Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis.
BACKGROUND
The 3' untranslated region (UTR) of p53 mRNA contains two conserved U-rich sequences resembling cytoplasmic polyadenylation elements (CPE). It is not known if these sequences regulate p53 expression by post-transcriptional mechanisms.


METHODS
Stable p53 3'UTR reporter HaCaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy.


RESULTS
The wild-type p53 3'UTR reduced mRNA steady state levels of the reporter gene and point mutations in the CPEs rescued the mRNA steady state levels in the MCF-7 cells, but not in the HaCaT cells. In both cell lines, the CPEs had a significant effect on translation of the reporter and influenced the effect of UV irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs.


CONCLUSIONS
Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non-irradiated as well as irradiated cells. GAPDH, hnRNP D and hnRNP A/B bind specifically to the p53 CPEs and could potentially be involved in the post-transcriptional regulation of p53.
Rosenstierne
Maiken W
MW
Diabetes Research Group, Department of Biomedical Sciences, University of Copenhagen, Denmark.
Vinther
Jeppe
J
Mittler
Gerhard
G
Larsen
Louise
L
Mann
Matthias
M
Norrild
Bodil
B
D020413:3' Untranslated Regions; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D017930:Genes, Reporter; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000528:radiation effects; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000528:radiation effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism*
D020413:3' Untranslated Regions; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D039221:mRNA Cleavage and Polyadenylation Factors