Abstract

BackgroundTranslation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can also be used as start sites at low levels in Saccharomyces 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.ResultsUsing 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.ConclusionsOur data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Highlights

  • Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can be used as start sites at low levels in Saccharomyces cerevisiae

  • Growth temperature affects the efficiency of using nonAUG start codons in reporter mRNAs in yeast We previously developed and validated a dual-luciferase assay to calculate the efficiency of utilization of nearcognate codons (NCCs) as translational start sites in yeast [20]

  • We examined which of these 6061 putative upstream open reading frames (uORFs) show evidence of translation in our ribosome profiling data using a different identification tool, RibORF [36], which is based on the criteria of 3-nt periodicity and a uniform distribution of reads across uORF codons

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Summary

Introduction

Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can be used as start sites at low levels in Saccharomyces cerevisiae. The recognition of the start codon in an mRNA during cap-dependent translation initiation is thought to occur by the scanning mechanism [1] It begins with the formation of a ternary complex (TC) that consists of translation initiation factor 2 (eIF2) in its GTP-bound form along with the methionyl initiator tRNA (Met-tRNAi). Upon recognition of the start codon, sequential events occur in the PIC that eventually leads to joining of the large (60S) ribosomal subunit and commencement of the elongation phase of protein synthesis.

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