Unusual −1 Ribosomal Frameshift Caused by Stable RNA G‑Quadruplex in Open Reading Frame

Tertiary structures formed by mRNAs impact the efficiency of the translation reaction. Ribosomal frameshift is a well-characterized recoding process that occurs during translation elongation. Pseudoknot and stem-loop structures may stimulate frameshifting by causing a translational halt at a slippery sequence. In this study, we evaluated the efficiency of an unusual −1 frameshift caused by a noncanonical RNA G-quadruplex structure in mammalian cells. The reporter gene construct consisting of a fluorescent protein and Luciferase enabled evaluation of apparent and absolute values of the −1 frameshift efficiency and revealed significant increase of the efficiency by G-quadrupex forming potential sequence. In addition, berberine, a small molecule that binds to and stabilizes G-quadruplex structures, further increased the frameshift efficiency. These results indicate that the stable G-quadruplex structure stimulates the unusual −1 frameshift and has a potential to regulate the frameshift with its ligand

The interaction of initial RNA libraries and the Tat-peptide.

<p>(A, B) Fluorescence intensities (FI) of the TMR-Tat at 590 nm mixed with G0 RNA libraries in a buffer containing 20 mM phosphate (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 20 ng/ µL tRNA, and 0.005% (v/v) Tween 20 at 37°C. TMR-Tat was mixed with varying concentrations of (A) th5N-G0 and (B) tc5N-G0 in the absence (blue) or presence of 3 mM theophylline (red) or 100 µM tetracycline (green).</p

Selection of RNAs for Constructing “Lighting-UP” Biomolecular Switches in Response to Specific Small Molecules

<div><p>RNA and protein are potential molecules that can be used to construct functional nanobiomaterials. Recent findings on riboswitches emphasize on the dominative function of RNAs in regulating protein functions through allosteric interactions between RNA and protein. In this study, we demonstrate a simple strategy to obtain RNAs that have a switching ability with respect to protein function in response to specific target molecules. RNA aptamers specific for small ligands and a trans-activation-responsive (TAR)-RNA were connected by random RNA sequences. RNAs that were allosterically bound to a trans-activator of transcription (Tat)-peptide in response to ligands were selected by repeated negative and positive selection in the absence and presence of the ligands, respectively. The selected RNAs interacted with artificially engineered <i>Renilla</i> Luciferase, in which the Tat-peptide was inserted within the Luciferase, in the presence of the specific ligand and triggered the “Lighting-UP” switch of the engineered Luciferase.</p> </div

Observed association constants between RNA libraries and TMR-Tat at 37°C.

<p>Observed association constants between RNA libraries and TMR-Tat at 37°C.</p

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

Conformational transitions of biomolecules in response to specific stimuli control many biological processes. In natural functional RNA switches, often called riboswitches, a particular RNA structure that has a suppressive or facilitative effect on gene expression transitions to an alternative structure with the opposite effect upon binding of a specific metabolite to the aptamer region. Stability of RNA secondary structure (−Δ<i>G</i>°) can be predicted based on thermodynamic parameters and is easily tuned by changes in nucleobases. We envisioned that tuning of a functional RNA switch that causes an allosteric interaction between an RNA and a peptide would be possible based on a predicted switching energy (ΔΔ<i>G</i>°) that corresponds to the energy difference between the RNA secondary structure before (−Δ<i>G</i>°_before) and after (−Δ<i>G</i>°_after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔ<i>G</i>° values ranging from 5.6 to 12.2 kcal mol^–1. We then demonstrated a simple strategy to rationally convert the functional RNA switch to switches responsive to natural metabolites thiamine pyrophosphate, <i>S</i>-adenosyl methionine, and adenine based on the predicted ΔΔ<i>G</i>° values. The ΔΔ<i>G</i>° values of the designed RNA switches proportionally correlated with interaction energy (Δ<i>G</i>°_interaction) between the RNA and peptide, and we were able to tune the sensitivity of the RNA switches for the trigger molecule. The strategy demonstrated here will be generally applicable for construction of functional RNA switches and biosensors in which mechanisms are based on conformational transition of nucleic acids

Detection of target molecules by the Lighting-UP switch.

<p>(A, B) Relative luminescence intensities (LI) of the engineered Luciferase in buffer containing 20 mM phosphate (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 100 ng/ µL tRNA, and 0.005% Tween 20 at 37°C. An aliquot (0.04 µL) of the <i>in vitro</i> translation product was mixed with varying concentrations of (A) th5N-G6 or (B) tc5N-G6 in the absence (blue) or presence of 3 mM theophylline (red) or 100 µM tetracycline (green). tRNA was added at 100 ng/ µL to improve the signal to noise ratio. (C) Theophylline (red) or caffeine (purple) at the indicated concentrations was mixed with the <i>in vitro</i> translation product in the presence of 25 nM th5N-G6. (D) Tetracycline (green) or doxycycline (orange) at indicated concentrations was mixed with the <i>in vitro</i> translation product in the presence of 50 nM tc5N-G6. (E) Chemical structures of target molecules and analogs.</p

Choline Dihydrogen Phosphate Destabilizes G‑Quadruplexes and Enhances Transcription Efficiency <i>In Vitro</i> and in Cells

G-quadruplexes in disease-related genes are associated with various biological processes and regulate disease progression. Although methods involving ligands and other techniques are available to stabilize G-quadruplexes, approaches for destabilizing G-quadruplexes remain limited. Here, we evaluated whether G-quadruplexes can be destabilized using choline dihydrogen phosphate (choline dhp), a highly biocompatible hydrated ionic liquid. Circular dichroism spectral measurements at increasing temperatures revealed that choline dhp destabilized G-quadruplexes more effectively than did KCl-containing solutions. Thermodynamic analysis indicated that destabilization occurred via an entropic contribution, suggesting that choline ions did not coordinate with the G-quartets, because of their large radii. Subsequently, plasmid DNAs containing G-quadruplexes were constructed, and transcription reactions were performed in nuclear extracts from living cells. G-quadruplexes repressed transcription, whereas the addition of choline dhp increased transcription. Although ionic liquids often inactivate biomolecules, choline dhp can be used to culture various cells. Furthermore, the transcription of template DNA containing the G-quadruplex was greatly enhanced in living MDA-MD-231 cells (aggressive human breast cancer cells) cultured with choline dhp. Our results show that choline dhp destabilizes G-quadruplexes in cells, indicating that choline dhp can regulate gene expression. Thus, choline dhp may be useful for regulating target disease-related genes

Synchronized Translation for Detection of Temporal Stalling of Ribosome during Single-Turnover Translation

Arrhythmic translation caused by temporal stalling of ribosome during translation elongation is essential for gene expression and protein folding. To analyze the positions of the temporarily stalled ribosome and length of the stalling, the ribosomes must be synchronized during translation elongation. In this study, we designed a two-step translation reaction to synchronize the ribosome during a single-turnover translation. First, ribosomes decoding mRNA were artificially and specifically halted before isoleucine codon by reducing isoleucyl-tRNA synthetase from reaction mixture of in vitro translation. Then, translation elongation was restarted simultaneously to synchronize the translation. It enabled evaluation of translation elongation with time resolving capacity shorter than ever before. In addition, position-specific incorporation of fluorescent amino acid and mass spectrometry analyses enabled trace of translation elongation after gel electrophoresis and accurate determination of ribosome positions temporarily stalled before rare codons, respectively. The synchronized translation demonstrated here would be useful to evaluate trans- and cis-elements that affect rate of the translation elongation

Effects on RNA polymerase elongation by structures in template DNA (a–d) and illustration of the template DNA (e, f).

<p>(a) An unstructured template, (b) a template with a slippage site, (c) a template with a pause site, and (d) a template with an arrest site. (e) The region denoted by the box marked with an X contains the sequence designed to form a random coil or non-canonical structure. (f) Sequence names and sequences of X regions. Sequences expected to form non-canonical structures are highlighted by italic and bold.</p

Destabilization of DNA G‑Quadruplexes by Chemical Environment Changes during Tumor Progression Facilitates Transcription

DNA G-quadruplex formation is highly responsive to surrounding conditions, particularly K⁺ concentration. Malignant cancer cells have a much lower K⁺ concentration than normal cells because of overexpression of a K⁺ channel; thus, G-quadruplexes may be unstable in cancer cells. Here, we physicochemically investigated how changes in intracellular chemical environments <i>in vitro</i> and in cells influence G-quadruplex formation and transcription during tumor progression. <i>In vitro</i>, the stable G-quadruplex formation inhibits transcription in a solution containing 150 mM KCl (normal condition). As K⁺ concentration decreases, which decreases G-quadruplex stability, transcript production from templates with G-quadruplex-forming potential increases. In normal cells, the trend in transcript productions was similar to that in <i>in vitro</i> experiments, with transcription efficiency inversely correlated with G-quadruplex stability. Interestingly, higher transcript levels were produced from templates with G-quadruplex-forming potential in Ras-transformed and highly metastatic breast cancer cells (MDA-MB-231) than in nontransformed and control MCF-7 cells. Moreover, the amount of transcript produced from G-quadruplex-forming templates decreased upon addition of siRNA targeting <i>KCNH1</i> mRNA, which encodes a potassium voltage-gated channel subfamily H member 1 (K_V10.1). Importantly, G-quadruplex dissociation during tumor progression was observed by immunofluorescence using a G-quadruplex-binding antibody in cells. These results suggest that in normal cells, K⁺ ions attenuate the transcription of certain oncogenes by stabilizing G-quadruplex structures. Our findings provide insight into the novel mechanism of overexpression of certain G-rich genes during tumor progression