Temperature-dependent regulation of upstream open reading frame translation in s. cerevisiae

Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.真核生物的信使RNA（messenger RNA，mRNA）翻译大多以AUG作为起始位点。在酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）中，UUG、ACG、AUU等近同源密码子（near-cognate codons，NCCs）也可作为低频率使用的翻译起始位点。在mRNA的5'非翻译区（5’-untranslated regions，UTRs）内，由NCCs或AUG启动的翻译可产生上游开放阅读框（upstream open reading frames，uORFs），这类uORFs可调控主开放阅读框（main ORF，mORF）的基因表达。尽管已有间接证据表明uORFs的翻译可受环境条件影响，但目前对生长温度变化如何调控该过程仍知之甚少。本研究通过报告基因检测实验（reporter assays）发现，生长温度改变可影响酵母细胞内基于NCC起始位点的翻译，这提示温度可通过改变不同uORF起始密码子的使用情况来调控基因表达。本研究进一步利用核糖体谱分析（ribosome profiling）技术，证实生长温度可调控酿酒酵母中近200个uORFs的翻译效率。在这些uORFs中，多数以AUG为起始密码子的序列在37℃时的翻译效率相较于30℃时有所提升，而在20℃时翻译效率则出现下降。对于以NCCs为起始密码子且受翻译调控的uORFs，本研究未观察到其调控方向随温度变化的通用规律，这提示mRNA的特异性特征可决定温度依赖性调控的模式。与该结论一致的是，uORFs在5'先导序列中相对于5'帽结构及主开放阅读框起始密码子的位置，与uORF翻译的温度依赖性调控方向存在相关性。本研究已鉴定出多个新型案例，其中uORF翻译的变化与下游主开放阅读框的翻译效率变化呈负相关。本研究数据表明，这类mRNA的翻译受温度依赖性、uORFs介导的调控机制影响。综上，本研究数据证实，温度对特定uORF翻译的调控可影响酿酒酵母的基因表达。