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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

Modulation of the p53 and Δ40p53 translation initiation process by antisense oligomers complementary to the U180-U228 region in HepG2 upon stress conditions.Cell transfection with oligomers No. 7, 7a, 7b, and control oligomer followed by (A) oxidative and (B) genotoxic treatment, respectively. Endogenous p53, Δ40p53 and Hdm2 proteins’ level was analyzed by Western blot as described in Fig 3. The GAPDH level was used to normalize the data. The levels of p53 and Δ40p53 proteins in the presence of selected 2′-OMe oligomers were compared to the value obtained with the control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. (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.
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pone.0141676.g005: Modulation of the p53 and Δ40p53 translation initiation process by antisense oligomers complementary to the U180-U228 region in HepG2 upon stress conditions.Cell transfection with oligomers No. 7, 7a, 7b, and control oligomer followed by (A) oxidative and (B) genotoxic treatment, respectively. Endogenous p53, Δ40p53 and Hdm2 proteins’ level was analyzed by Western blot as described in Fig 3. The GAPDH level was used to normalize the data. The levels of p53 and Δ40p53 proteins in the presence of selected 2′-OMe oligomers were compared to the value obtained with the control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. (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.

Mentions: We showed that the antisense oligonucleotides affected either FLp53 in MCF-7 cells, or Δ40p53 in HT-29 cells, in which both forms of p53 protein are present, but FLp53 is mutated and overexpressed. We were curious to know whether the oligomers were able to simultaneously affect both of the wild-type p53 isoforms. Antisense oligomers No. 7, 7a and 7b targeting the U180-U228 region were applied in hepatocellular carcinoma cells, HepG2, in which the wild type FLp53 and its Δ40p53 isoform are expressed under normal conditions [11]. The presence of both isoforms of p53 was confirmed by PAB1801 and DO1 antibodies (Fig 5). Under oxidative stress, only modest effects of the oligomers on the translation were observed (Fig 5A). In order to make sure that this was not due to inefficient oligomer incorporation into the cell, a DNA oligomer labelled with fluorescein (FAM) was applied in our assay. Transfection of FAM-conjugated oligomer proved no limitation at this stage of the experiment (data not shown). We observed that under oxidative stress, oligomer No. 7b was most effective in inhibiting both FLp53 and Δ40p53 synthesis by about 10–20%. No change in Hdm2 levels occurred (Fig 5A).


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)

Modulation of the p53 and Δ40p53 translation initiation process by antisense oligomers complementary to the U180-U228 region in HepG2 upon stress conditions.Cell transfection with oligomers No. 7, 7a, 7b, and control oligomer followed by (A) oxidative and (B) genotoxic treatment, respectively. Endogenous p53, Δ40p53 and Hdm2 proteins’ level was analyzed by Western blot as described in Fig 3. The GAPDH level was used to normalize the data. The levels of p53 and Δ40p53 proteins in the presence of selected 2′-OMe oligomers were compared to the value obtained with the control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. (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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141676.g005: Modulation of the p53 and Δ40p53 translation initiation process by antisense oligomers complementary to the U180-U228 region in HepG2 upon stress conditions.Cell transfection with oligomers No. 7, 7a, 7b, and control oligomer followed by (A) oxidative and (B) genotoxic treatment, respectively. Endogenous p53, Δ40p53 and Hdm2 proteins’ level was analyzed by Western blot as described in Fig 3. The GAPDH level was used to normalize the data. The levels of p53 and Δ40p53 proteins in the presence of selected 2′-OMe oligomers were compared to the value obtained with the control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. (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.
Mentions: We showed that the antisense oligonucleotides affected either FLp53 in MCF-7 cells, or Δ40p53 in HT-29 cells, in which both forms of p53 protein are present, but FLp53 is mutated and overexpressed. We were curious to know whether the oligomers were able to simultaneously affect both of the wild-type p53 isoforms. Antisense oligomers No. 7, 7a and 7b targeting the U180-U228 region were applied in hepatocellular carcinoma cells, HepG2, in which the wild type FLp53 and its Δ40p53 isoform are expressed under normal conditions [11]. The presence of both isoforms of p53 was confirmed by PAB1801 and DO1 antibodies (Fig 5). Under oxidative stress, only modest effects of the oligomers on the translation were observed (Fig 5A). In order to make sure that this was not due to inefficient oligomer incorporation into the cell, a DNA oligomer labelled with fluorescein (FAM) was applied in our assay. Transfection of FAM-conjugated oligomer proved no limitation at this stage of the experiment (data not shown). We observed that under oxidative stress, oligomer No. 7b was most effective in inhibiting both FLp53 and Δ40p53 synthesis by about 10–20%. No change in Hdm2 levels occurred (Fig 5A).

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