Loss-of-function genetic screening identifies a cluster of ribosomal proteins regulating p53 function.
Introduction of conditional murine p53 (p53val135) and oncogenic ras into double p53/p21-null mouse embryonic fibroblasts (MEFs) showed that p21waf1 was not required for combined ras/p53-induced senescent-like growth arrest. We used this cellular system to identify key players in the ras-p53-induced senescence in the absence of p21. Applying a retroviral-based genetic screen, we obtained mRNA antisense fragments against a cluster of 14 different ribosomal proteins which loss of function bypasses p53-induced growth arrest. The expression of the ribosomal protein antisense fragments reduced the transcriptional activity of p53. Experiments with eGFP-p53 chimeras suggest that the effect is mediated by a reduction of p53. To study whether p53 was downregulated by MDM2-dependent degradation, we tested the effect of the RP antisenses in double p53/MDM2-null MEFs and observed that in the absence of MDM2, reduction of the RP levels also decreases p53 levels. Therefore, although we cannot discard other unknown mechanism, we suggest that the decrease in the levels of ribosomal proteins might inhibit p53-specific translation. Finally, quantitative analysis comparing levels of mRNA in tumours versus mRNA in normal tissue of the same organ and patient showed that a variable percentage of lung, prostate or colon tumours have reduced levels of the RPs tested. Interestingly, in most cases, the reduction of ribosomal protein mRNAs occurs only to 50%. Our data suggest that ribosomal protein imbalance might contribute to p53 regulation through the ribosomal biogenesis checkpoint.
Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncológicas, C/ Melchor Fernández Almagro, 3, 28029 Madrid, Spain.
Maria E
ME
Castro
Juan F M
JF
Leal
Matilde E
ME
Lleonart
Santiago
S
Ramon Y Cajal
Amancio
A
Carnero
D000818:Animals; D048708:Cell Growth Processes / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*
D016372:RNA, Antisense; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D016159:Tumor Suppressor Protein p53