Hypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here, we describe a workflow for measurement of ribosome load of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells reveals extensive translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation are limited, and we do not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage are associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR reveal that specific classes of HIF1A and HIF2A transcriptional target gene manifest different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer.

缺氧诱导因子 (HIF) 和哺乳动物雷帕霉素靶标 (mTOR) 通路协调对氧气和营养可用性的反应。这些通路在癌症中经常失调，但人们对它们之间的相互作用知之甚少，部分原因是同时测量整体翻译和 mRNA 特异性翻译存在困难。在这里，我们描述了测量由转录起始位点 (TSS) 解析的 mRNA 核糖体负载的工作流程。其在肾癌细胞中的应用揭示了 mTOR 的广泛翻译重编程，强烈影响许多代谢酶和途径。相比之下，HIF 对翻译的整体影响有限，并且我们没有观察到 HIF2A 的翻译激活报告。相比之下，TSS 使用中 HIF 依赖性的改变与基因子集中翻译效率的剧烈变化相关。对 HIF 和 mTOR 相互作用的分析表明，特定类别的 HIF1A 和 HIF2A 转录靶基因对 mTOR 表现出不同的敏感性，这支持联合使用 HIF2A 和 mTOR 抑制剂治疗肾癌。