 A260 units of each extract were resolved in 7–50% sucrose gradients and fractionated. RNA was extracted from each fraction and analysed by Northern blotting using probes c, h and b, which reveal 20S pre-rRNA and mature 25S and 18S rRNAs, respectively. The position of free 40S and 60S ribosomal subunits, 80S ribosomes and polysomes are shown. T stands for RNA from total extract.
Figure 5.
Translation rate modulates the levels of 20S pre-rRNA in rpl3[W255C] cells.
A. Inhibition of translation by cycloheximide treatment partially suppresses the 20S pre-rRNA processing defect of the rpl3[W255C] mutant. The wild-type strain and the rpl3[W255C] mutant were exponentially grown in YPD at 23°C. Cycloheximide (final concentration, 0.8 µg/ml) was added to the cultures and cells were harvested at the indicated times after the addition. B. Inhibition of translation initiation partially suppresses the 20S pre-rRNA processing defect of the rpl3[W255C] mutant. The indicated strains were grown exponentially in YPD medium at 30°C. C. The levels of 20S pre-rRNA are reduced in slowly growing rpl3[W255C] cells. The wild-type strain and the rpl3[W255C] mutant were grown at 23°C in either rich medium containing glucose (YPD), rich medium containing galactose (YPGal), minimal medium containing glucose (SD) or minimal medium containing glycerol and lactate as carbon source (SGly). In all cases, RNA was extracted and equal amounts of RNA (5 µg) subjected to Northern blot hybridisation as described in the legend of Figure 2. To quantify the relative amounts of pre-40S r-particles in the different conditions and mutants, the signal intensities for 20S pre-rRNA and 25S rRNA were measured by phosphorimager scanning. The 20S/25S ratios calculated were calculated and normalised to that obtained for the wild-type strain under the same conditions.
Figure 6.
The rpl3[W255C] mutation does not significantly impair the association of Fun12 to pre-40S ribosomal particles and mature 60S ribosomal subunits.
Immunoprecipitation was carried out using IgG-Sepharose in isogenic W303-1A strain (Fun12 Wild type), DY121 (Fun12-TAP Wild type) and JDY1025 (Fun12-TAP W255C). Cells were grown at 23°C in YPD to mid-log phase, lysed and total extracts were subjected to immunoprecipitation. A. Protein corresponding to 0.1% of each total extract (lanes T) and 1% of the immunoprecipitates (lanes IP) were subjected to SDS-PAGE and then analysed by Western blotting using specific antibodies. B. RNA was also extracted and 1% of each total extract (T) and 45% of the immunoprecipitates (IP) were subjected to Northern analysis. Pre-rRNAs and mature rRNAs were analysed by Northern blot hybridisation as described in the legend of Figure 2.
Figure 7.
In vitro processing of 20S pre-rRNA is impaired in the rpl3[W255C] mutant.
In vitro cleavage assays were performed with pre-ribosomal particles purified via PTH-tagged Nob1 from different strains: wild-type (blue, circle), rpl3[W255C] (red, square), rpl3[K30E] (green, square) and rsa3Δ (purple, triangle). Purified particles were incubated in reaction buffer containing 1 mM ATP (A and B) or 1 mM GTP (C and D) for the indicated times (0, 2, 5, 10 and 30 min). RNA was extracted and cleavage at site D was analysed by primer extension with probe c' (Figure S1 and Table S3). Representative primer extension analyses are shown (A and C). The strong upper stops result from termination at sites of 18S rRNA base-dimethylation at A1781 and A1782. These modifications precede site D cleavage in vivo. The black arrow indicates site D. Filled and empty dots indicate non-relevant primer extensions stops that were observed in some experiments (for further discussion, see [26]). Signal intensities were measured by phosphoimager scanning; values were corrected for RNA loading using the dimethylation signals as internal standards, normalised to the sample at the zero time-point, arbitrarily set at 1.0, and plotted (B and D). The average of 2 (B) and 4 (D) independent experiments is shown; the error bars indicate the standard deviation.
Figure 8.
Synthetic enhancement of the slow-growth phenotype of the rpl3[W255C] mutant by the NOB1-TAP allele.
The strains YKL207 (NOB1) and YKL233 (NOB1-TAP) harbour the rpl3 null allele complemented by the pHT4467Δ-RPL3 plasmid and a wild-type NOB1 or NOB1-TAP allele, respectively. The NOB1-TAP allele expresses a C-terminally TAP-tagged Nob1 protein. These strains were transformed with YCplac111 plasmids that carry either the wild-type RPL3 or the indicated mutant rpl3 alleles. After 5-FOA shuffling, cells were spotted in 10-fold serial dilution steps onto YPD plates, which were incubated for 2 days at 30°C or 3 days at 23°C. Note that the NOB1-TAP allele specifically synthetically enhances the growth defect of the rpl3[W255C] mutant.
Figure 9.
Model for the conversion of 20S pre-rRNA into mature 18S rRNA within cytoplasmic pre-40S r-particles.
A. In wild-type cells, immature pre-40S and pre-60S r-particles are transported through the channel of nuclear pore complexes to the cytoplasm. L3, which is incorporated into early pre-ribosomal particles in the nucleolus, is highlighted in pre-60S particles. Few assembly factors (AFs) remain associated with the particles as they enter the cytoplasm (e.g. Tif6 and Nob1). The release and recycling of these factors is concomitant to the assembly of a few remaining r-proteins (RPs). The translation initiation factor eIF5B/Fun12 binds pre-40S r-particles and stimulates joining of the 60S r-subunits. Formation of the resulting 80S-like particle triggers Fun12 to hydrolyse its bound GTP, thereby presumably promoting its dissociation. We assume that GTP hydrolysis by Fun12 generates a relative movement of the head to the body of pre-40S r-particles that triggers the activation of Nob1 and, thus, the endonucleolytic cleavage of ITS1 at site D (discussed in [26]). Nob1 activity is also stimulated by a still unknown ATPase [26]. Wild-type 80S-like particles are not competent for translation; thus, the alternative fate to maturation is degradation. Final maturation of pre-60S r-particles includes 6S pre-rRNA processing to mature 5.8S rRNA (not shown). Note that the precise timing of most of the events shown in this cartoon has not yet been defined. B. In rpl3[W255C] cells, the conformational changes caused by the mutation W255C in L3 (white dot on the L3 drawing) might impair the GTP-dependent stimulation of 20S pre-rRNA processing exerted by Fun12/eIF5B within 80S-like particles. Our data also show that the 80S-like r-particles containing the L3[W255C] protein efficiently translate, thereby preventing their degradation.