title: Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches.
abstract: Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism, termed -1 programmed ribosomal frameshifting (-1 PRF), to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient -1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling -1 PRF stimulatory elements to RNA aptamers using rational design and directed evolution in Saccharomyces cerevisiae. We demonstrate that -1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, -1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and they establish -1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes.
authors.affiliation: Department of Chemistry, Columbia University, New York, New York, USA.|Department of Systems Biology, Columbia University, New York, New York, USA.
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Anzalone
authors.affiliation: Department of Chemistry, Columbia University, New York, New York, USA.|Department of Systems Biology, Columbia University, New York, New York, USA.
authors.forename: Annie J
authors.initials: AJ
authors.lastname: Lin
authors.affiliation: Department of Systems Biology, Columbia University, New York, New York, USA.
authors.forename: Sakellarios
authors.initials: S
authors.lastname: Zairis
authors.affiliation: Department of Systems Biology, Columbia University, New York, New York, USA.|Department of Biomedical Informatics, Columbia University College of Physicians and Surgeons, New York, New York, USA.
authors.forename: Raul
authors.initials: R
authors.lastname: Rabadan
authors.affiliation: Department of Chemistry, Columbia University, New York, New York, USA.|Department of Systems Biology, Columbia University, New York, New York, USA.
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
mesh_terms: D001709:Biotechnology / Q000379:methods*; D065150:Cellular Reprogramming / Q000235:genetics*; D019020:Directed Molecular Evolution; D018965:Frameshifting, Ribosomal / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008024:Ligands; D014176:Protein Biosynthesis / Q000235:genetics*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry; D058928:Riboswitch / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D008024:Ligands; D012331:RNA, Fungal; D012333:RNA, Messenger; D058928:Riboswitch