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An approach to analyse the specific impact of rapamycin on mRNA-ribosome association

机译：分析雷帕霉素对mRNA-核糖体结合的特异性影响的方法

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Background Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background. Methods We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out). For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation. Results High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug. Conclusion The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of a particular drug in a living cell.
机译：背景技术最近使用细胞培养模型系统和肿瘤来源的细胞系的工作表明，mRNA群体多核糖体的差异募集可能足以启动和维持肿瘤的形成。因此，正在进行大量努力来使用高密度微阵列图谱来建立暴露于确定药物方案的细胞的分子指纹。这些药物基因组学方法的目的是提供有关药物如何在限定的细胞背景内影响翻译读数的新信息。方法我们描述了一种方法，该方法可以在短时间暴露于药物的体内分析de-novo mRNA-核糖体缔合。它结合了高渗休克，多核糖体分级分离和高通量分析，以提供翻译反应性转录本的分子表型。与先前的翻译谱分析研究相比，由于消除了药物的次级作用（例如，对转录读出），该方法提供了更高的特异性。对于该试验性的“原理验证”试验，我们选择雷帕霉素药物是因为其对翻译起始的广泛研究影响。结果对轻和重多态性部分的高通量分析已鉴定出mRNA，其重新招募至游离核糖体后对短时间暴露于雷帕霉素有反应。使用实时RT-PCR已经证实了微阵列的结果。 TOP转录本的选择性下调也与以前使用该药物进行的翻译谱研究一致。结论本手稿中概述的技术进步为对影响翻译起始的mRNA特性提供了新的见解，并提供了在任何细胞类型中和存在多种目标药物的情况下，转录本-核糖体缔合的分子指纹。临床前定义的此类分子表型可能最终影响活细胞中特定药物的评估。

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