Small molecule-mediated inhibition of translation by targeting a native RNA G-quadruplex

Abstract

Herein, we show that a naturally occurring RNA G-quadruplex element within the 5' UTR of the human NRAS proto-oncogene is a target for a small molecule that inhibits translation in vitro. The present study provides a first demonstration that natural 5' UTR mRNA G-quadruplexes have potential as molecular targets for small molecules that modulate translation.

Fig. 1

Schematic representation of NRAS UTR(−)Q and NRAS UTR(+)Q firefly luciferase reporter mRNAs.

Fig. 2

Effect of RR82 on the in vitro translation efficiency of NRAS UTR(−)Q and NRAS UTR(+)Q mRNAs. (A) Chemical structure of RR82. (B) Relative translation efficiencies of NRAS UTR(−)Q and NRAS UTR(+)Q mRNAs in the presence of the indicated amounts of RR82, as measured by luciferase activities. For each construct, the measured luciferase activity was normalized to the activity obtained in the absence of the ligand, which was set as 100%. Experiments were performed at least three times using at least two separate batches of RNA. The average values are presented along with the standard error on the mean.

Fig. 3

Effect of RR110 on the in vitro translation efficiency of mRNAs comprising a G-quadruplex motif in their 5′ UTR. (A) Chemical structure of RR110. (B) Relative in vitro translation efficiencies of NRAS UTR(−)Q, NRAS UTR(+)Q and NRAS UTR(+120)Q mRNAs in the presence of the indicated amounts of RR110, as measured by luciferase activities. For each construct, the measured luciferase activity was normalized to the activity obtained in the absence of the ligand, which was set as 100%. Experiments were performed at least three times using at least two separate batches of RNA. The average values are presented along with the standard error on the mean.

Fig. 4

Effect of RR110 (10 μM) on the in vitro translation efficiency of NRAS UTR(+)Q mRNA in the presence (1 or 10 μM) of double-stranded DNA, hairpin RNA and NRQ RNA G-quadruplex nucleic acid competitors (see Experimental section for sequences). Measured luciferase activities were normalized to activities obtained in the absence of the ligand, which were set as 100%. Experiments were performed at least three times using at least two separate batches of RNA. The average values are presented along with the standard error on the mean.

Fig. 5

¹H NMR spectroscopic analysis of the interaction between the RNA G-quadruplex (NRQ) and G-quadruplex ligand RR110. (A) ¹H NMR titration of the NRQ element with RR110. The resonances arising from RR110 are indicated by open circles. While several aromatic protons from the RNA nucleobases exhibit negligible change in chemical shift (indicated by dashed lines), marked upfield shift changes are observed in the imino proton region at a ligand-to-NRQ ratio of three and higher. (B) HDX kinetics of the hydrogen-bonded imino proton resonances of the G-tetrads in the absence (open red diamond) and in the presence of 5 molar equivalent of RR110 (open black circle). The fitting results are shown in green lines with the residuals shown below.

Fig. 6

mRNA stability of the NRAS UTR(+)Q construct translated in the absence and in the presence of RR110 (10 μM). (A) A representative autoradiograph from one experiment. The time points and the absence or presence of RR110 is indicated above the panel. (B) Summary of the experiments. Values were normalized to the value at the beginning of the experiment (t = 0 min), which set at 100%. Experiments were performed at least three times using at least two separate batches of RNA. The average values are presented along with the standard error on the mean.