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Translation initiation site profiling reveals widespread synthesis of non-AUG-initiated protein isoforms in yeast

ABSTRACT: Genomic analyses in budding yeast have helped to define the foundational principles of eukaryotic gene expression, but have systematically excluded specific classes of potential coding regions, including those with non-AUG start codons. Without methods to define coding regions empirically, the prevalence of these non-canonical coding regions has been impossible to assess. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified a class of 149 genes that encode N-terminally extended alternate protein isoforms that result from translation initiation at non-AUG codons upstream of the annotated AUG start codon. These alternate isoforms are produced in concert with canonical isoforms and are translated with a high degree of specificity, resulting from initiation at only a small subset of possible start codons in 5’ leader regions. Their translation is enriched during meiosis, and is induced by low eIF5A levels, which are observed in this context. These findings reveal widespread production of non-canonical protein isoforms and, more generally, show unexpected complexity to the rules by which the budding yeast genome is decoded.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE150375 | GEO | 2020/07/24

REPOSITORIES: GEO

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Project description: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.

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Translation initiation site profiling reveals widespread synthesis of non-AUG-initiated protein isoforms in yeast

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