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.

Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5′-Untranslated Region

“…On the other hand, G-quadruplex signature motifs occur predominantly at the 5Ј end of the 5Ј-UTR, which suggests that G-quadruplex secondary structures are involved in translational regulation either by inhibiting the formation of the initiation complex or by inhibition of the scanning ribosome (30). In agreement with this hypothesis, a series of recent reports demonstrate that the 5Ј-UTRs of N-ras, Zic-1, membrane-type matrix metalloproteinase 3, ESR-1, Bcl-2, and others contain a G-quadruplex motif that inhibits the translation of their downstream genes (35)(36)(37)(38)(39)(40)(41)(42).…”

“…3A). 24 h after transfection of the resulting plasmids in HEK293 cells, we performed Dual-Luciferase reporter assays as described (36,37,39). Strikingly, for both mutant variants, GQ-mut1 and GQ-mut2, we observed a significant increase in Renilla luciferase activity normalized to firefly luciferase activity ratios (Fig.…”

“…The ADAM10 G-quadruplex Motif Inhibits Translation of a Luciferase Reporter in Vivo-Recently, it was shown that G-quadruplex motifs within 5Ј-UTRs inhibit translation of their downstream gene in vitro and in vivo (35)(36)(37)(38)(39)(40)(41)(42). Moreover, artificially introduced RNA-G-quadruplex motifs near the ribosomal binding site suppress bacterial gene expression (58).…”

“…The basic structural core motif of G-quadruplexes consists of a series of G-quartet planes, each of which consists of four guanines connected by Hoogsteen-hydrogen bonds and stabilized by monovalent ions, particularly potassium ions (31-34). There is growing evidence that the formation of G-quadruplex secondary structures within 5Ј-UTRs results in translational repression of a number of different mRNAs, including N-ras, Zic-1, membrane-type matrix metalloproteinase 3, ESR-1, and Bcl-2 (35)(36)(37)(38)(39)(40)(41)(42). Similar to the IRE stem loop, the stability of G-quadruplex structures could be modulated by RNA binding proteins such as FMRP or members of the hnRNP A family (43,44).…”

An RNA G-quadruplex in the 3' UTR of the proto-oncogene PIM1 represses translation

“…[1][2][3][4][5][6][7][8][9][10][11] In addition, RNA G-quadruplex structures can also stimulate mitochondrial transcription termination 12 and regulate alternative splicing in TP53 intron 3. 13 In addition, a G-quadruplex close to a cleavage sites in the 3' UTR of insulinlike growth factor II mRNA has been proposed and its structure formation has been demonstrated in vitro.…”