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The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3-MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells.

Morris MJ, Wingate KL, Silwal J, Leeper TC, Basu S - Nucleic Acids Res. (2012)

Bottom Line: Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5'-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex.However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct.Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, USA.

ABSTRACT
We report that the cationic porphyrin TmPyP4, which is known mainly as a DNA G-quadruplex stabilizer, unfolds an unusually stable all purine RNA G-quadruplex (M3Q) that is located in the 5'-UTR of MT3-MMP mRNA. When the interaction between TmPyP4 and M3Q was monitored by UV spectroscopy a 22-nm bathochromic shift and 75% hypochromicity of the porphin major Soret band was observed indicating direct binding of the two molecules. TmPyP4 disrupts folded M3Q in a concentration-dependent fashion as was observed by circular dichroism (CD), 1D (1)H NMR and native gel electrophoresis. Additionally, when TmPyP4 is present during the folding process it inhibits the M3Q RNA from adopting a G-quadruplex structure. Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5'-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex. However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct. Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.

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(A) CD spectra of 4 µM pre-folded (in 100 mM KCl) M3Q in the absence and presence of increasing concentrations of TmPyP4. The chemical structure of TmPyP4 is shown in the inset. The arrow defines the decrease in CD signal as a function of increasing TmPyP4 concentration. (B) Plot of calculated fraction folded versus TmPyP4 concentration.
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gkr1308-F1: (A) CD spectra of 4 µM pre-folded (in 100 mM KCl) M3Q in the absence and presence of increasing concentrations of TmPyP4. The chemical structure of TmPyP4 is shown in the inset. The arrow defines the decrease in CD signal as a function of increasing TmPyP4 concentration. (B) Plot of calculated fraction folded versus TmPyP4 concentration.

Mentions: It has been previously shown that a G-quadruplex located in the 5′-UTR of MT3-MMP (M3Q) mRNA forms an extremely stable quadruplex structure and inhibits translation of a reporter gene in eukaryotic cells (3). The physiological processes related to up-regulation of MT3-MMP make it a target to find small molecules that could selectively interact with the M3Q motif and affect translation. To determine the effect of TmPyP4 on the folded M3Q G-quadruplex, several biochemical and biophysical techniques were utilized. As shown in Figure 1A, the CD spectrum of folded M3Q in 100 mM KCl shows a peak at 263 nm and a trough at 240 nm, which is characteristic of a parallel RNA quadruplex (3,4). Titrating increasing concentrations of TmPyP4 resulted in a decrease in the CD signal at 263 nm, reflecting a disappearance of the quadruplex structure presumably due to its unfolding. According to previously published results, at this concentration of K+, M3Q was unable to be unfolded even at a temperature as high as 95°C (3). Despite the extreme stability of this quadruplex structure under these conditions, it was remarkable that TmPyP4 was able to unfold M3Q. The possibility that the reduction in the CD signal could result from non-specific associations of the added ligand to the RNA and not due to a destabilization of the quaduplex was considered. To ensure that this was not the case and to distinguish between the two possibilities, CD spectra were taken at different wavelengths to check if there was a negative induced circular dichoism (ICD), but there was no evidence of such phenomenon indicating that the reduction at 263 nm was a direct effect of the quadruplex being destabilized (Supplementary Figure S1) (42,43). The results from Figure 1A at different concentrations of TmPyP4 were normalized and graphed against CD signal by assuming that the signal at 263 nm for M3Q in the absence of TmPyP4 corresponds to 100% folded while that at 100 µM TmPyP4 represented 0% folded. As shown in Figure 1B, the data fit well with an exponential function which permitted interpolation for a value of TmPyP4 corresponding to that for 50% unfolding (11 μM). Additionally, the presence of 20 µM TmPyP4 prevents M3Q quadruplex formation when present during the folding of the G-quadruplex, as indicated by the complete lack of any structural features in the CD spectrum (Supplementary Figure S2) (42,43). A possible explanation for the lack of spectral features of a quadruplex structure when TmPyP4 is added before folding of the M3Q motif might be that TmPyP4 binds to the unfolded form of M3Q RNA (44) thus preventing the formation of the quadruplex structure.Figure 1.


The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3-MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells.

Morris MJ, Wingate KL, Silwal J, Leeper TC, Basu S - Nucleic Acids Res. (2012)

(A) CD spectra of 4 µM pre-folded (in 100 mM KCl) M3Q in the absence and presence of increasing concentrations of TmPyP4. The chemical structure of TmPyP4 is shown in the inset. The arrow defines the decrease in CD signal as a function of increasing TmPyP4 concentration. (B) Plot of calculated fraction folded versus TmPyP4 concentration.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351169&req=5

gkr1308-F1: (A) CD spectra of 4 µM pre-folded (in 100 mM KCl) M3Q in the absence and presence of increasing concentrations of TmPyP4. The chemical structure of TmPyP4 is shown in the inset. The arrow defines the decrease in CD signal as a function of increasing TmPyP4 concentration. (B) Plot of calculated fraction folded versus TmPyP4 concentration.
Mentions: It has been previously shown that a G-quadruplex located in the 5′-UTR of MT3-MMP (M3Q) mRNA forms an extremely stable quadruplex structure and inhibits translation of a reporter gene in eukaryotic cells (3). The physiological processes related to up-regulation of MT3-MMP make it a target to find small molecules that could selectively interact with the M3Q motif and affect translation. To determine the effect of TmPyP4 on the folded M3Q G-quadruplex, several biochemical and biophysical techniques were utilized. As shown in Figure 1A, the CD spectrum of folded M3Q in 100 mM KCl shows a peak at 263 nm and a trough at 240 nm, which is characteristic of a parallel RNA quadruplex (3,4). Titrating increasing concentrations of TmPyP4 resulted in a decrease in the CD signal at 263 nm, reflecting a disappearance of the quadruplex structure presumably due to its unfolding. According to previously published results, at this concentration of K+, M3Q was unable to be unfolded even at a temperature as high as 95°C (3). Despite the extreme stability of this quadruplex structure under these conditions, it was remarkable that TmPyP4 was able to unfold M3Q. The possibility that the reduction in the CD signal could result from non-specific associations of the added ligand to the RNA and not due to a destabilization of the quaduplex was considered. To ensure that this was not the case and to distinguish between the two possibilities, CD spectra were taken at different wavelengths to check if there was a negative induced circular dichoism (ICD), but there was no evidence of such phenomenon indicating that the reduction at 263 nm was a direct effect of the quadruplex being destabilized (Supplementary Figure S1) (42,43). The results from Figure 1A at different concentrations of TmPyP4 were normalized and graphed against CD signal by assuming that the signal at 263 nm for M3Q in the absence of TmPyP4 corresponds to 100% folded while that at 100 µM TmPyP4 represented 0% folded. As shown in Figure 1B, the data fit well with an exponential function which permitted interpolation for a value of TmPyP4 corresponding to that for 50% unfolding (11 μM). Additionally, the presence of 20 µM TmPyP4 prevents M3Q quadruplex formation when present during the folding of the G-quadruplex, as indicated by the complete lack of any structural features in the CD spectrum (Supplementary Figure S2) (42,43). A possible explanation for the lack of spectral features of a quadruplex structure when TmPyP4 is added before folding of the M3Q motif might be that TmPyP4 binds to the unfolded form of M3Q RNA (44) thus preventing the formation of the quadruplex structure.Figure 1.

Bottom Line: Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5'-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex.However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct.Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, USA.

ABSTRACT
We report that the cationic porphyrin TmPyP4, which is known mainly as a DNA G-quadruplex stabilizer, unfolds an unusually stable all purine RNA G-quadruplex (M3Q) that is located in the 5'-UTR of MT3-MMP mRNA. When the interaction between TmPyP4 and M3Q was monitored by UV spectroscopy a 22-nm bathochromic shift and 75% hypochromicity of the porphin major Soret band was observed indicating direct binding of the two molecules. TmPyP4 disrupts folded M3Q in a concentration-dependent fashion as was observed by circular dichroism (CD), 1D (1)H NMR and native gel electrophoresis. Additionally, when TmPyP4 is present during the folding process it inhibits the M3Q RNA from adopting a G-quadruplex structure. Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5'-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex. However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct. Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.

Show MeSH
Related in: MedlinePlus