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

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.