Abstract
Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the trans isoform of a stiff-stilbene (amino-tSS)-a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than the cis photoisoform (amino-cSS). In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS. Photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. In contrast, amino-cSS supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible photoregulation of gene expression using a genetically encoded RNA will likely facilitate high-resolution spatiotemporal analysis of complex RNA processes.
Highlights
Optogenetic techniques have transformed the biomedical sciences by controlling biological events with high spatial and temporal resolution through triggering signal transduction pathways via lightsensing proteins (Fenno et al, 2011; Moglich and Moffat, 2010; Motta-Mena et al, 2014; Cambridge et al, 2009); there is a need for photoactive molecules that can quickly and reversibly regulate cellular events at the RNA level
To isolate a new aptamer fused to a functional expression platform, we constructed an RNA pool derived from a bacterial SAM-I riboswitch (Winkler et al, 2003) by replacing its ligand-binding domain with a 45-nucleotide random sequence, partially randomizing its anti-terminator and terminator hairpins, and retaining its translation initiation sequences (Figure 1—figure supplement 1)
We hypothesized that a pool of amino-trans stiff stilbene (tSS)–binding aptamers would include motifs that do not bind the cis photoisoform of the ligand and that the tSS–binding conformation stabilizes the expression platform in a single state that affects either transcription or translation of a downstream open reading frame (ORF)
Summary
Optogenetic techniques have transformed the biomedical sciences by controlling biological events with high spatial and temporal resolution through triggering signal transduction pathways via lightsensing proteins (Fenno et al, 2011; Moglich and Moffat, 2010; Motta-Mena et al, 2014; Cambridge et al, 2009); there is a need for photoactive molecules that can quickly and reversibly regulate cellular events at the RNA level. Riboswitches, cellular ligand-dependent regulatory RNAs that modulate transcription and translation in bacteria, and splicing, translation, and RNA stability in eukaryotes (Breaker, 2011) represent an attractive platform for the development of photosensitive regulatory RNAs. Riboswitches typically consist of an aptamer domain, which bind cellular targets, such as cofactors and metabolites, and an expression platform, which regulates gene expression by forming structures that affect downstream events, such as ribosome binding (Fuchs et al, 2007; Breaker, 2012). Riboswitches typically consist of an aptamer domain, which bind cellular targets, such as cofactors and metabolites, and an expression platform, which regulates gene expression by forming structures that affect downstream events, such as ribosome binding (Fuchs et al, 2007; Breaker, 2012) Many riboswitches sense their ligands during transcription; ligand-dependent conformational changes occur on the timescales of synthesis of these RNAs (Greenleaf et al, 2008; Uhm et al, 2018). Our results demonstrate how a genetically encoded light-responsive RNA can reversibly regulate gene expression using light, providing a new optogenetic tool to broaden the analysis of complex RNA processes in living cells (You and Jaffrey, 2015; Liang et al, 2011; Jaschke, 2012; Szymanski et al, 2013b)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
