Limits...
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

The p53 and Δ40p53 translation efficiency in the presence of antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA under conditions of with and without oxidative stress in HT-29 cells.The cells were transfected with oligomers No. 1, 4, 7, 7a, 7b, and the control oligomer, respectively. Subsequently, the cells were exposed to stress conditions for 6 hours and then harvested. (A) Endogenous p53 and Δ40p53 level was determined by Western blot using two specific monoclonal antibodies p53: Pab 1801 (two panels are shown—with short and long exposures of the gel) and DO1. The long exposure revealed lower band, corresponding to the Δ40p53 isoform. The α-tubulin level was used as a loading control. (B) The Δ40p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. (C) The p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. White and grey bars indicate normal and stress conditions, respectively. (*) p-values were calculated using Student’s t-test. (D) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from cells after transfection with 2′-OMe oligomers and incubation of the cells in the absence; (-) and the presence (+) of hydrogen peroxide. At least two independent experiments were done to verify that no changes in the p53 mRNA level takes place.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4626026&req=5

pone.0141676.g003: The p53 and Δ40p53 translation efficiency in the presence of antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA under conditions of with and without oxidative stress in HT-29 cells.The cells were transfected with oligomers No. 1, 4, 7, 7a, 7b, and the control oligomer, respectively. Subsequently, the cells were exposed to stress conditions for 6 hours and then harvested. (A) Endogenous p53 and Δ40p53 level was determined by Western blot using two specific monoclonal antibodies p53: Pab 1801 (two panels are shown—with short and long exposures of the gel) and DO1. The long exposure revealed lower band, corresponding to the Δ40p53 isoform. The α-tubulin level was used as a loading control. (B) The Δ40p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. (C) The p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. White and grey bars indicate normal and stress conditions, respectively. (*) p-values were calculated using Student’s t-test. (D) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from cells after transfection with 2′-OMe oligomers and incubation of the cells in the absence; (-) and the presence (+) of hydrogen peroxide. At least two independent experiments were done to verify that no changes in the p53 mRNA level takes place.

Mentions: To evaluate the role of the 5'-terminal region of p53 mRNA in the translation initiation process under stress conditions selected antisense oligomers were applied in the HT-29 colorectal adenocarcinoma cell line. These cells contain a G to A point mutation in codon 273 of the p53 gene, which results in an arginine—histidine substitution (R273H). This mutation results in an overexpression of p53 protein [32]. The employed oligomers bore methyl groups at the 2' hydroxyl positions of each of the ribose residues to prevent triggering the RNase H activity present in the cells and subsequent degradation of the p53 mRNA [29]. This excluded RNase H-dependent effects from analysis. Additionally, we previously showed that p53 mRNA levels were unchanged even after 10 hours after 2′-OMe oligomer transfection [27]. This indicates that 2′-OMe oligomer may disturb the translation process via structurally disordering the RNA, but RNA transcription and stability are unaffected. The level of p53 synthesis in the presence of antisense oligonucleotides was analysed under normal conditions and conditions of oxidative stress. Hydrogen peroxide, at a final concentration of 200 μM, was added to the cell culture 4 hours post-transfection and the cells were subsequently incubated for additional 6 hours. The monoclonal antibody PAB1801, which recognizes amino acid residues 46–55 of the N-terminal region of p53, revealed two bands on a Western blot (Fig 3A, two panels show short and long exposure of the autoradiogram). The major band corresponds to the full-length p53 protein, while the minor one is its isoform, with a molecular weight of approx. 44–46 kDa. The monoclonal antibody DO1 only detected the upper band on the gel (Fig 3A). This antibody only recognizes FLp53 and its isoforms which have the entire N-terminal region, since the antibody is directed against amino acid residues 21–25 of the N-terminus of the p53 protein [18]. It has been previously shown that in HT-29 cells not only FLp53, but also its isoforms, can be detected by Western blotting using the polyclonal antibody CM1 [2]. Thus, most likely, the lower migrating band on the gel, detected by PAB1801 but not detected by DO1, corresponds to the Δ40p53 isoform.


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)

The p53 and Δ40p53 translation efficiency in the presence of antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA under conditions of with and without oxidative stress in HT-29 cells.The cells were transfected with oligomers No. 1, 4, 7, 7a, 7b, and the control oligomer, respectively. Subsequently, the cells were exposed to stress conditions for 6 hours and then harvested. (A) Endogenous p53 and Δ40p53 level was determined by Western blot using two specific monoclonal antibodies p53: Pab 1801 (two panels are shown—with short and long exposures of the gel) and DO1. The long exposure revealed lower band, corresponding to the Δ40p53 isoform. The α-tubulin level was used as a loading control. (B) The Δ40p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. (C) The p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. White and grey bars indicate normal and stress conditions, respectively. (*) p-values were calculated using Student’s t-test. (D) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from cells after transfection with 2′-OMe oligomers and incubation of the cells in the absence; (-) and the presence (+) of hydrogen peroxide. At least two independent experiments were done to verify that no changes in the p53 mRNA level takes place.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141676.g003: The p53 and Δ40p53 translation efficiency in the presence of antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA under conditions of with and without oxidative stress in HT-29 cells.The cells were transfected with oligomers No. 1, 4, 7, 7a, 7b, and the control oligomer, respectively. Subsequently, the cells were exposed to stress conditions for 6 hours and then harvested. (A) Endogenous p53 and Δ40p53 level was determined by Western blot using two specific monoclonal antibodies p53: Pab 1801 (two panels are shown—with short and long exposures of the gel) and DO1. The long exposure revealed lower band, corresponding to the Δ40p53 isoform. The α-tubulin level was used as a loading control. (B) The Δ40p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. (C) The p53 level in the presence of selected 2′-OMe oligomers was compared to the value obtained with control oligomer under normal conditions, which was defined as 100%. The bar graphs show averages and standard deviations for at least three independent experiments. White and grey bars indicate normal and stress conditions, respectively. (*) p-values were calculated using Student’s t-test. (D) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from cells after transfection with 2′-OMe oligomers and incubation of the cells in the absence; (-) and the presence (+) of hydrogen peroxide. At least two independent experiments were done to verify that no changes in the p53 mRNA level takes place.
Mentions: To evaluate the role of the 5'-terminal region of p53 mRNA in the translation initiation process under stress conditions selected antisense oligomers were applied in the HT-29 colorectal adenocarcinoma cell line. These cells contain a G to A point mutation in codon 273 of the p53 gene, which results in an arginine—histidine substitution (R273H). This mutation results in an overexpression of p53 protein [32]. The employed oligomers bore methyl groups at the 2' hydroxyl positions of each of the ribose residues to prevent triggering the RNase H activity present in the cells and subsequent degradation of the p53 mRNA [29]. This excluded RNase H-dependent effects from analysis. Additionally, we previously showed that p53 mRNA levels were unchanged even after 10 hours after 2′-OMe oligomer transfection [27]. This indicates that 2′-OMe oligomer may disturb the translation process via structurally disordering the RNA, but RNA transcription and stability are unaffected. The level of p53 synthesis in the presence of antisense oligonucleotides was analysed under normal conditions and conditions of oxidative stress. Hydrogen peroxide, at a final concentration of 200 μM, was added to the cell culture 4 hours post-transfection and the cells were subsequently incubated for additional 6 hours. The monoclonal antibody PAB1801, which recognizes amino acid residues 46–55 of the N-terminal region of p53, revealed two bands on a Western blot (Fig 3A, two panels show short and long exposure of the autoradiogram). The major band corresponds to the full-length p53 protein, while the minor one is its isoform, with a molecular weight of approx. 44–46 kDa. The monoclonal antibody DO1 only detected the upper band on the gel (Fig 3A). This antibody only recognizes FLp53 and its isoforms which have the entire N-terminal region, since the antibody is directed against amino acid residues 21–25 of the N-terminus of the p53 protein [18]. It has been previously shown that in HT-29 cells not only FLp53, but also its isoforms, can be detected by Western blotting using the polyclonal antibody CM1 [2]. Thus, most likely, the lower migrating band on the gel, detected by PAB1801 but not detected by DO1, corresponds to the Δ40p53 isoform.

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