The ‘stalk’ is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.
- ribosomal P1 and P2 proteins
- RNA interference (RNAi)
- subunit joining
Eukaryotic ribosomes contain four RNA molecules, and approx. 80 proteins make up the small 40 S and large 60 S subunit. As key components of the translation machinery, ribosomes are central to many cellular processes . In both prokaryotes and eukaryotes, a lateral protuberance projects from the large subunit known as the ‘ribosomal stalk’ . This structure is an active ribosomal domain that directly interacts with the soluble translation factors, regulating their activity during the course of protein synthesis . The eukaryotic ribosomal stalk is composed of ribosomal P proteins that usually exist as phosphoproteins in the cell. A 32 kDa protein P0 is located at the stalk base, and it forms a complex with several highly conserved 12 kDa proteins with very acidic (3–4) pI values. On the basis of primary sequence similarities, these proteins are classified as P1 and P2 proteins , and an additional P3 group is also found in plants . The stalk structure is formed by two heterodimers (P1/P2)2 [6,7], with P1 anchoring the complex to the P0 core ribosomal protein [8–10], which is, in turn, attached to 28 S RNA . These acidic ribosomal P proteins are the only molecules in the ribosome that exist in multiple units, although they are also found in a free state in the cytoplasm. In yeast [12,13], plants  and mammals , the free proteins have been shown to participate in an exchange process between the ribosome-bound P1/P2 and a cytoplasmic pool of these proteins. Indeed, this exchange seems to be connected with ribosome-modulating mechanisms in which the ribosomal stalk participates in yeast . Therefore the stalk can exist in multiple configurations [17–19], and altering the P1/P2 composition of the stalk, mainly through its acidic P-protein content  and/or the phosphorylation of these components [21,22], can influence the activity of the ribosome and provoke global changes in protein synthesis [19,23].
Most information on the possible regulatory functions of the ribosomal stalk has been obtained in Saccharomyces cerevisiae (baker's yeast). Intrinsic problems in performing genetic manipulation in higher eukaryotes has made it difficult to investigate whether the stalk fulfils similar roles in mammalian cells. However, gene silencing with RNAi (RNA interference) enables the function of specific proteins to be investigated in established cell lines. Thus permanent transfected cell lines expressing plasmid-encoded shRNAs (short-hairpin RNAs) can lead to partial silencing of genes.
Using direct transient transfection of carcinoma cell lines with antisense oligonucleotides, approx. 60% silencing of the P2 protein has been achieved . However, this level of inhibition is probably not sufficient to perform a convincing analysis of the function of the silenced proteins. In the present study the importance of the stalk acidic proteins in translation has been investigated by generating stable human cell lines in which the P2 protein could be conditionally suppressed by up to 95% with RNAi. Using this approach, we show that RNAi expression directly reduces P2 mRNA and protein levels, which also results in a parallel decrease in the ribosomal P1 protein. Moreover, conditional depletion of P2 mRNA and protein also reduces the growth rate of the cells. We present evidence that the phenotype produced by P1/P2 depletion is due to the effects on the capacity of the 60 S ribosomal subunit to associate with the 40 S subunit.
MATERIALS AND METHODS
Cell lines and culture conditions
HEK-293T (human embryonic kidney-293T) cell line (from A.T.C.C., Manassas, VA, U.S.A.) and the newly generated TTR4 and SP23 cell lines (see below) were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with 10% (v/v) heat-inactivated foetal bovine serum, 100 i.u./ml penicillin G and 100 μg/ml streptomycin. The cells were maintained in a humidified incubator at 37 °C with 5% (v/v) CO2, and they were passaged regularly at subconfluence.
The target-specific siRNA (small interfering RNA) duplexes were designed with the online siRNA Target Finder software (Ambion; http://www.ambion.com/techlib/misc/siRNA_finder.html), and the sequence selected to generate siRNA corresponding to the 369–387 region of the P2 mRNA sequence . The pSU-PERIOR.puro (OligoEngine, Seattle, WA, U.S.A.) vector for inducible expression of siRNA was digested with BglII and HindIII, and the oligonucleotides (GATCCCCGGAGGAGTCTGAAGAGTCATTCAAGAGATGACTCTTCAGACTCCTCCTTTTTA and AGCTTAAAAAGGAGGAGTCTGAAGAGTCATCTCTTGAATGACTCTTCAGACTCCTCCGGG) were ligated into the vector to generate pSUPERIOR.puro-P2. The 19-nucleotide target sequence for P2 is indicated in bold typeface within the oligonucleotide sequences.
Generation of a stable TTR4 cell line
Human HEK-293T cells were transfected with the TetR (tetracycline repressor)-expressing vector pcDNA6/TR (Invitrogen), which contains a blasticidin selection marker, using the jetPEI™ (Qbiogene, Carlsbad, CA, U.S.A.) transfection reagent according to the manufacturer's instructions. After 48 h, the cells were transferred to medium containing 10 μg/ml blasticidin hydrochloride (Sigma–Aldrich, St Louis, MO, U.S.A.) for 2 weeks. Single clones were then isolated and expanded for an additional 2 months in medium containing 10 μg/ml blasticidin, then analysed by PCR.
Generation of Dox (doxycycline; Sigma–Aldrich)-inducible P2 protein silencing cell line (SP23 clones)
TTR4 cells were transfected with pSUPERIOR.puro-P2 plasmid and selected with 1 μg/ml puromycin (Sigma–Aldrich) for 2 weeks. Single clones were isolated and expanded for an additional 2 months in the presence of the antibiotic and analysed by PCR. The cells (stable clones) were treated with different concentrations of Dox for the times indicated, and the P1 and P2 mRNA and proteins they expressed were then analysed.
After treatment with and without Dox for the different times indicated, subconfluent cell monolayers were washed twice with ice-cold PBS before they were removed from the dish by trypsinization. The cells were resuspended in a buffer containing 15 mM Tris/HCl, pH 7.4, 80 mM KCl, 5 mM MgCl2, 1% (v/v) Triton X-100 and protease inhibitors [2.5 μg/ml each leupeptin, pepstatin, bestatin, aprotinin, chymostatin and antipain (Sigma–Aldrich), and 1 mM PMSF], and they were disrupted by ten passages through a 20-gauge syringe needle. SDS/12.5%-(w/v)-PAGE gels were loaded with 10 μg of protein or 20 μl of the sucrose fractions, and the separated proteins were then transferred to PVDF membranes (Immobilon-P; Millipore, Milford, MA, U.S.A.), which were probed with a monoclonal antibody, 3BH5, specific to the highly conserved C-terminus of eukaryotic acidic P-protein . A horseradish-peroxidase-conjugated rabbit anti-mouse antiserum was used as a secondary antibody. The amounts of P0, P1 and P2 protein were quantified from the Western blots by scanning densitometry using NIH Image version 1.29 software. All results were calculated as the percentage of protein expression (P1 or P2 protein normalized to the P0 protein) in Dox-induced cells versus control cells.
Real-time quantitative RT (reverse transcription)–PCR analysis
Total RNA was isolated using a RNeasy mini kit (Qiagen, Valencia, CA, U.S.A.) according to the manufacturer's instructions, and the RNA recovered was quantified by measuring the absorbance at 260/280 nm using a Nanodrop™ ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, U.S.A.). An equal amount of purified RNA was examined in triplicate from each sample in each experiment. Real-time quantitative RT–PCR was performed using LightCycler® RNA Master SYBR Green I (Roche, Basel, Switzerland) on the LightCycler® 2.0 System (Roche). The primers used in the present study were those retrieved from PrimerBank [http://pga.mgh.harvard.edu/primerbank/; PrimerBank IDs: P0 (16933546a1), P1 (4506669a1) and P2 (4506671a1)] and they were purchased from Isogen Life Science (IJsselstein, The Netherlands). The reaction was carried out in a 20 μl volume, and it included an initial reverse transcription at 61 °C for 20 min, followed by an incubation at 95 °C for 30 s and 45 amplification cycles of: 1 s at 95 °C followed by 5 s at 60 °C and finally 7 s at 72 °C. The products amplified from each reaction were analysed using LightCycler Relative Quantification Software v.1.0 (Roche). PCR efficiencies (E) were calculated based on the slope of the relationship between the log input RNA versus Ct (the threshold-cycle value, defined as the point where the fluorescence exceeds the background threshold level and it is determined as the second derivative maximum): The relative expression between Dox-treated and control cells was quantified by the Pfaffl method  using P0 as a reference gene: The following equation was used to determine the relative quantification of a target gene in comparison with a reference gene (P0) in control cells:
To analyse cell proliferation, 5×105 cells were cultured for 3 days before exposing them to different concentrations of Dox. The number of cells (N) was counted daily using a Neubauer cell counter, and the DT (doubling time) was calculated from the regression analysis of the growth curves: where t indicates the length of time, N denotes the cell number, N0 is the number of cells at zero time (t=0) and k is a proliferation rate coefficient that was estimated individually for each exponentially growth curve.
The effect of silencing P2 by Dox induction on cell viability was assessed using a Trypan Blue exclusion assay. Prior to trypsinization, medium was collected from all the cultures in order to include any cells that had detached during treatment. Cells were then combined with 0.4% Trypan Blue at a 1:1 ratio and counted to assess their viability.
Cells were harvested after the treatments and time periods indicated and were analysed by flow cytometry. The cells were fixed with methanol, treated with 50 μg/ml RNase A and stained with 50 μg/ml of propidium iodide (PD Pharmigen). Their DNA content was analysed on a FACSCalibur flow cytometer (BD Biosciences). For each sample, 20000 cells were used, and the data were analysed with the CELLQuest software (Beckton–Dickinson). The percentage of cells in each phase of the cell cycle was determined as a ratio of the fluorescence area of the appropriate peaks to the total fluorescence area.
Analysis of polysomes
Ribosome profiles were prepared from a total of 2×107 cells at 80% confluence, which were washed with ice-cold PBS containing 100 μg/ml cycloheximide (Sigma) to block ribosomes in the elongation step. Cells were lysed with buffer A (15 mM Tris/HCl, pH 7.4, 80 mM KCl, 5 mM MgCl2 and 100 μg/ml cycloheximide), containing 1% (v/v) Triton X-100, 40 units/ml RNasin (a recombinant mammalian RNase inhibitor) and protease inhibitors (see above). Cytoplasmic extracts were obtained after centrifugation at 15000 g for 30 min at 4 °C, they were loaded on to a linear 10–50% (w/v) sucrose gradient in buffer A and then centrifuged at 39000 rev./min in an SW40 Ti rotor (Beckman Coulter) for 2 h 15 min at 4 °C. Gradients were fractionated by upward displacement with 87% (v/v) glycerol in an ISCO density-gradient fractionator, and the A260 was monitored continuously using an ISCO UA-5 UV monitor. The area under the profile in each peak (subunits, monosomes and polysomes) was calculated and the background signal was subtracted. The overall translation efficiency was calculated as the area under the curve representing two or more ribosomes (polysomal RNA) divided by the total area under the curve . The average number of ribosomes per mRNA in the polysomes (with two or more ribosomes attached) was estimated from the polysome profiles, summing the product of the area of each peak multiplied by the number of ribosomes at that peak and dividing by the total area of polysomes. The relative ribosome content was calculated as the area under the curve representing ribosomes (one or more ribosomes) divided by the area under the subunit peaks (40 S and 60 S).
In ribosome-dissociation experiments, cycloheximide was omitted from all the buffers, and the KCl concentration in the cytoplasmic extracts obtained after centrifugation was increased to 0.5 M (high salt). Ribosomal subunits were resolved by centrifugation at 39000 rev./min in an SW 40 Ti rotor (Beckman Coulter, Fullerton, CA, U.S.A.) for 3 h 30 min at 4 °C on a 10–30% (w/v) sucrose gradient containing 15 mM Tris/HCl, pH 7.4, 500 mM KCl and 5 mM MgCl2.
In the steady-state subunit-joining analysis, the cells were lysed with buffer B (15 mM Tris/HCl, pH 7.4, 80 mM KCl and 0, 1, 2.5 or 5 mM MgCl2), containing 1% (v/v) Triton X-100, 40 units/ml RNasin and protease inhibitors. The lysates were loaded on to a linear 10–30% sucrose gradient in buffer B, centrifuged at 39000 rev./min in an SW Ti 40 rotor for 3 h 30 min at 4 °C and fractionated. To quantify the association between ribosomal subunits from either control and P1/P2-depleted ribosomes, the peak areas from the sucrose gradient profiles were calculated thus:
The results were analysed using Student's t test, and P values of less than 0.05 were considered statistically significant.
Selection of stable Dox-inducible P-protein-silencing cell lines
TTR4 cells transfected with pSUPERIOR.puro vector (Oligo-Engine) were selected with puromycin. Stably transfected SP14 (see the supplementary online material at http://www.BiochemJ.org/bj/413/bj4130527add.htm) and SP23 cells containing the P1 and P2 constructs were assayed by exposing them to Dox (10 μg/ml) for 2 days. Among the SP14 transfectants, only one clone showed a ∼60% decrease in P1 protein and a parallel decrease in P2 protein of ∼40% (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/413/bj4130527add.htm). By contrast, three SP23 transfectants showed a decrease in P2 protein levels after Dox exposure above 90% (clones SP23-5, SP23-8 and SP23-10; Figure 1A) and, notably, the amount of P1 proteins was also decreased when P2 was silenced in these clones. The effect of different Dox concentrations on P2 accumulation was tested by Western blotting of SP23-5 cells (Figure 1B) and, accordingly, Dox concentrations as low as 0.05 μg/ml significantly decreased P2 accumulation in these cells. Thus the SP23-5 clone was used in the following experiments as a Dox-inducible P2-silencing cell line to induce P1/P2 protein depletion.
In human cells, P1 Protein is unstable in the absence of P2
To quantify stalk-protein depletion, SP23-5 cells were treated with 0 (control), 0.1 or 10 μg/ml Dox for 4 days and the accumulation of P0, P1 and P2 proteins was assessed. Since the amount of P0 protein remained unchanged, this protein was subsequently used to normalize the values of P1 and P2 protein. A mean decrease in P2 protein of 90 and 95% was observed in cells induced with either 0.1 or 10 μg/ml Dox respectively (Figure 1C). Significantly, in these same cells, the amount of P1 protein also fell by 70% or 85% respectively (Figure 1C; see also Figures 1A and 1B). Thus both acidic stalk proteins were depleted in these cells following Dox induction.
To confirm that P2 expression was indeed silenced, the individual mRNAs present were measured by real-time RT–PCR (Figure 1D). Within 4 days of Dox induction, there was a dramatic decrease in P2 mRNA transcripts (Figure 1D), with a depletion of P2 mRNA above 70% in the presence of 0.1 μg/ml Dox and of around 90% with 10 μg/ml Dox. Therefore, the loss of the P2 protein was correlated with a decrease in the P2 mRNA available following exposure to Dox. By contrast, the amount of P1 mRNA expressed, which is notably higher than that of P2 mRNA in the controls (P1 mRNA/P0 mRNA 10.4±5.8; P2 mRNA/P0 mRNA 0.7±0.3), is even higher after Dox treatment, suggesting that cells try to compensate for P1 protein depletion by increasing the transcription of RPLP1, the gene coding for P1.
Depletion of P1/P2 proteins reduces the proliferative capacity of the induced cells
The number of cells was monitored in order to analyse the effects of suppression of P1/P2 on cell proliferation (Figure 2). When the SP23-5 cells were cultured with Dox at different concentrations (from 0.1 to 10 μg/ml), their capacity to proliferate was significantly diminished when compared with the control uninduced cells. Moreover, on the removal of Dox, the proliferation of the SP23-5 cells recovered to control levels (Figure 2A). In order to quantify these changes, the DT of the cells was calculated from the growth curves (Figure 2B). Thus the DT of the SP23-5 cells increased from 21.9±0.2 h to 44.7±2.9 h following P1/P2 depletion on exposure to 0.1 μg/ml Dox, the concentration used in the following experiments. This low concentration of Dox (0.1 μg/ml) had no effect on the proliferation of TTR4 cells (results not shown). When Dox was removed from the medium, the DT returned to 29.4±1.6 h after 6 days or to 22.4±1.9 h after 12 days, indicating that the effect of P1/P2 depletion on the rate of cell growth was reversible. To test whether the suppression of the acidic stalk proteins induced by Dox could also be reverted, a time-course study of P2 silencing was performed. The kinetics of P2 silencing was examined in cells by Western blotting and was found to follow a time course similar to that of the cell-growth data (Figure 2C). Therefore, P2 depletion and its effect on cell growth are inducible and reversible.
Depletion of P1/P2 proteins does not affect cell viability or the proportion of cells in the different phases of the cell cycle
The decreased growth of SP23-5 cells after P1/P2 depletion prompted us to evaluate the viability of the cells after Dox induction. At all concentrations and time points evaluated, the viability of P1/P2-depleted cells remained above 90%, and this did not differ significantly from that of the control cells (91±4%; Figure 3A). Thus there was no indication that P1 and P2 protein depletion induced cell death.
The effect of P1/P2 depletion on the cell cycle was also evaluated after a 4-day induction of SP23-5 cells with 0.1 μg/ml Dox. There was clearly no difference between the number of cells in the different phases of the cell cycle in control and P1/P2-depleted cells (Figure 3B). Therefore, acidic-stalk-protein depletion did not affect the distribution of the cells in the distinct phases of the cell cycle. Furthermore, the apoptotic cells identified (sub-G0 population) were ∼6% in both groups, confirming that there was no increase in cell death following P1 and P2 protein depletion.
Effect of P1/P2 depletion on translation efficiency
The polysome profiles were examined to determine whether the decreased growth rate of P1/P2-depleted cells was due to a defect in some aspect of ribosome synthesis, assembly or function. In these assays, the number of ribosomes found within the polysomal mRNA fraction (mRNA containing two or more ribosomes) is a reflection of de novo protein synthesis . The control ribosomal profile shows the translating polysomes (the 80 S monosome peak harbours translating single 80 S ribosomes and inactive couples), and free 60 S and 40 S subunits respectively (Figure 4A, left panel). In comparison with the control cells, the P1/P2-depleted cells show a decrease in the total amount of monosomes and polysomes, but not in the number and the relative proportion of the polysome peaks (Figure 4A, right panel). Furthermore, a decrease in the 80 S peak was accompanied by the appearance of half-mers [29,30] and an increase in the amount of free 60 S subunits. These results, especially the obvious decrease in the amount of polysomes, suggest that the decreased growth rate of P1/P2-depleted cells was compatible with the diminished rate of translation initiation .
The proteins extracted from gradient fractions were resolved by SDS/PAGE and analysed in Western blots (Figure 4B). The P0 protein clearly co-sedimented with free 60 S, 80 S and polysome fractions and was absent from cytosolic and free 40 S fractions from control and Dox-treated cells. By contrast, free P1 and P2 proteins were detected in the cytosolic fractions as well as in the particles where P0 was detected. P1 and P2 proteins were depleted in all ribosome-containing fractions (4–10) in Dox-induced cells when compared with control cells, indicating the presence of active ribosomes lacking P1 and/or P2 proteins in the polyribosomes of P1/P2-depleted cells. It is noteworthy that the amount of P1 protein found at the top of the gradient, which corresponds to the free cytoplasmic pool, is much less affected in the Dox-treated cells.
The overall translation efficiency is defined as the proportion of rRNA participating in polysomes . Thus, to determine whether depletion of P1/P2 proteins altered translational efficiency, the amount of rRNA actively involved in translation (associated with polysomes) was measured. In control cells, 54±3% of RNA was in polysomes, and this value decreased to 43±4% after acidic-protein depletion (Figure 4C). Thus, there was a reduction in global translation of ∼20%.
An analysis of the polysome profiles showed that the depletion in P1/P2 proteins did not change the distribution of the polysomal mRNA (Figure 4A). Thus the average number of ribosomes per translated transcript (mRNAs containing two or more ribosomes) was not altered (5.0±0.1 in both cases; Figure 4D). By contrast, the relative ribosomal content, estimated from the polysome profiles as the ratio of ribosomes (monosomes and polysomes) and subunits (40 S and 60 S), decreased from 5.2±0.3 to 3.4±0.4 (Figure 4E), indicating a defect in the equilibrium between ribosomes and subunits.
Depletion in P1/P2 proteins makes ribosomal subunit joining less efficient
The total amount of 40 S and 60 S ribosomal subunits was analysed in control and P1/P2-depleted cells after treatment with 0.1 μg/ml Dox for 4 days. The cell extracts were treated with high K+ (500 mM KCl) in the absence of cycloheximide in order to fully dissociate the 80 S ribosomes and, in this way, the total amount of the 40 S and 60 S ribosomal subunits could be estimated . P1/P2 protein depletion did not affect the steady-state levels of 40 S and 60 S ribosomal subunits (Figure 5A).
The decrease in the relative ribosome content (Figure 4E), the production of half-mers in P1/P2-depleted cells and the accumulation of free 60 S subunits (Figure 4A), while the overall subunit stoichiometry remained unchanged (Figure 5A), strongly suggested that the low levels of P1 and P2 proteins made subunit joining less efficient. To confirm this suggestion, the steady-state association capacity of 40 S and 60 S ribosomal subunits in control and P1/P2-depleted cell extracts was tested by sucrose-gradient centrifugation at different concentrations of Mg2+ (Figure 5B). As expected, 80 S ribosomes totally dissociated in the absence of Mg2+ in both cell types tested. The 40 S/60 S ratio showed average values of 2.8±0.3 and 2.6±0.3 for control and Dox-treated cells respectively, confirming that the steady-state ratio of 40 S and 60 S subunits was not affected by P1/P2 depletion. Increasing the Mg2+ concentration in the gradients from 1 to 5 mM highlighted the different extents of subunit association, which was always lower in the Dox-treated sample than in the corresponding controls (Figure 5B). This effect was most evident when the proportion of associated subunits was plotted as a function of the Mg2+ concentration (Figure 5C). As expected, the extent of association increases as a function of the Mg2+ concentration for both control and P1/P2-depleted cells. However, the association capacity of the ribosomes from P1/P2-depleted cells was notably lower (∼30%) than that of the control cells. Hence, it seems clear that the lack of P1 and P2 proteins clearly diminished the capacity of the 60 S subunit to form 80 S ribosomes in the cell extracts.
Through the Dox-induced expression of shRNAs targeted to mRNA encoding stalk ribosomal P2 protein, both P2 transcripts and protein have been decreased in stably transfected human cells to levels below 10% of those in control cells. This depletion of P2 was associated with a similar decrease in its partner ribosomal stalk component, the P1 protein, without a parallel decrease in P1 mRNA, which, rather, increased significantly. These results indicate that P1 was unstable in the absence of P2 in human cells, consistent with data obtained in S. cerevisiae. In yeast, free P1 protein is highly unstable, with a half-life of a few minutes, whereas P2 has a half-life of several hours. The yeast P1 protein is degraded by a proteosome-independent mechanism, and its association with P2 protein protects it from degradation . However, although P1 protein is not detected in yeast strains that lack the P2 protein , in human cells a significant amount of P1 is found in the supernatant when P2 is silenced in cells, considerably more than the residual amount of P2 detected (Figure 4B). This difference could be attributed to a higher intrinsic resistance to degradation of the human protein, possibly due to the lack of a serine residue at the second position in the N-terminus of human P1, an important residue in the yeast P1 degradation signal (; G. Nusspaumer, V. Briceño, M. Remacha and J. P. G. Ballesta, unpublished work). Alternatively, the large excess of P1 mRNA detected in the SP23 cell line (P1mRNA>P0mRNA≈P2mRNA), which is not seen in yeast , could result in an excessive accumulation of protein that might saturate the degradation system. The fact that P1 protein can be detected in a yeast strain lacking P2 protein when overexpressed, but not when it is expressed only from the endogenous gene , supports this possibility. However, further experiments will be required to determine how this phenomenon can be explained. Indeed, in contrast with yeast, where P2 proteins were not affected by P1 deficiency , the amount of P2 protein in human cells was decreased when P1 was depleted (Supplementary Figure S1), indicating that P2 was less stable in absence of P1.
As in yeast , human ribosomal P proteins preferentially form P1–P2 heterodimers  that attach to P0 through the P1 protein , forming the pentameric P0–(P1–P2)2 structure on the ribosome [20,36]. The interaction between P1 and P2 to form the P1–P2 heterodimer seems to be a crucial step for efficient assembly of the functional GTPase-associated centre, and P1 or P2 alone fails to bind efficiently to P0 . The results presented here are consistent with the assembly model whereby the soluble heterodimers P1–P2 bind to P0 on the ribosome. For this reason, P1 that is more resistant to degradation accumulates in the cytosol when the levels of P2 are too low to form heterodimers, even though the ribosomes are defective in P1/P2 proteins.
As found previously in S. cerevisiae , we can conclude that the ribosomal stalk P1 and P2 proteins are not a prerequisite for ribosome activity and protein synthesis in human cells. Indeed, particles lacking both proteins are incorporated into polysomes (Figures 4A and 4B, right panels). However, the absence of these ribosomal components does have consequences for the cell. Recent studies have shown that ribosome synthesis is intimately linked with cell growth and the regulation of the cell cycle . Although the details of this association are not yet understood, it is clear that the quality and quantity of ribosomes directly determine the growth rate of cells and, by extension, the timing of cell division. Indeed, a decrease in the cell growth rate was the most obvious consequence of the absence of acidic P proteins in cells, with doubling times increasing from 22 to 45 h. This inhibition was not correlated with a loss of cell viability, and there was no evidence of cell death when the sub-G0 phase in the cell cycle was evaluated, indicating that the decrease in cell growth was not due to cytotoxicity. Moreover, there was no arrest of P1/P2-depleted cells at any specific phase of the cell cycle, but, rather, they seemed to progress through it at a lower rate. It is therefore conceivable that the overall reduction in mRNA translation observed in cells lacking P1/P2 proteins affects genes involved in the cell cycle. This would explain the decreased rate of cell-cycle progression. However, these effects are reversible, and upon re-establishing P2 expression, the growth rate returns to normal.
After P1/P2 protein depletion, the polysome profiles show an increase in the 60 S peak as well as a reduction in 80 S and polysome peaks, without any apparent effect on the average number of ribosome per mRNA transcript. In addition, half-mers appear in polyribosomes, owing to a late-stage translational initiation defect in which 43 S preinitiation complexes are attached to an mRNA species that already contains at least one translating 80 S ribosome, but lacks the corresponding 60 S subunit . These structures were first described in vitro in the rabbit reticulocyte translation system after NaF treatment  and in vivo in Ehrlich ascites cells treated with anisomycin . Half-mers may reflect a stoichiometric ribosomal subunit imbalance caused by a diminished supply of 60 S subunits, resulting in an excess of 40 S over 60 S subunits [41–43]. Alternatively, half-mers may be generated without altering the subunit balance from impaired translation initiation, whereby inhibitors of translation initiation  or defective ribosomal components [30,31,44,45] prevent subunit joining. Thus half-mers are indicative of a defect in ribosomal subunit joining  and, coupled with the decrease in the amount of polysomes, their presence is compatible with defects at the level of translation initiation caused by depletion in P1/P2 proteins. However, the decrease in the average number of ribosomes per translated transcript when initiation is blocked [28,47,48] was not detected in our system, and thus it is possible that acidic-protein depletion also affects other translation steps. This is not unexpected, since bacterial stalk acidic proteins influence the activity of initiation, elongation and termination factors [3,49].
The ratio between free 60 S and 40 S ribosomal subunits in polyribosome profiles revealed an increase in the amount of free 60 S ribosomal subunits relative to free 40 S subunits in P1/P2-depleted cells. However, when the 40 S/60 S ratio was more closely examined after ribosome dissociation on sucrose gradients with either high K+ or in the absence of Mg2+, no changes were detected. These results indicate that the biogenesis of the subunits was not affected by P1/P2 protein depletion, and thus a subunit imbalance is unlikely to be the cause of half-mer formation. Furthermore, the polysome profile of P1/P2-depleted cells showed a significant decrease in the relative ribosome content (accumulation of subunits). Consequently, the absence of P1/P2 proteins could produce defective steady-state subunit joining and, indeed, a decrease in ribosomal subunit association in P1/P2-depleted cells was directly confirmed by sedimentation analysis at different Mg2+ concentrations. Thus 60 S subunits depleted of acidic proteins were ∼30% less efficient in joining to 40 S subunits and forming 80 S ribosomes at all Mg2+ concentrations tested. This illustrates the importance of the contribution of P1/P2 proteins to the subunit-joining activity. In addition, a global decrease in translation efficiency that would be expected to contribute to a depletion of many proteins could account for the reduced growth rate of the P1/P2-depleted cells. This presumably results from a decrease in the rate at which ribosomal subunits can be loaded on to mRNA as a consequence of the impaired association of ribosomes depleted of P1/P2 proteins. Thus the P1/P2 content of the stalk could modulate the joining of the 60 S ribosomal subunit. Although it is widely accepted that translation initiation is the rate-limiting step for translation, and our results show that depletion of P1/P2 proteins significantly affect the ribosomal subunit joining, we cannot rule out an additional effect of the P1/P2 depletion on the elongation step of translation.
Data obtained from S. cerevisiae strongly suggest that the ribosomal stalk could function as a translational regulator of specific mRNAs . The results presented here indicate that this regulation could be achieved by modulating ribosomal subunit association. In agreement with this notion, several studies have indicated that translation can be regulated at the subunit-joining stage [50–52].
We thank Mr M. C. Fernández Moyano for expert technical assistance. This work was supported by the Ministerio de Educación y Ciencia, Spain (grant BFU2006-00365 to J. P. G. B. and grant BFU2004-03079 to M. R.) and by an institutional grant from the Fundación Ramón Areces, Spain (a grant to the Centro de Biologia Molecular ‘Severo Ochoa’).
Abbreviations: Dox, doxycycline; HEK-293T, human embryonic kidney-293T; RNAi, RNA interference; RT-PCR, reverse transcription–PCR; shRNA, short-hairpin RNA; siRNA, small interfering RNA
- © The Authors Journal compilation © 2008 Biochemical Society