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. 2016 Jun 6;7:11390.
doi: 10.1038/ncomms11390.

Involvement of Human Ribosomal Proteins in Nucleolar Structure and p53-dependent Nucleolar Stress

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Free PMC article

Involvement of Human Ribosomal Proteins in Nucleolar Structure and p53-dependent Nucleolar Stress

Emilien Nicolas et al. Nat Commun. .
Free PMC article

Abstract

The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology.

Conflict of interest statement

A patent (EP16168087.1) naming D.L.J.L., C.D.V., P.P. and E.N. as inventors has been filed (EP16168087.1) by the Université Libre de Bruxelles and Université Catholique de Louvain that protects a method for the quantitative and qualitative analysis of nucleolar structure and the index of nucleolar disruption (iNo) index.

Figures

Figure 1
Figure 1. Systematic screening of human r-proteins reveals that uL5 (RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar structure maintenance.
(a) Experimental strategy: all 80 r-proteins were depleted one by one in human cells by use of specific siRNAs. The nucleolar structure (fluorescence microscopy), the accumulation of mature 18S and 28S rRNAs (electropherograms), pre-rRNA processing (high-resolution northern blotting), and steady-state accumulation of p53 (fluorescent western blotting) were monitored. (b) PCA showing a classification of r-proteins according to their requirement for nucleolar structure maintenance. Each r-protein was depleted in three knockdown experiments, each performed with a different siRNA. The image-processing algorithm that we designed for this analysis involves selecting five discriminant shape and textural features, computing five dk values, and reducing the five dimensions to two by PCA. In the resulting plot, each coloured dot represents one population of cells treated with one siRNA. Dot colour is indicative of the targeted protein: green for SSU r-proteins and magenta for LSU r-proteins. The mean of three populations of cells treated with a non-targeting control siRNA (SCR) is shown in red. Blue symbols represent the six calibration controls (FBL, GFP, nucleolin, nucleophosmin, MOCK and TIF1A, see Supplementary Fig. 1). Insets show images of the nuclei of cells depleted of representative proteins with the DNA stained in blue and the nucleoli appearing in green (FBL). For a few representative examples, a specific symbol is used (for example, a diamond for uL5). RPL, r-proteins of the LSU; RPS, r-proteins of the SSU. (c) r-proteins and calibration controls classified according to the severity of nucleolar disruption caused by their absence. The iNo was defined as the sum of the dk values of the five most discriminant shape and textural features identified in this work (Methods section). Higher iNo correspond to more severe disruption. Colour-coding as in b. The coloured dots are the means of three individual experiments (shown in grey). Note: the r-proteins are named according to a recently revised nomenclature where the ‘e' prefix stands for eukaryote-specific and ‘u' for universal (present in bacteria, archaea and eukaryotes).
Figure 2
Figure 2. Late-assembling r-proteins of the LSU are the strongest contributors to nucleolar structure maintenance and p53 homeostasis.
Three-dimensional (3-D) models of human ribosomal subunits based on protein data bank (PDB) entries 3J3D, 3J3A, 3J3F and 3J3B. The r-proteins are colour-coded according to the impact of their depletion on nucleolar structure (iNo values) (a), pre-rRNA processing (b) or the p53 steady-state level (c). Left, subunit interface views; right, solvent-exposed views. The aminoacyl (A), peptidyl (P) and exit (E) transfer RNA (tRNA) sites are indicated. Morphological features of the subunits are highlighted. On the LSU: the L1-stalk, CP and phospho-stalk (P-stalk). On the SSU, the beak (Be), head (H), platform (Pt), body (Bd), left foot (Lf) and right foot (Rf).
Figure 3
Figure 3. Quantitative monitoring of nucleolar morphology in different human cell lines based on detection of endogenous PES1.
The data show, for a selection of eight representative r-proteins, that the r-proteins contributing weakly or strongly to nucleolar structure maintenance are largely the same in multiple cell lines. (a) The indicated r-proteins were depleted with an siRNA for 3 days in two cervical carcinoma cell lines (HeLa-GFP-FBL, engineered to express green fluorescent FBL, and genetically unmodified HeLa), one colon carcinoma cell line (HCT116) and two lung carcinoma cell lines (A549 and H1944). Endogenous PES1 was detected by immunostaining with a specific antibody (Methods section). As a control, cells were treated with a non-targeting control siRNA (SCR) and depleted of nucleophosmin (NPM; Supplementary Fig. 1). (b) Values of the nucleolar disruption index (iNo) obtained after 3 days of siRNA-mediated depletion of the indicated r-protein as calculated on the basis of the endogenous PES1 signal.
Figure 4
Figure 4. Involvement of human r-proteins in pre-rRNA processing.
(a) The 28S/18S ratio calculated from Agilent bioanalyzer electropherograms. Data are shown for the two different siRNAs used (siRNA #1 and #2). (b) Major pre-rRNA intermediates and probes used in this work. Three of the four rRNAs are produced by RNA Pol I as a long 47S primary transcript. The 18S, 5.8S and 28S rRNAs are separated by noncoding external (ETS) and internal (ITS) transcribed spacers. Probes a, b and c are the oligonucleotides LD1844, LD1827 and LD1828, respectively (Methods section). (c) Pre-rRNA processing inhibitions after depletion of SSU r-proteins. On the northern blots (www.RibosomalProteins.com, Supplementary Figs 5,6 and 11), all RNA species were quantified with a Phosphorimager, normalized with respect to the non-targeting control (SCR), and their abundances represented on a heatmap using the colour code indicated. The heatmap profiles were clustered with ‘R' and the corresponding proteins grouped in classes of r-proteins affecting the same or similar processing steps. The different siRNAs used are indicated (#). Asterisks (*) refer to r-proteins assigned to two groups according to the siRNA used. (d) As in c for LSU r-proteins.
Figure 5
Figure 5. The central protuberance assembly factors BXDC1 and RRS1 are required for nucleolar structure integrity.
(a) Cells expressing FBL fused to the GFP were treated for 3 days with an siRNA targeting transcripts encoding the indicated protein. Two independent siRNAs (#1 and #2) were used in each case. Cells treated with a non-targeting (SCR) siRNA control are shown for reference. (b) For each depletion, the nucleolar disruption index (iNo) was calculated (see Fig. 1 and Methods section).
Figure 6
Figure 6. Involvement of human r-proteins in p53 homeostasis.
(a) Steady-state level of p53 determined by quantitative fluorescent western blotting. Western blots analysis are shown for representative r-proteins, with the p53 level indicated underneath as a mean of biological triplicates obtained after treatment of cells with the same siRNA (i, ii and iii). The siRNA used was selected on the basis of its proven efficacy in the processing and nucleolar screens (Figs 1 and 4). The p53 signal corrected for loading (using β-actin as reference) was expressed with respect to the level observed in cells treated with a non-targeting siRNA control (p53+/+). Red signal, p53; green signal, β-actin. A complete data set for all 80 r-proteins is available at www.RibosomalProteins.com and in Supplementary Fig. 11. As loading control we used HCT116 p53+/+ cells transfected with a non-targeting siRNA (p53+/+) providing the basal level of p53 or with an antisense oligonucleotide suppressing the activity of the box C/D snoRNA U8 (#U8), thereby stimulating p53 accumulation up to sixfold (D.L.J.L. submitted). As background control, we used a matched isogenic HCT116 cell line that does not express p53 (HCT116 p53−/−, (ref. 39)) treated with a non-targeting siRNA (p53−/−). (b) r-proteins classified according to their impact on the p53 steady-state level. The non-targeting (SCR) control is shown in red, the SSU r-proteins in green, and the LSU r-proteins in magenta. The histogram bars are the means of triplicates with s.d. r-proteins whose depletion leads to a fivefold increase in p53 level are highlighted in a grey box.

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