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Modulation of p53 expression using antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA in vitro and in the living cells.

Gorska A, Swiatkowska A, Dutkiewicz M, Ciesiolka J - PLoS ONE (2013)

Bottom Line: Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells.A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins.One of these oligomers might be used in the future as a support treatment in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5'-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2'-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5'-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.

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Structure probing of the 5′-terminal region of p53 mRNA in a model RNA construct in rabbit reticulocyte lysate using DMS.(A) Bars represent normalized DMS reactivity as a function of nucleotide position. Bars are coloured using the scheme shown in panel B. (B) The secondary structure of ΔNp53utr in the model RNA construct containing Renilla luciferase coding sequence. Nucleotides modified by DMS are coloured according to their reactivity.
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pone-0078863-g002: Structure probing of the 5′-terminal region of p53 mRNA in a model RNA construct in rabbit reticulocyte lysate using DMS.(A) Bars represent normalized DMS reactivity as a function of nucleotide position. Bars are coloured using the scheme shown in panel B. (B) The secondary structure of ΔNp53utr in the model RNA construct containing Renilla luciferase coding sequence. Nucleotides modified by DMS are coloured according to their reactivity.

Mentions: The structure of the ΔNp53utr-Luc transcript was probed in vitro using Pb2+-induced cleavage [33], [34], SHAPE, and chemical modification method with DMS (Fig. S1). The results revealed no changes in folding of the 5′ part of the transcript in comparison with the 5′-terminal region of the full-length p53 mRNA [16]. Then, the structure of this region was determined in RRL. It turned out that DMS was particularly suitable for probing the RNA structure in the lysate. As shown in Figure 2 the modification sites are in good agreement with the earlier proposed secondary structure model of the 5′-terminal region of p53 mRNA [16]. Modified A and C residues are located in the bulges and apical loops of hairpins G56-C169 and U180-A218, and in the small hairpin G23-C34. Additionally, the nucleotide stretches spanning positions 170–180 and 219–229 were fully accessible to chemical modification with DMS. This confirms the lack of stable structural elements in these regions. Therefore, we concluded that folding of the 5′-terminal part of ΔNp53utr-Luc in RRL is similar to that proposed for the corresponding region of the full-length p53 mRNA. Further, we envisage that the same folding of the 5′-terminal region of p53 mRNA is likely preserved in endogenous p53 mRNA in the living cell.


Modulation of p53 expression using antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA in vitro and in the living cells.

Gorska A, Swiatkowska A, Dutkiewicz M, Ciesiolka J - PLoS ONE (2013)

Structure probing of the 5′-terminal region of p53 mRNA in a model RNA construct in rabbit reticulocyte lysate using DMS.(A) Bars represent normalized DMS reactivity as a function of nucleotide position. Bars are coloured using the scheme shown in panel B. (B) The secondary structure of ΔNp53utr in the model RNA construct containing Renilla luciferase coding sequence. Nucleotides modified by DMS are coloured according to their reactivity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824000&req=5

pone-0078863-g002: Structure probing of the 5′-terminal region of p53 mRNA in a model RNA construct in rabbit reticulocyte lysate using DMS.(A) Bars represent normalized DMS reactivity as a function of nucleotide position. Bars are coloured using the scheme shown in panel B. (B) The secondary structure of ΔNp53utr in the model RNA construct containing Renilla luciferase coding sequence. Nucleotides modified by DMS are coloured according to their reactivity.
Mentions: The structure of the ΔNp53utr-Luc transcript was probed in vitro using Pb2+-induced cleavage [33], [34], SHAPE, and chemical modification method with DMS (Fig. S1). The results revealed no changes in folding of the 5′ part of the transcript in comparison with the 5′-terminal region of the full-length p53 mRNA [16]. Then, the structure of this region was determined in RRL. It turned out that DMS was particularly suitable for probing the RNA structure in the lysate. As shown in Figure 2 the modification sites are in good agreement with the earlier proposed secondary structure model of the 5′-terminal region of p53 mRNA [16]. Modified A and C residues are located in the bulges and apical loops of hairpins G56-C169 and U180-A218, and in the small hairpin G23-C34. Additionally, the nucleotide stretches spanning positions 170–180 and 219–229 were fully accessible to chemical modification with DMS. This confirms the lack of stable structural elements in these regions. Therefore, we concluded that folding of the 5′-terminal part of ΔNp53utr-Luc in RRL is similar to that proposed for the corresponding region of the full-length p53 mRNA. Further, we envisage that the same folding of the 5′-terminal region of p53 mRNA is likely preserved in endogenous p53 mRNA in the living cell.

Bottom Line: Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells.A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins.One of these oligomers might be used in the future as a support treatment in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5'-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2'-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5'-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.

Show MeSH
Related in: MedlinePlus