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.

Figure 1

Design of ligand-responsive −1 PRF switches. (a) Translation control scheme. The protein output of an mRNA is dictated by the translation reading frame. −1 PRF switches direct the ribosome’s translation reading frame depending on the presence or absence of a ligand. (b) Methodological approach to build −1 PRF switches. Active frameshift stimulatory elements are discovered from large RNA libraries using a functional in vitro selection. Frameshift stimulatory elements (purple) are then coupled to RNA aptamer modules (gold) by rational design to create frameshift switches. Lastly, frameshift switch devices are optimized by in vivo directed evolution using a frameshift-dependent growth selection.

Figure 2

In vitro selection for −1 PRF stimulatory elements. (a) The mRNA display selection. The display construct encodes an N-terminal FLAG tag, a heptanucleotide slippery site, an in-frame stop codon, the stimulatory element library, and a C-terminal His₆-tag encoded in the −1 frame. The selection cycle comprises four stages: (i) RNA is in vitro transcribed from DNA library templates, then ligated to the puromycin adaptor; (ii) mRNA display templates are translated in vitro with rabbit reticulocyte lysate; (iii) mRNA-peptide fusions are isolated from non-fused RNA by affinity purification of the peptide tags; (iv) enriched sequences are reverse transcribed and PCR amplified for subsequent rounds of selection. (b) Translation reprogramming selection principle. Ribosomes that terminate translation upstream of the designated fusion point will fail to produce mRNA-peptide fusions (upper). Frameshifting enables bypass of encoded stop codons (lower). (c) Dual-FP reporter assay in S. cerevisiae. The frameshift variant is cloned between a green fluorescent protein (GFP) and the red fluorescent protein variant mCherry. The ratio of FP signals reflects bulk −1 PRF efficiency. Flow cytometry of individual clones harboring -1 PRF stimulatory elements of varying efficiencies is shown. (d) NGS analysis workflow for library selection products. Selected sequences are grouped into pseudoknot (PK) families, analyzed for post-selection enrichment, and clustered based on primary sequence identity. Motifs can be analyzed by comparative analysis, or individual sequences can be analyzed for single and pair-wise nucleotide changes (see Supplementary Fig. 5).

Figure 3

Rational design of frameshift switches. (a) Architecture of frameshift switch devices. Each device contains a 5′ heptanucleotide slippery site, the pseudoknot switch, and a 3′ insulator sequence. (b) OFF-switch design. In the absence of ligand, the stimulatory pseudoknot (purple) is energetically dominant, producing high frameshift levels. Ligand binding induces aptamer (gold) folding, which disrupts the pseudoknot structure leading to lowered frameshift levels. (c) ON-switch design. A switching hairpin (gray) is installed to disrupt the pseudoknot and lower basal frameshifting. In the presence of ligand, the aptamer folds and destabilizes the switching hairpin, allowing the pseudoknot to re-fold and restore frameshift activity. (d) The ligand responsiveness of four −1 PRF switches assayed in the dual-FP reporter. Error bars represent the s.e.m. for n = 3 biological replicates derived from individual yeast transformants. Theophylline was used at a concentration of 40 mM; neomycin was used at a concentration of 550 μM. (e) Flow cytometry of yeast harboring the Theo-ON-5 switch in the absence and presence of theophylline (40 mM).

Figure 4

Construction of logic gates and an apoptosis module with layered −1 PRF switches. For all constructs, translation in the absence of a ligand follows the black path; theophylline directs translation down the red path; neomycin directs translation down the blue path. (a) The NOR gate is composed of Theo-OFF-3 and Neo-OFF-DE switches. (b) The AND gate is composed Theo-ON-5 and Neo-ON-4 switches. For both gates, mCherry is encoded in the -2 frame with respect to GFP. Gate function was assessed within the dual-FP reporter in yeast by flow cytometry. Theophylline was used at a concentration of 40 mM; neomycin was used at a concentration of 550 μM. (c) The apoptosis module construct encodes Puma under the control of a theophylline-responsive ON-switch and Bcl-xL under the control of the Neo-OFF-DE switch. 2A peptides encoded 5′ to the Puma and Bcl-xL open reading frames cleave the functional proteins from the nonsense translation products of the switch devices and alternative reading frames. The latter products are targeted for degradation by an N-degron signal at the N-terminus of the polypeptide. Relative production of Puma and Bcl-xL controls the ability of Bax to induce cell death. Cells expressing Bax and the apoptosis module were grown in various concentrations of neomycin (0 μM, 44 μM, 165 μM, 715 μM) and theophylline (0 mM, 1 mM, 5 mM, 20 mM) and assessed for viability by plating efficiency, reported as the mean of three technical replicates.