Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches

Nat Methods. 2016 May;13(5):453-8. doi: 10.1038/nmeth.3807. Epub 2016 Mar 21.

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.

MeSH terms

  • Biotechnology / methods*
  • Cellular Reprogramming / genetics*
  • Directed Molecular Evolution
  • Frameshifting, Ribosomal / genetics*
  • Gene Expression Regulation, Fungal
  • Ligands
  • Protein Biosynthesis / genetics*
  • RNA, Fungal / chemistry
  • RNA, Fungal / genetics
  • RNA, Messenger / chemistry
  • Riboswitch / genetics*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism

Substances

  • Ligands
  • RNA, Fungal
  • RNA, Messenger
  • Riboswitch