Abstract
The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5′ terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3′ end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5′ terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.
Highlights
The p53 protein is one of the major transcription factors responsible for cell homeostasis and cell stress-response [1,2]
As heterogeneous nuclear ribonucleoprotein K (hnRNP K) belongs to the family of poly(C)-binding proteins (PCBPs), we wondered whether others protein members of that family could affect the p53 expression profile, via interaction with the 5 terminus of p53 mRNA
Since poly(C)-binding protein 2 (PCBP2) was among the top 30 proteins with the highest MS score and it has been shown to bind to the 5 terminus of viral and cellular mRNAs we decided to focus on this protein in our further analysis
Summary
The p53 protein is one of the major transcription factors responsible for cell homeostasis and cell stress-response [1,2]. Recent research focuses on how the expression of p53 protein is modulated and connected to other processes in the cell [4] It emerges that regulation of the p53 expression is based on an intricate network of direct and indirect interactions of many protein and nucleic acid factors with the TP53 gene, p53 protein and p53 mRNA. Interactions of the 5 -terminal region of p53 mRNA with protein factors seem to be crucial for the regulation of p53 expression. We have applied RNA-centric chromatography combined with mass spectrometry analysis to identify new proteins which can modulate the p53 expression via interacting with the 5 -terminal region of p53 mRNA [6]. We treated PCBP2-overexpressed cells with rapamycin and a mix of rapamycin and doxorubicin to gain more details about the PCBP2 regulatory function for p53 translation
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