title: Deciphering the rules by which 5'-UTR sequences affect protein expression in yeast.
abstract: The 5'-untranslated region (5'-UTR) of mRNAs contains elements that affect expression, yet the rules by which these regions exert their effect are poorly understood. Here, we studied the impact of 5'-UTR sequences on protein levels in yeast, by constructing a large-scale library of mutants that differ only in the 10 bp preceding the translational start site of a fluorescent reporter. Using a high-throughput sequencing strategy, we obtained highly accurate measurements of protein abundance for over 2,000 unique sequence variants. The resulting pool spanned an approximately sevenfold range of protein levels, demonstrating the powerful consequences of sequence manipulations of even 1-10 nucleotides immediately upstream of the start codon. We devised computational models that predicted over 70% of the measured expression variability in held-out sequence variants. Notably, a combined model of the most prominent features successfully explained protein abundance in an additional, independently constructed library, whose nucleotide composition differed greatly from the library used to parameterize the model. Our analysis reveals the dominant contribution of the start codon context at positions -3 to -1, mRNA secondary structure, and out-of-frame upstream AUGs (uAUGs) to phenotypic diversity, thereby advancing our understanding of how protein levels are modulated by 5'-UTR sequences, and paving the way toward predictably tuning protein expression through manipulations of 5'-UTRs.
authors.affiliation: Departments of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Shlomi
authors.initials: S
authors.lastname: Dvir
authors.forename: Lars
authors.initials: L
authors.lastname: Velten
authors.forename: Eilon
authors.initials: E
authors.lastname: Sharon
authors.forename: Danny
authors.initials: D
authors.lastname: Zeevi
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Carey
authors.forename: Adina
authors.initials: A
authors.lastname: Weinberger
authors.forename: Eran
authors.initials: E
authors.lastname: Segal
mesh_terms: D020121:5' Untranslated Regions; D001483:Base Sequence; D018387:Codon, Initiator; D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D018387:Codon, Initiator; D017931:DNA Primers; D005656:Fungal Proteins; D012333:RNA, Messenger
keywords: AUG sequence context; computational prediction; mRNA folding; post-transcriptional regulation; upstream start codons