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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) Schematic of dual luciferase bi-cistronic constructs (3). (B) Histogram showing percentage of activity of the translation of the Renilla gene as a function of TmPyP4 concentration.
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gkr1308-F5: (A) Schematic of dual luciferase bi-cistronic constructs (3). (B) Histogram showing percentage of activity of the translation of the Renilla gene as a function of TmPyP4 concentration.

Mentions: The possibility that TmPyP4 could unfold the M3Q motif in eukaryotic cells and affect translation efficiency was addressed by using a dual luciferase reporter construct in which the M3Q sequence was placed just before a Renilla luciferase gene, while the upstream firefly luciferase was under the control of a herpes simplex virus thymidine kinase promoter (p-M3Q, Figure 5A). It has been shown previously that the quadruplex has an inhibitory effect on the translation of the Renilla mRNA in this particular plasmid (3). This plasmid was transfected in HeLa cells and incubated with 0, 50 or 100 µM TmPyP4, respectively for 24 h. The cells where inspected prior to the luciferase assay and no detectable change in the cell number or morphology in any of the treatment groups was observed. As can be seen in Figure 5B, 50 µM TmPyP4 had a moderate effect on translation with an increase of 15 ± 4%. However, when the concentration of TmPyP4 was increased to 100 µM it had an increase of 35 ± 2% in translation. In order to increase translation of Renilla mRNA, the TmPyP4 would have to relieve the repressive effect of the quadruplex, presumably by destabilizing the quadruplex structure. It was also of interest to compare these results with those found from having the M3Q sequence in the context of the entire 282 nucleotide 5′-UTR of MT3-MMP mRNA (wt-UTR). A similar increase in activity would suggest that TmPyP4 is binding to the quadruplex embedded within the entire 5′-UTR and is modulating its activity. The entire 5′-UTR was placed in front of the Renilla gene and again assayed after the cells where incubated in the presence of 0, 50 or 100 µM TmPyP4, respectively for 24 h. As shown in Figure 5B, the data for the two constructs correlate well with wt-UTR increasing activity 22 ± 4% in the presence of 50 µM of TmPyP4 and 37 ± 5% when the cells were treated with 100 µM TmPyP4. While evaluating the enhancement of translation by TmPyP4 it should be taken into account that the translation was repressed by the G-quadruplex alone (no ligand) by 55%. We performed qRT-PCR experiments that showed no change in mRNA levels (Supplementary Figure S4) of the wt-UTR construct at 0 and 100 µM concentration of TmPyP4 which shows that the gain in activity was at the translational level. We then tested the effect of TmPyP4 on a mutant of wt-UTR in which the M3Q sequence was deleted (del-UTR). The strategy of using the deletion mutant as a control was based upon two previous independent reports (26,45) where the quadruplex forming segment deleted to test the functional consequence of small-molecule G-quadruplex interactions. Figure 5B shows that at concentrations up to 100 µM there is no increase in Renilla luciferase activity. The lack of any change in the level of translation of the deletion mutant can be explained by presuming that TmPyP4 is specifically disrupting the M3Q quadruplex in vivo to significantly mitigate its repressive effect on translation.Figure 5.


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) Schematic of dual luciferase bi-cistronic constructs (3). (B) Histogram showing percentage of activity of the translation of the Renilla gene as a function of TmPyP4 concentration.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3351169&req=5

gkr1308-F5: (A) Schematic of dual luciferase bi-cistronic constructs (3). (B) Histogram showing percentage of activity of the translation of the Renilla gene as a function of TmPyP4 concentration.
Mentions: The possibility that TmPyP4 could unfold the M3Q motif in eukaryotic cells and affect translation efficiency was addressed by using a dual luciferase reporter construct in which the M3Q sequence was placed just before a Renilla luciferase gene, while the upstream firefly luciferase was under the control of a herpes simplex virus thymidine kinase promoter (p-M3Q, Figure 5A). It has been shown previously that the quadruplex has an inhibitory effect on the translation of the Renilla mRNA in this particular plasmid (3). This plasmid was transfected in HeLa cells and incubated with 0, 50 or 100 µM TmPyP4, respectively for 24 h. The cells where inspected prior to the luciferase assay and no detectable change in the cell number or morphology in any of the treatment groups was observed. As can be seen in Figure 5B, 50 µM TmPyP4 had a moderate effect on translation with an increase of 15 ± 4%. However, when the concentration of TmPyP4 was increased to 100 µM it had an increase of 35 ± 2% in translation. In order to increase translation of Renilla mRNA, the TmPyP4 would have to relieve the repressive effect of the quadruplex, presumably by destabilizing the quadruplex structure. It was also of interest to compare these results with those found from having the M3Q sequence in the context of the entire 282 nucleotide 5′-UTR of MT3-MMP mRNA (wt-UTR). A similar increase in activity would suggest that TmPyP4 is binding to the quadruplex embedded within the entire 5′-UTR and is modulating its activity. The entire 5′-UTR was placed in front of the Renilla gene and again assayed after the cells where incubated in the presence of 0, 50 or 100 µM TmPyP4, respectively for 24 h. As shown in Figure 5B, the data for the two constructs correlate well with wt-UTR increasing activity 22 ± 4% in the presence of 50 µM of TmPyP4 and 37 ± 5% when the cells were treated with 100 µM TmPyP4. While evaluating the enhancement of translation by TmPyP4 it should be taken into account that the translation was repressed by the G-quadruplex alone (no ligand) by 55%. We performed qRT-PCR experiments that showed no change in mRNA levels (Supplementary Figure S4) of the wt-UTR construct at 0 and 100 µM concentration of TmPyP4 which shows that the gain in activity was at the translational level. We then tested the effect of TmPyP4 on a mutant of wt-UTR in which the M3Q sequence was deleted (del-UTR). The strategy of using the deletion mutant as a control was based upon two previous independent reports (26,45) where the quadruplex forming segment deleted to test the functional consequence of small-molecule G-quadruplex interactions. Figure 5B shows that at concentrations up to 100 µM there is no increase in Renilla luciferase activity. The lack of any change in the level of translation of the deletion mutant can be explained by presuming that TmPyP4 is specifically disrupting the M3Q quadruplex in vivo to significantly mitigate its repressive effect on translation.Figure 5.

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