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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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Visible absorption spectra of TmPyP4 in the absence and presence of increasing concentration of pre-folded M3Q. The initial concentration of TmPyP4 was 4 µM (100 µl) to which 0.21 nmoles of the pre-folded M3Q RNA quadruplex was added in 0.5 µl increments. The experiment was performed at 100 mM KCl, 0.1 mM EDTA and 10 mM Tris–HCl (pH. 7.5). The arrow pointing downward indicates the decrease in the λmax 424 nm of TmPyP4 with the addition of M3Q and the arrow pointing upward indicates the shifted Soret band.
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gkr1308-F4: Visible absorption spectra of TmPyP4 in the absence and presence of increasing concentration of pre-folded M3Q. The initial concentration of TmPyP4 was 4 µM (100 µl) to which 0.21 nmoles of the pre-folded M3Q RNA quadruplex was added in 0.5 µl increments. The experiment was performed at 100 mM KCl, 0.1 mM EDTA and 10 mM Tris–HCl (pH. 7.5). The arrow pointing downward indicates the decrease in the λmax 424 nm of TmPyP4 with the addition of M3Q and the arrow pointing upward indicates the shifted Soret band.

Mentions: The binding of M3Q and TmPyP4 was also monitored via UV–Vis spectroscopy. Folded M3Q was titrated into a solution of TmPyP4 and the Soret band was monitored as a function of M3Q concentration. As the concentration of M3Q was increased there was substantial hypochromicity (maximum 75%) as well as a rather unusually large bathochromic shift of 22 nm (from λmax 424 to 446 nm), which is indicative of binding of TmPyP4 to the M3Q RNA G-quadruplex (Figure 4) (51). A sharp isosbestic point was observed at 436 nm. When mut-M3Q was titrated, no shift or hypochromicity was apparent even after addition of up to five equivalents of TmPyP4, which suggests specificity of TmPyP4 interaction with the M3Q RNA (Supplementary Figure S3). These results correlate well with the native gel electrophoresis data on the specificity of the interaction between M3Q and TmPyP4.Figure 4.


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)

Visible absorption spectra of TmPyP4 in the absence and presence of increasing concentration of pre-folded M3Q. The initial concentration of TmPyP4 was 4 µM (100 µl) to which 0.21 nmoles of the pre-folded M3Q RNA quadruplex was added in 0.5 µl increments. The experiment was performed at 100 mM KCl, 0.1 mM EDTA and 10 mM Tris–HCl (pH. 7.5). The arrow pointing downward indicates the decrease in the λmax 424 nm of TmPyP4 with the addition of M3Q and the arrow pointing upward indicates the shifted Soret band.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351169&req=5

gkr1308-F4: Visible absorption spectra of TmPyP4 in the absence and presence of increasing concentration of pre-folded M3Q. The initial concentration of TmPyP4 was 4 µM (100 µl) to which 0.21 nmoles of the pre-folded M3Q RNA quadruplex was added in 0.5 µl increments. The experiment was performed at 100 mM KCl, 0.1 mM EDTA and 10 mM Tris–HCl (pH. 7.5). The arrow pointing downward indicates the decrease in the λmax 424 nm of TmPyP4 with the addition of M3Q and the arrow pointing upward indicates the shifted Soret band.
Mentions: The binding of M3Q and TmPyP4 was also monitored via UV–Vis spectroscopy. Folded M3Q was titrated into a solution of TmPyP4 and the Soret band was monitored as a function of M3Q concentration. As the concentration of M3Q was increased there was substantial hypochromicity (maximum 75%) as well as a rather unusually large bathochromic shift of 22 nm (from λmax 424 to 446 nm), which is indicative of binding of TmPyP4 to the M3Q RNA G-quadruplex (Figure 4) (51). A sharp isosbestic point was observed at 436 nm. When mut-M3Q was titrated, no shift or hypochromicity was apparent even after addition of up to five equivalents of TmPyP4, which suggests specificity of TmPyP4 interaction with the M3Q RNA (Supplementary Figure S3). These results correlate well with the native gel electrophoresis data on the specificity of the interaction between M3Q and TmPyP4.Figure 4.

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