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5'-UTR RNA G-quadruplexes: translation regulation and targeting.

Bugaut A, Balasubramanian S - Nucleic Acids Res. (2012)

Bottom Line: It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation.We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target.Finally, we close the review with a perspective on the future challenges in the field of 5'-UTR RNA G-quadruplex-mediated translation regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK. ab605@cam.ac.uk

ABSTRACT
RNA structures in the untranslated regions (UTRs) of mRNAs influence post-transcriptional regulation of gene expression. Much of the knowledge in this area depends on canonical double-stranded RNA elements. There has been considerable recent advancement of our understanding of guanine(G)-rich nucleic acids sequences that form four-stranded structures, called G-quadruplexes. While much of the research has been focused on DNA G-quadruplexes, there has recently been a rapid emergence of interest in RNA G-quadruplexes, particularly in the 5'-UTRs of mRNAs. Collectively, these studies suggest that RNA G-quadruplexes exist in the 5'-UTRs of many genes, including genes of clinical interest, and that such structural elements can influence translation. This review features the progresses in the study of 5'-UTR RNA G-quadruplex-mediated translational control. It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation. We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target. Finally, we close the review with a perspective on the future challenges in the field of 5'-UTR RNA G-quadruplex-mediated translation regulation.

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Chemical structures of synthetic molecules that have been demonstrated to exert selective RNA G-quadruplex mediated translation inhibition.
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gks068-F3: Chemical structures of synthetic molecules that have been demonstrated to exert selective RNA G-quadruplex mediated translation inhibition.

Mentions: Using our previously developed NRAS 5′-UTR luciferase system, we evaluated the translational effect of several small molecules from our ligand collection that had previously been established to exhibit selective binding to DNA G-quadruplexes as compared to double-stranded DNA. In this study, we compared the in vitro translation efficiencies of both the wild-type NRAS 5′-UTR and a control 5′-UTR in the presence of increasing concentration of the G-quadruplex ligands (117). The control mRNA was derived from the wild-type NRAS 5′-UTR by deleting the 18-nt G-quadruplex sequence from the wild-type NRAS 5′-UTR. In an initial screening study, we found several molecules that had no effect on translation and others that inhibited translation in a G-quadruplex-independent manner. For example, the well-studied G-quadruplex ligand TMPyP4 showed translational inhibition without any RNA G-quadruplex specificity in this assay, probably because of its poor selectivity for G-quadruplex versus other nucleic acids structures (118,119). However, we also identified molecules that displayed selective translational inhibition depending on the presence of the RNA G-quadruplex. Among these, a pyridine-2,6-bis-quinolino-dicarboxamide derivative (RR82, Figure 3) reduced the translational efficiency of the NRAS 5′-UTR by ∼50% at 1.25 µM concentration; whereas under the same conditions the control still exhibited ∼80% translation efficiency. We then tested structural variants of this molecule and found that a para-fluorophenyl substituent at pyridine C4 (RR110, Figure 3) considerably improved G-quadruplex selectivity. At 10 µM concentration, RR110 had no effect on the translational efficiency of the control mRNA, but inhibited that NRAS 5′-UTR translation by ∼40%. This inhibitory effect was retained in the presence of large excess of double-stranded DNA or hairpin RNA competitors, and it also compared favorably with studies based on in vitro selected RNA aptamers (115,116). Using hydrogen-deuterium exchange followed by 1H-NMR, we demonstrated that RR110 stabilizes the NRAS RNA G-quadruplex and we performed an mRNA stability assay to show that the small molecule did not exert its effect by altering the mRNA degradation rate (117). In addition, we also showed that an RNA G-quadruplex motif that did not display any intrinsic translational inhibitory activity could become a translational inhibitory element upon binding to a G-quadruplex ligand, suggesting that a G-quadruplex-binding small molecule can trigger a translational effect (117). Collectively, these observations support a mechanism whereby stabilization of the NRAS RNA G-quadruplex affects initiation of the translation process and provided proof-of-concept that a small molecule G-quadruplex ligand is able to modulate translation via selectively binding to a 5′-UTR RNA G-quadruplex.Figure 3.


5'-UTR RNA G-quadruplexes: translation regulation and targeting.

Bugaut A, Balasubramanian S - Nucleic Acids Res. (2012)

Chemical structures of synthetic molecules that have been demonstrated to exert selective RNA G-quadruplex mediated translation inhibition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367173&req=5

gks068-F3: Chemical structures of synthetic molecules that have been demonstrated to exert selective RNA G-quadruplex mediated translation inhibition.
Mentions: Using our previously developed NRAS 5′-UTR luciferase system, we evaluated the translational effect of several small molecules from our ligand collection that had previously been established to exhibit selective binding to DNA G-quadruplexes as compared to double-stranded DNA. In this study, we compared the in vitro translation efficiencies of both the wild-type NRAS 5′-UTR and a control 5′-UTR in the presence of increasing concentration of the G-quadruplex ligands (117). The control mRNA was derived from the wild-type NRAS 5′-UTR by deleting the 18-nt G-quadruplex sequence from the wild-type NRAS 5′-UTR. In an initial screening study, we found several molecules that had no effect on translation and others that inhibited translation in a G-quadruplex-independent manner. For example, the well-studied G-quadruplex ligand TMPyP4 showed translational inhibition without any RNA G-quadruplex specificity in this assay, probably because of its poor selectivity for G-quadruplex versus other nucleic acids structures (118,119). However, we also identified molecules that displayed selective translational inhibition depending on the presence of the RNA G-quadruplex. Among these, a pyridine-2,6-bis-quinolino-dicarboxamide derivative (RR82, Figure 3) reduced the translational efficiency of the NRAS 5′-UTR by ∼50% at 1.25 µM concentration; whereas under the same conditions the control still exhibited ∼80% translation efficiency. We then tested structural variants of this molecule and found that a para-fluorophenyl substituent at pyridine C4 (RR110, Figure 3) considerably improved G-quadruplex selectivity. At 10 µM concentration, RR110 had no effect on the translational efficiency of the control mRNA, but inhibited that NRAS 5′-UTR translation by ∼40%. This inhibitory effect was retained in the presence of large excess of double-stranded DNA or hairpin RNA competitors, and it also compared favorably with studies based on in vitro selected RNA aptamers (115,116). Using hydrogen-deuterium exchange followed by 1H-NMR, we demonstrated that RR110 stabilizes the NRAS RNA G-quadruplex and we performed an mRNA stability assay to show that the small molecule did not exert its effect by altering the mRNA degradation rate (117). In addition, we also showed that an RNA G-quadruplex motif that did not display any intrinsic translational inhibitory activity could become a translational inhibitory element upon binding to a G-quadruplex ligand, suggesting that a G-quadruplex-binding small molecule can trigger a translational effect (117). Collectively, these observations support a mechanism whereby stabilization of the NRAS RNA G-quadruplex affects initiation of the translation process and provided proof-of-concept that a small molecule G-quadruplex ligand is able to modulate translation via selectively binding to a 5′-UTR RNA G-quadruplex.Figure 3.

Bottom Line: It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation.We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target.Finally, we close the review with a perspective on the future challenges in the field of 5'-UTR RNA G-quadruplex-mediated translation regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK. ab605@cam.ac.uk

ABSTRACT
RNA structures in the untranslated regions (UTRs) of mRNAs influence post-transcriptional regulation of gene expression. Much of the knowledge in this area depends on canonical double-stranded RNA elements. There has been considerable recent advancement of our understanding of guanine(G)-rich nucleic acids sequences that form four-stranded structures, called G-quadruplexes. While much of the research has been focused on DNA G-quadruplexes, there has recently been a rapid emergence of interest in RNA G-quadruplexes, particularly in the 5'-UTRs of mRNAs. Collectively, these studies suggest that RNA G-quadruplexes exist in the 5'-UTRs of many genes, including genes of clinical interest, and that such structural elements can influence translation. This review features the progresses in the study of 5'-UTR RNA G-quadruplex-mediated translational control. It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation. We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target. Finally, we close the review with a perspective on the future challenges in the field of 5'-UTR RNA G-quadruplex-mediated translation regulation.

Show MeSH