RESUMO
Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE-like elements was performed by luciferase reporter assays, qPCR, and poly(A) assays. Herein, we report the down regulation of a luciferase reporter fused to the p53 3'-UTR, when human CPE-binding protein 1 (hCPEB1) is overexpressed. This inhibition is partially rescued when hCPEB1fused to hGLD-2 [a human cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR-based poly(A) test and in vitro poly(A) assay. Taken together, our results suggest that hCPEB1 and hGLD-2 are antagonizing factors regulating p53 mRNA stability.
Assuntos
Citoplasma/metabolismo , Poliadenilação , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Fatores de Poliadenilação e Clivagem de mRNA/fisiologia , Regiões 3' não Traduzidas/genética , Humanos , Polinucleotídeo Adenililtransferase , Estabilidade de RNA , Fatores de Transcrição/fisiologiaRESUMO
All human papillomavirus type 16 (HPV-16) early mRNAs are polyadenylated at the poly(A) signal within the early 3' untranslated region (3'UTR). The 3'end of the early E5 open reading frame and the 3'UTR of HPV-16 is very AU-rich, with five regions similar to cytoplasmic polyadenylation elements (CPEs). We show here that a fragment of the early 3'end comprising four of the five CPE-like regions when inserted downstream of a reporter gene confers regulation of the gene expression. A key protein involved in cytoplasmic polyadenylation is CPEB. We show that the human CPEB1 can repress the activity of the reporter construct containing the HPV-16 early sequences. This repression can be counteracted by a human cytoplasmic poly(A) polymerase, hGLD-2 fused to CPEB1. The hGLD-2/CPEB1 fusion protein facilitates furthermore poly(A) elongation of early HPV transcripts.
Assuntos
Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/fisiologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Transcrição Gênica , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Regiões 3' não Traduzidas , Fusão Gênica Artificial , Humanos , Luciferases/genética , Luciferases/metabolismo , Polinucleotídeo Adenililtransferase , Fatores de Transcrição/metabolismoRESUMO
The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regiões 3' não Traduzidas , Animais , Composição de Bases , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Retroalimentação Fisiológica , Genes p53 , Humanos , Camundongos , Células NIH 3T3 , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , RNA Mensageiro/química , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Estresse FisiológicoRESUMO
Human papillomavirus type 16 (HPV-16) has the capacity to transform human primary keratinocytes. Maintenance of the transformed phenotype requires constitutive expression of the oncoproteins E6 and E7. The low-risk HPV types express E7 from monocistronic mRNA, but for the high-risk types, no mRNA that encodes E7 as the first open reading frame (ORF) has been identified. We recently identified a transcription initiation site within the E6 ORF of HPV-16 at nt 542. In the present study we have characterized the P542 promoter, which putatively controls monocistronic expression of E7. The monocistronic mRNA is not very abundant, but we have shown that an E7-luciferase fusion protein can be expressed in SiHa cells from a monocistronic HPV-16 transcript initiated at nt 542. The monocistronic mRNA expresses E7-luciferase more efficiently than the most abundant in vivo-like mRNA E6*IE7, initiated by P97 and spliced from nt 226 to 409. Furthermore, the translation initiation of E7 is most abundant from the monocistronic mRNA. We have also shown that the P542 promoter is downregulated by the transcription factor activator protein 4 (AP-4) and the differentiation-dependent factor hSkn-1a, both binding downstream of the transcription initiation site. In conclusion, we have found that P542 is a relatively weak promoter compared with P97 and may be downregulated in differentiated epithelial cells.
