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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) Native gel shift assay of M3Q (final concentration of ∼250 nM) in the absence and presence of increasing concentrations of TmPyP4. M denotes mut-M3Q. Concentrations used are 0, 1, 5, 10, 20 and 50 µM TmPyP4. (B) Native gel shift assay of mut-M3Q in the absence and presence of increasing concentrations of TmPyP4. M denotes the single stranded RNA marker mut-M3Q. Concentrations of TmPyP4 are the same as in A.
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gkr1308-F2: (A) Native gel shift assay of M3Q (final concentration of ∼250 nM) in the absence and presence of increasing concentrations of TmPyP4. M denotes mut-M3Q. Concentrations used are 0, 1, 5, 10, 20 and 50 µM TmPyP4. (B) Native gel shift assay of mut-M3Q in the absence and presence of increasing concentrations of TmPyP4. M denotes the single stranded RNA marker mut-M3Q. Concentrations of TmPyP4 are the same as in A.

Mentions: To determine whether TmPyP4 can interact and influence the folding and subsequent migration patterns of M3Q RNA, native gel electrophoresis was performed. As shown in the first two lanes of Figure 2A, M3Q has a greater mobility than a mutated version of this sequence (mut-M3Q) which has been previously shown not to form any structure (3). Since the two oligonucleotides have the same charge, the greater mobility of M3Q can be ascribed to formation of a quadruplex structure (45). Therefore, if TmPyP4 unfolds the M3Q quadruplex, then a species with lower mobility should be observed. At total concentrations of TmPyP4 from 5 to 50 μM, an RNA species emerges that has a lower mobility than mut-M3Q which may represent a complex of TmPyP4 with unfolded M3Q. It has been previously shown by the Fry group that the mobility of an RNA sequence that presumably adopts a quadruplex structure can be retarded by TmPyP4 (45). The mut-M3Q sequence shows no change in mobility in the presence of up to 50 µM of TmPyP4 indicating the specificity of the interaction between TmPyP4 and the M3Q sequence. The lower mobility of the M3Q band resulting in the unfolding of the sequence can be corroborated with CD experiments (Figure 1), which indicate that the M3Q quadruplex is almost completely unfolded in the presence of 50 μM TmPyP4. In addition, the binding of TmPyP4 with unfolded M3Q was evidenced by spectral shifts of its Soret absorption band (see below). Interestingly, at concentrations of the ligand from 1 to 10 μM, a band with intermediate mobility was observed, which may reflect the existence of a transient, intermediate complex of TmPyP4 with partially unfolded M3Q. While this may be an intermediate structure, its exact identity cannot be established at this point. It should also be noted that the different levels of retardation in mobility of the M3Q band observed may also be due to binding of the cationic TmPyP4 molecule resulting in a diminished net negative charge on the RNA which would cause its lower mobility. Considering the prevailing dynamic, non-equilibrium experimental condition where RNA anions and TmPyP4 cations are being separated by electrophoresis, the cause of different lower mobilities cannot be determined unambiguously. Overall, the CD and native gel electrophoresis data suggest a change in nature of the M3Q quadruplex structure in the presence of TmPyP4 presumably due to unfolding of M3Q.Figure 2.


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) Native gel shift assay of M3Q (final concentration of ∼250 nM) in the absence and presence of increasing concentrations of TmPyP4. M denotes mut-M3Q. Concentrations used are 0, 1, 5, 10, 20 and 50 µM TmPyP4. (B) Native gel shift assay of mut-M3Q in the absence and presence of increasing concentrations of TmPyP4. M denotes the single stranded RNA marker mut-M3Q. Concentrations of TmPyP4 are the same as in A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1308-F2: (A) Native gel shift assay of M3Q (final concentration of ∼250 nM) in the absence and presence of increasing concentrations of TmPyP4. M denotes mut-M3Q. Concentrations used are 0, 1, 5, 10, 20 and 50 µM TmPyP4. (B) Native gel shift assay of mut-M3Q in the absence and presence of increasing concentrations of TmPyP4. M denotes the single stranded RNA marker mut-M3Q. Concentrations of TmPyP4 are the same as in A.
Mentions: To determine whether TmPyP4 can interact and influence the folding and subsequent migration patterns of M3Q RNA, native gel electrophoresis was performed. As shown in the first two lanes of Figure 2A, M3Q has a greater mobility than a mutated version of this sequence (mut-M3Q) which has been previously shown not to form any structure (3). Since the two oligonucleotides have the same charge, the greater mobility of M3Q can be ascribed to formation of a quadruplex structure (45). Therefore, if TmPyP4 unfolds the M3Q quadruplex, then a species with lower mobility should be observed. At total concentrations of TmPyP4 from 5 to 50 μM, an RNA species emerges that has a lower mobility than mut-M3Q which may represent a complex of TmPyP4 with unfolded M3Q. It has been previously shown by the Fry group that the mobility of an RNA sequence that presumably adopts a quadruplex structure can be retarded by TmPyP4 (45). The mut-M3Q sequence shows no change in mobility in the presence of up to 50 µM of TmPyP4 indicating the specificity of the interaction between TmPyP4 and the M3Q sequence. The lower mobility of the M3Q band resulting in the unfolding of the sequence can be corroborated with CD experiments (Figure 1), which indicate that the M3Q quadruplex is almost completely unfolded in the presence of 50 μM TmPyP4. In addition, the binding of TmPyP4 with unfolded M3Q was evidenced by spectral shifts of its Soret absorption band (see below). Interestingly, at concentrations of the ligand from 1 to 10 μM, a band with intermediate mobility was observed, which may reflect the existence of a transient, intermediate complex of TmPyP4 with partially unfolded M3Q. While this may be an intermediate structure, its exact identity cannot be established at this point. It should also be noted that the different levels of retardation in mobility of the M3Q band observed may also be due to binding of the cationic TmPyP4 molecule resulting in a diminished net negative charge on the RNA which would cause its lower mobility. Considering the prevailing dynamic, non-equilibrium experimental condition where RNA anions and TmPyP4 cations are being separated by electrophoresis, the cause of different lower mobilities cannot be determined unambiguously. Overall, the CD and native gel electrophoresis data suggest a change in nature of the M3Q quadruplex structure in the presence of TmPyP4 presumably due to unfolding of M3Q.Figure 2.

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