Identification and characterization of upstream open reading frames (uORF) in the 5′ untranslated regions (UTR) of genes in Saccharomyces cerevisiae
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We have taken advantage of recently sequenced hemiascomycete fungal genomes to computationally identify additional genes potentially regulated by upstream open reading frames (uORFs). Our approach is based on the observation that the structure, including the uORFs, of the post-transcriptionally uORF regulated Saccharomyces cerevisiae genes GCN4 and CPA1 is conserved in related species. Thirty-eight candidate genes for which uORFs were found in multiple species were identified and tested. We determined by 5′ RACE that 15 of these 38 genes are transcribed. Most of these 15 genes have only a single uORF in their 5′ UTR, and the length of these uORFs range from 3 to 24 codons. We cloned seven full-length UTR sequences into a luciferase (LUC) reporter system. Luciferase activity and mRNA level were compared between the wild-type UTR construct and a construct where the uORF start codon was mutated. The translational efficiency index (TEI) of each construct was calculated to test the possible regulatory function on translational level. We hypothesize that uORFs in the UTR of RPC11, TPK1, FOL1, WSC3, and MKK1 may have translational regulatory roles while uORFs in the 5′ UTR of ECM7 and IMD4 have little effect on translation under the conditions tested.
KeywordsYeast Translation Expression Post-transcription mRNA
We thank John E.G. McCarthy at UMIST for providing YCp22FL series plasmids. We thank Joe Heitman for providing yeast strains and Miguel Arevalo-Rodriguez, Shihua Lu, and Carol Gallione for valuable technical help and advice. We are grateful to Joe Heitman, John McCusker, Douglas Marchuk, and Bryan Cullen and for generously providing laboratory facilities and comments on the whole project. The authors also thank Mark DeLong for his careful reading and revising of the manuscript.
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