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The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach.

Swiatkowska A, Zydowicz P, Gorska A, Suchacka J, Dutkiewicz M, Ciesiołka J - PLoS ONE (2015)

Bottom Line: In the 5'-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression.The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors.These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland.

ABSTRACT
The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5'-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2'-OMe modified antisense oligomers hybridizing to the 5'-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers' binding were monitored by the Pb2+-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.

No MeSH data available.


Related in: MedlinePlus

Impact of antisense oligomers hybridized to the U180-A218 hairpin on FLp53 translation in MCF-7 cells under conditions of oxidative and genotoxic stress.After oligomer transfection the cells were treated with (A) hydrogen peroxide at a final concentration of 100 μM and (B) 25 μM etoposide. Western blot analysis was performed with a monoclonal antibody (Pab 1801) to detect p53 levels. The GAPDH level was used as a loading control. The experiments were repeated at least twice. Representative Western blots are displayed in the figure. (C) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after 2′-OMe oligomers transfection and incubation the cells in the absence; c(-) and the presence (+) of hydrogen peroxide, respectively. At least two independent experiments were performed, which showed no changes in p53 mRNA level.
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pone.0141676.g004: Impact of antisense oligomers hybridized to the U180-A218 hairpin on FLp53 translation in MCF-7 cells under conditions of oxidative and genotoxic stress.After oligomer transfection the cells were treated with (A) hydrogen peroxide at a final concentration of 100 μM and (B) 25 μM etoposide. Western blot analysis was performed with a monoclonal antibody (Pab 1801) to detect p53 levels. The GAPDH level was used as a loading control. The experiments were repeated at least twice. Representative Western blots are displayed in the figure. (C) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after 2′-OMe oligomers transfection and incubation the cells in the absence; c(-) and the presence (+) of hydrogen peroxide, respectively. At least two independent experiments were performed, which showed no changes in p53 mRNA level.

Mentions: To elucidate which structural elements of the 5'-terminal region of p53 mRNA are important for FLp53 translation efficiency in the presence of selected stress factors, the antisense oligonucleotides were applied to MCF-7 breast carcinoma cells. The cells were exposed to 100 μM hydrogen peroxide after oligomer transfection, and subsequently, incubated for 6 hours. The antibody Pab1801 detected only one band, corresponding to FLp53 (Fig 4A). This is consistent with previously published data demonstrating a lack of Δ40p53 detection/activation upon oxidative stress in MCF-7 cells [11]. Hydrogen peroxide caused an elevation of the FLp53 protein level, as expected (Fig 4A, lanes c and c(-)). In the presence of oligomers No. 7a and 7b, hybridizing to the U180-A218 hairpin and the A219-U228 single-stranded stretch, respectively, decreases in FLp53 translation were observed. Slight translation inhibition was also observed with oligomer No. 7. In contrast oligomers No. 1 and 4 barely affected the efficiency of the FLp53 synthesis (Fig 4A). RT-PCR analysis showed no changes in the level of p53 mRNA in the presence of 2′-OMe oligomers (Fig 4C).


The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach.

Swiatkowska A, Zydowicz P, Gorska A, Suchacka J, Dutkiewicz M, Ciesiołka J - PLoS ONE (2015)

Impact of antisense oligomers hybridized to the U180-A218 hairpin on FLp53 translation in MCF-7 cells under conditions of oxidative and genotoxic stress.After oligomer transfection the cells were treated with (A) hydrogen peroxide at a final concentration of 100 μM and (B) 25 μM etoposide. Western blot analysis was performed with a monoclonal antibody (Pab 1801) to detect p53 levels. The GAPDH level was used as a loading control. The experiments were repeated at least twice. Representative Western blots are displayed in the figure. (C) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after 2′-OMe oligomers transfection and incubation the cells in the absence; c(-) and the presence (+) of hydrogen peroxide, respectively. At least two independent experiments were performed, which showed no changes in p53 mRNA level.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626026&req=5

pone.0141676.g004: Impact of antisense oligomers hybridized to the U180-A218 hairpin on FLp53 translation in MCF-7 cells under conditions of oxidative and genotoxic stress.After oligomer transfection the cells were treated with (A) hydrogen peroxide at a final concentration of 100 μM and (B) 25 μM etoposide. Western blot analysis was performed with a monoclonal antibody (Pab 1801) to detect p53 levels. The GAPDH level was used as a loading control. The experiments were repeated at least twice. Representative Western blots are displayed in the figure. (C) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after 2′-OMe oligomers transfection and incubation the cells in the absence; c(-) and the presence (+) of hydrogen peroxide, respectively. At least two independent experiments were performed, which showed no changes in p53 mRNA level.
Mentions: To elucidate which structural elements of the 5'-terminal region of p53 mRNA are important for FLp53 translation efficiency in the presence of selected stress factors, the antisense oligonucleotides were applied to MCF-7 breast carcinoma cells. The cells were exposed to 100 μM hydrogen peroxide after oligomer transfection, and subsequently, incubated for 6 hours. The antibody Pab1801 detected only one band, corresponding to FLp53 (Fig 4A). This is consistent with previously published data demonstrating a lack of Δ40p53 detection/activation upon oxidative stress in MCF-7 cells [11]. Hydrogen peroxide caused an elevation of the FLp53 protein level, as expected (Fig 4A, lanes c and c(-)). In the presence of oligomers No. 7a and 7b, hybridizing to the U180-A218 hairpin and the A219-U228 single-stranded stretch, respectively, decreases in FLp53 translation were observed. Slight translation inhibition was also observed with oligomer No. 7. In contrast oligomers No. 1 and 4 barely affected the efficiency of the FLp53 synthesis (Fig 4A). RT-PCR analysis showed no changes in the level of p53 mRNA in the presence of 2′-OMe oligomers (Fig 4C).

Bottom Line: In the 5'-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression.The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors.These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland.

ABSTRACT
The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5'-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2'-OMe modified antisense oligomers hybridizing to the 5'-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers' binding were monitored by the Pb2+-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.

No MeSH data available.


Related in: MedlinePlus