Conjunction of potential G-quadruplex and adjacent cis-elements in the 5′ UTR of hepatocyte nuclear factor 4-alpha strongly inhibit protein expression

Abstract

Hepatocyte nuclear factor 4-alpha (HNF4α) is a well established master regulator of liver development and function. We identified the in vitro presence of a stable secondary structure, G-quadruplex (G4) in the 5′ UTR of P1-HNF4A, the predominant HNF4α isoform(s) in adult liver. Our data suggest that the cooperation of G4 and the adjacent putative protein-binding sites within the 5′ UTR was necessary and sufficient to mediate a strong translational repression. This was supported by analysis of deleted/mutated 5′UTRs and two native regulatory single-nucleotide polymorphisms in the 5′UTR. Additional results indicated that G4 motifs in the 5′ UTRs of other liver-enriched transcription factors also inhibited protein expression. Moreover, pyridostatin, a G4 ligand, specifically potentiated the translational suppressing effect of P1-HNF4A-5′ UTR. In summary, the present study provides the first evidence of the presence of G4 in human P1-HNF4A-5′ UTR in vitro, and establishes a novel working model of strong inhibition of protein translation via interactions of G4 with potential RNA-binding proteins (RBPs). The protein expression of the tumor suppressor HNF4α may be inhibited by interactions of RBPs with the G4 motif in the 5′ UTR to promote cell proliferation during liver development and carcinogenesis.

Novel mechanisms of regulation of the expression and transcriptional activity of hepatocyte nuclear factor 4α

“…We recently discovered the presence of G-quadruplex within the 5ʹ-UTR of human HNF4A that induces a strong translational suppression. 27 In addition, our results indicate that the reporter activity of P1-5ʹ-UTR activated by the P1-HNF4α is comparable with the basal activity of the P1 promoter without the 5ʹ-UTR (data not shown). Therefore, HNF4α may shift the transcriptional starting site (TSS) forward, which consequently shortens the 5′-UTR of HNF4A mRNA and thus attenuates the inhibitory effect by the 5′-UTR.…”

“…We in the first place wanted to reproduce the self‐stimulation of HNF4α. Our recent study discovered a critical role of the HNF4α 5′‐untranslated region (5′‐UTR) in inhibiting the protein expression of HNF4α . To further investigate the mechanism of the self‐stimulation of P1‐HNF4A, we generated multiple‐luciferase reporter vectors carrying the P1 promoter (nucleotide [nt] –985) and/or the coding sequence of 5′‐UTR (nt +89).…”

Translational Inhibition of α-Neurexin 2

“…The fluorescence intensity was measured by Synergy H1 micro-plate reader (BioTek). The excitation spectra were set at 410 nm and the emission spectra was from 550 to 700 nm 46,47 .…”

Colocalization of m6A and G-Quadruplex-Forming Sequences in Viral RNA (HIV, Zika, Hepatitis B, and SV40) Suggests Topological Control of Adenosine N6-Methylation

“…13 In RNA, the literature is incongruent with respect to the importance of these folds, 2,14 although experimental evidence has built a strong case supporting G4 folding in cells 2,15 even though they may not persist long-term. In RNA, G4s can alter mRNA expression, 16,17 regulate pre-mRNA processing (splicing and polyadenylation), 16 are a component of stress granules, 18 and function in microRNAs. 19,20 Thus, G4s in DNA and RNA are critical for regulating the complex human cellular network, and similar functions have been reported in other eukaryotes, 21 plants, 22 prokaryotes, 23,24 and viruses.…”