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.
Highlights
The nucleolus is a potent disease biomarker and a target in cancer therapy
Ribosomopathies are cancer predisposition syndromes caused by ribosome biogenesis dysfunction[7], due to mutations in r-proteins or ribosomal assembly factors. r-proteins are intimately linked to tumourigenesis, being directly involved in regulating the steadystate level of the anti-tumor protein p53
In an attempt to correlate the effects of r-protein depletion on nucleolar structure with defects in ribosome biogenesis, we determined which r-proteins are essential to pre-rRNA processing (Fig. 4)
Summary
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. A second group, beneath the SCR control, comprises proteins whose depletion did not alter the nucleolar structure but reduced the fluorescence intensity (for example, control cells treated with siRNAs against GFP or FBL, see Supplementary Fig. 1). We selected eight representative r-proteins, depleted them for 3 days in each of the five cell lines and monitored nucleolar structure by immunostaining of endogenous PES1 and iNo score computation (Fig. 3).
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