Predictable suppression of gene expression by 5'-UTR-based RNA quadruplexes

Abstract

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure-function relationship of synthetic quadruplex sequences in 5'-UTRs in multiple mammalian cell-lines. We establish a universal 'translational suppressor' effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of 'GGG'-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.

Figure 1.

Effect of 5′-UTR RNA quadruplexes on gene expression. (A) Scheme of an RNA quadruplex located in the 5′-UTR (black) of the reporter gene. A typical RNA quadruplex structure comprising all-parallel strand orientation is shown. (B) The expression of various quadruplex-containing constructs and the respective controls (Table 1) in HEK293 cells, measured using a dual luciferase assay and transient transfection, is shown. Error bars represent standard deviation of three independent experiments.

Figure 2.

In vitro stability and in vivo abundancy of the quadruplex motifs. The CD scans (black) show a positive band around 264 nm and negative maxima around 240 nm for 4G₃U (A), 4G₃U₂ (B) and 4G₃U₃ (C), characteristic for a parallel stranded quadruplex motif, while the respective control sequences; con4G₃U (A), con4G₃U₂ (B) and con4G₃U₃ (C) does not show any quadruplex characteristic peaks (grey). In CD melting experiments (D), denaturing (hollow) and annealing (solid) curves are almost identical and show a T_m of >95, 73 and 61°C for 4G₃U (dark grey), 4G₃U₂ (grey) and 4G₃U₃ (light grey) RNA-quadruplex motifs, respectively. (E) Relative hRluc mRNA levels for different constructs determined by real-time PCR assays [hRluc(C_T)] is normalized to hluc mRNA levels [hluc(C_T)], as reported earlier (35). Error bars represent standard deviation of three independent experiments.

Figure 3.

Positional effect and naturally occurring RNA quadruplexes. (A) Normalized luminescence of hRluc luciferase of 4G₃U, 6G₃U, con4G₃U or con6G₃U construct at different positions in the 5′-UTR. Numbers in parentheses indicate the insertion position from the 5′-end. (B) Normalized luminescence of hRluc luciferase of naturally occurring RNA G4 motifs and respective control sequences (Supplementary Table 2). Error bars are standard deviations of three independent experiments.