Loss-of-function genetic screening identifies a cluster of ribosomal proteins regulating p53 function
Loss-of-function genetic screening identifies a cluster of ribosomal proteins regulating p53 function
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DOI：
10.1093/carcin/bgm302
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发表时间：
2008-07-01
期刊：
影响因子：
4.7
通讯作者：
Carnero, Amancio
中科院分区：
文献类型：
--
作者：
Castro, Maria E.;Leal, Juan F. M.;Carnero, Amancio
条件小鼠p53（p53 val 135）和致癌ras双p53/p21-null小鼠胚胎成纤维细胞（MEFs）的介绍表明，p21 waf 1是不需要联合ras/p53诱导衰老样生长停滞。我们使用这种细胞系统来识别在p21不存在的情况下ras-p53诱导衰老的关键参与者。应用逆转录病毒为基础的遗传筛选，我们获得了针对一组14种不同的核糖体蛋白的mRNA反义片段，这些蛋白的功能丧失绕过了p53诱导的生长停滞。核糖体蛋白反义片段的表达降低了p53的转录活性。用eGFP-p53嵌合体进行的实验表明，这种作用是由p53的减少介导的。为了研究p53是否被MDM 2依赖性降解下调，我们测试了RP反义在双p53/MDM 2缺失MEFs中的作用，并观察到在不存在MDM 2的情况下，RP水平的降低也降低了p53水平。因此，尽管我们不能排除其他未知的机制，我们认为核糖体蛋白水平的降低可能会抑制p53特异性翻译。最后，比较肿瘤中mRNA水平与相同器官和患者的正常组织中mRNA水平的定量分析显示，不同百分比的肺、前列腺或结肠肿瘤具有降低的测试RP水平。有趣的是，在大多数情况下，核糖体蛋白mRNA的减少仅发生到50%。我们的数据表明，核糖体蛋白质的不平衡可能有助于p53的调节，通过核糖体生物发生检查点。
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 down-regulated 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.