Temperature-dependent regulation of upstream open reading frame translation in s. cerevisiae. 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. Overall design: We examined the effect of changes in culture temperature on usage of alternate start sites upstream of the annotated start codon AUG (translation of upstream open reading frame i.e. uORF). We calculated global translational efficiencies (TEs) by ribosome footprint profiling of BY4741 cells cultured at multiple temperatures. The study includes 12 samples, comprised of 6 mRNA-Seq samples and 6 ribosome footprint profiling samples, derived from 2 biological replicates of cells cultured at 20 °C, 30 °C. and 37 °C. The ribo-seq data generated in this study are previously published (Martin-Marcos et al., 2017, eLife 2017;6:e31250 DOI: 10.7554/eLife.31250). The ribo-seq data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSM2895484, GSM2895485, GSM2895488, GSM2895489, GSM2895490, GSM2895491.

真核生物的mRNA翻译大多以AUG作为起始密码子。在酿酒酵母（S. cerevisiae）中，UUG、ACG、AUU这类近同源密码子（near-cognate codons，NCCs）也会以低频率充当翻译起始位点。mRNA的5'非翻译区（5'-untranslated regions，UTRs）内的NCC或AUG起始翻译时，会生成上游开放阅读框（upstream open reading frames，uORFs），这类uORFs可能调控主开放阅读框（main ORF，mORF）的基因表达。尽管已有若干间接证据表明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uORF翻译仍知之甚少。
本研究通过报告基因实验发现，生长温度改变可影响酿酒酵母细胞中基于NCC起始位点的翻译，提示温度或可通过调控不同uORF起始密码子的使用来调节基因表达。本研究借助核糖体谱分析（ribosome profiling）证实，生长温度可调控酿酒酵母中近200个uORFs的翻译效率（translational efficiency，TE）。在这些uORFs中，绝大多数以AUG为起始密码子的uORFs在37℃时的翻译效率相较于30℃时有所提升，而在20℃时翻译效率则下降。对于以NCC为起始密码子且翻译受温度调控的uORFs，本研究未观察到统一的温度依赖调控趋势，这提示单个mRNA的特异性特征可决定其温度依赖调控模式。
与此结论一致的是，uORF在5'前导区中相对于5'帽结构及主ORF起始密码子的位置，与uORF翻译的温度依赖调控方向存在相关性。本研究还发现了若干新案例，其中uORF翻译的变化与下游主ORF的翻译效率变化呈负相关。数据表明，这类mRNA的翻译受温度依赖性、uORF介导的调控。
总体而言，本研究数据显示，温度对特定uORF翻译的改变可调控酿酒酵母的基因表达。
实验设计概述：本研究探究了培养温度变化对注释起始密码子AUG上游交替起始位点使用（即上游开放阅读框翻译）的影响。通过对在不同温度下培养的BY4741细胞进行核糖体足迹测序（ribosome footprint profiling），本研究计算了全局翻译效率。本研究共包含12个样本，分别来自20℃、30℃与37℃下培养的细胞的2个生物学重复，其中包括6个mRNA测序（mRNA-Seq）样本与6个核糖体足迹测序样本。
本研究产生的核糖体测序（ribo-seq）数据此前已发表（Martin-Marcos等人，2017，eLife 2017;6:e31250 DOI: 10.7554/eLife.31250）。本研究的核糖体测序数据已提交至NCBI基因表达综合数据库（Gene Expression Omnibus，GEO；http://www.ncbi.nlm.nih.gov/geo/），登录号为GSM2895484、GSM2895485、GSM2895488、GSM2895489、GSM2895490、GSM2895491。