title: Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants.
abstract: Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5' transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5'UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5'end can modulate protein levels up to 160%-300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for engineering synthetic gene expression in eukaryotes.
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.|b Department of Biological Chemistry ; Weizmann Institute of Science ; Rehovot, Israel.|c Department of Applied Mathematics and Computer Science ; Weizmann Institute of Science; Rehovot, Israel.|e These authors equally contributed to this work.
authors.forename: Tuval
authors.initials: T
authors.lastname: Ben-Yehezkel
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.|e These authors equally contributed to this work.
authors.forename: Shimshi
authors.initials: S
authors.lastname: Atar
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.
authors.forename: Hadas
authors.initials: H
authors.lastname: Zur
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.
authors.forename: Alon
authors.initials: A
authors.lastname: Diament
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.
authors.forename: Eli
authors.initials: E
authors.lastname: Goz
authors.affiliation: b Department of Biological Chemistry ; Weizmann Institute of Science ; Rehovot, Israel.
authors.forename: Tzipy
authors.initials: T
authors.lastname: Marx
authors.affiliation: b Department of Biological Chemistry ; Weizmann Institute of Science ; Rehovot, Israel.
authors.forename: Rafael
authors.initials: R
authors.lastname: Cohen
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dana
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.
authors.forename: Anna
authors.initials: A
authors.lastname: Feldman
authors.affiliation: b Department of Biological Chemistry ; Weizmann Institute of Science ; Rehovot, Israel.|c Department of Applied Mathematics and Computer Science ; Weizmann Institute of Science; Rehovot, Israel.
authors.forename: Ehud
authors.initials: E
authors.lastname: Shapiro
authors.affiliation: a Department of Biomedical Engineering ; Tel-Aviv University ; Tel-Aviv , Israel.|d Sagol School of Neuroscience; Tel-Aviv University ; Tel-Aviv , Israel.
authors.forename: Tamir
authors.initials: T
authors.lastname: Tuller
mesh_terms: D020121:5' Untranslated Regions; D001482:Base Composition; D003062:Codon; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D017930:Genes, Reporter; D006801:Humans; D016366:Open Reading Frames; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D000069456:Silent Mutation; D014158:Transcription, Genetic
chemical_list: D020121:5' Untranslated Regions; D003062:Codon; D012333:RNA, Messenger
keywords: codon usage bias; gene expression engineering; heterologous gene expression; mRNA folding; mRNA translation; ribosome; ribosome profiling; synonymous and silent mutation; synthetic biology; transcript evolution; viral protein expression