Mechanical insights into ribosomal progression overcoming RNA G-quadruplex from periodical translation suppression in cells

Abstract

G-quadruplexes formed on DNA and RNA can be roadblocks to movement of polymerases and ribosome on template nucleotides. Although folding and unfolding processes of the G-quadruplexes are deliberately studied in vitro, how the mechanical and physical properties of the G-quadruplexes affect intracellular biological systems is still unclear. In this study, mRNAs with G-quadruplex forming sequences located either in the 5' untranslated region (UTR) or in the open reading frame (ORF) were constructed to evaluate positional effects of the G-quadruplex on translation suppression in cells. Periodic fluctuation of translation suppression was observed at every three nucleotides within the ORF but not within the 5' UTR. The results suggested that difference in motion of ribosome at the 5' UTR and the ORF determined the ability of the G-quadruplex structure to act as a roadblock to translation in cells and provided mechanical insights into ribosomal progression to overcome the roadblock.

Figure 1. Design of reporter mRNAs.

(a) A G-rich sequence derived from E. coli eutE gene or a variant was inserted into the 5′ UTR or the ORF of an mRNA encoding T7-tagged Renilla luciferase. (b) Sequences of G-rich sequence variants. Wild-type and mutant G-rich sequence regions are underlined. Positions of substitutions from guanine to adenine in the mutant sequence are in italics. Amino acid sequences encoded by the G-rich sequences within the ORF are shown under the nucleotide sequences.

Figure 2. Analysis of NMM fluorescence in the presence of reporter mRNAs.

Reporter mRNAs (500 nM) were mixed with NMM (5 μM) in a buffer containing 30 mM HEPES-KOH (pH 6.8), 100 mM KCl, 0.1% DMSO, and 0.01% Tween 20. Florescence intensities were measured after incubation at 37 °C for 30 min using 400 nm excitation and 615 nm emission. mRNAs were purified and refolded by heating at 70 °C for 5 min and cooling to 25 °C at 1 °C min⁻¹ before addition of NMM.

Figure 3. Translation suppression in MCF7 cells caused by RNA G-quadruplexes located in the 5′ UTR and ORF.

(a) Translated products from reporter mRNAs detected by western blotting. (b) Translation efficiencies of 5′ UTR reporter (blue) and ORF reporter (red) mRNAs. The values were calculated by dividing relative R/F protein ratio by relative R/F mRNA ratio (see Figs S2b and S3, in the Supporting Information).

Figure 4

Dual luciferase assay in cell lysates from (a) MCF7, (b) Flp-Iin 293, (c) HeLa, and (d) HepG2 cells transfected with reporter and control plasmids. Luminescence signals of firefly (green) and Renilla (orange) luciferases (left axes) are plotted in bar graphs; R/F luminescence ratios of 5′ UTR reporter (blue) and ORF reporter (red) (right axes) are plotted as data points. Values are means ± S.D. obtained from 4 or 5 wells.

Figure 5

Schematic illustration of effects of RNA G-quadruplexes located at (a) wt + 0, wt + 3, and wt + 6, (b) wt + 1 and wt + 4, and (c) wt + 2 and wt + 5 positions of ORF on translation.