Synergetic regulation of translational reading-frame switch by ligand-responsive RNAs in mammalian cells

Abstract

Distinct translational initiation mechanisms between prokaryotes and eukaryotes limit the exploitation of prokaryotic riboswitch repertoire for regulatory RNA circuit construction in mammalian application. Here, we explored programmed ribosomal frameshifting (PRF) as the regulatory gene expression platform for engineered ligand-responsive RNA devices in higher eukaryotes. Regulation was enabled by designed ligand-dependent conformational rearrangements of the two cis-acting RNA motifs of opposite activity in -1 PRF. Particularly, RNA elements responsive to trans-acting ligands can be tailored to modify co-translational RNA refolding dynamics of a hairpin upstream of frameshifting site to achieve reversible and adjustable -1 PRF attenuating activity. Combined with a ligand-responsive stimulator, synthetic RNA devices for synergetic translational-elongation control of gene expression can be constructed. Due to the similarity between co-transcriptional RNA hairpin folding and co-translational RNA hairpin refolding, the RNA-responsive ligand repertoire provided in prokaryotic systems thus becomes accessible to gene-regulatory circuit construction for synthetic biology application in mammalian cells.

A general strategy to inhibiting viral −1 frameshifting based on upstream attenuation duplex formation

“…We have demonstrated that the formation of an upstream attenuator hairpin can be controlled by alternate base-pairing schemes to achieve −1 PRF activity regulation ( 31 ). Aiming for sequence-specific regulation of −1 PRF, we designed DNA oligonucleotides complementary to the 5′-half (6BPGC-5′-DNA) or the 3′-half (6BPGC-3′-DNA) sequences of the stem of a potent −1 PRF attenuator hairpin (6BPGC) ( 29 ) (Supplementary Figure S1A) to interfere with attenuation hairpin refolding.…”

“…Additionally, a variant of p2luc was engineered to facilitate radioactivity based −1 PRF activity analysis in vitro . The variant contains a premature −1 frame stop codon 33 nt downstream of the BamHI site of p2luc, and will be translated into a shortened −1 frame product in reticulocyte lysate ( 31 ).…”

Reprogramming the genetic code: The emerging role of ribosomal frameshifting in regulating cellular gene expression

“…In support of this, hybridization of antisense linked nucleic acids (LNAs) immediately 3 0 of heptameric slippery sequences was sufficient to promote efficient -1 frameshifting in the absence of any other stimulatory element [39]. Natural attenuation of -1 PRF by stem-loop structures immediately 5 0 of coronavirus slippery sequences has also been reported [40], a phenomenon that can be replicated by hybridization of oligonucleotides complementary to sequences lying just upstream of slippery sites [41]. These studies establish the role of ncRNAs in regulating translational fidelity and suggest that regulation of -1 PRF may not be limited to miRNAs.…”

Aptamer-based and aptazyme-based riboswitches in mammalian cells

“…A canonical -1PRF element consists of a 7-nt slippery sequence X XXY YYZ (trinucleotides XXY and YYZ representing the original frame and XXX YYY representing the -1 frameshift) followed by a stable secondary structure such as a pseudoknot or a stem-loop. The Chang group first controlled -1PRF in mammalian cells (Figure 3a) by incorporating theophylline and S-adenosyl-L-homocysteine (SAH) aptamers [28]. An ON/OFF ratio of 6 was reported but the frameshifting efficiency (FE) remained low.…”