RESUMO
The nonsense-mediated mRNA decay (NMD) mechanism is an evolutionarily conserved process ensuring the degradation of transcripts carrying premature termination codon(s). NMD is believed to prevent the synthesis of truncated proteins that could be detrimental to the cell. However, although numerous studies have assessed the efficiency of this mechanism at the mRNA level, data are lacking in regard to whether NMD fulfills its expected goal at the protein level. In this study, we have investigated whether endogenous alleles of breast cancer predisposing genes carrying nonsense codons were able to produce detectable amounts of truncated proteins in lymphoblastoid cell lines. A total of 20 truncating BRCA1 mutations were analyzed, along with the 1100delC CHEK2 and the 770delT TP53 mutations. All the studied alleles triggered NMD, the amount of mutant transcript ranging from 16 to 63% of that of the wild-type species. We found that BRCA1 and CHK2 truncated proteins could not be detected, even when NMD was inhibited. This suggests that BRCA1 and CHK2 truncated proteins are highly unstable. Conversely, the p53 protein encoded by the 770delT allele is as abundant as the wild-type protein, as removal of the C-terminal p53 domain leads to a stabilized mutant protein, whose abundance is markedly increased when NMD is inhibited. Therefore, our results show that it is not possible to infer the presence of truncated proteins in cells from carriers of a truncated mutation without experimental verification, as each case is expected to be different.
Assuntos
Proteína BRCA1/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Alelos , Androstadienos/farmacologia , Proteína BRCA1/genética , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Códon sem Sentido/metabolismo , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/genética , Estabilidade de RNA/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , WortmaninaRESUMO
BACKGROUND: The Nonsense-Mediated mRNA Decay (NMD) pathway detects and degrades mRNAs containing premature termination codons, thereby preventing the accumulation of potentially detrimental truncated proteins. Intertissue variation in the efficiency of this mechanism has been suggested, which could have important implications for the understanding of genotype-phenotype correlations in various genetic disorders. However, compelling evidence in favour of this hypothesis is lacking. Here, we have explored this question by measuring the ratio of mutant versus wild-type Men1 transcripts in thirteen tissues from mice carrying a heterozygous truncating mutation in the ubiquitously expressed Men1 gene. RESULTS: Significant differences were found between two groups of tissues. The first group, which includes testis, ovary, brain and heart, displays a strong decrease of the nonsense transcript (average ratio of 18% of mutant versus wild-type Men1 transcripts, identical to the value measured in murine embryonic fibroblasts). The second group, comprising lung, intestine and thymus, shows much less pronounced NMD (average ratio of 35%). Importantly, the extent of degradation by NMD does not correlate with the expression level of eleven genes encoding proteins involved in NMD or with the expression level of the Men1 gene. CONCLUSION: Mouse models are an attractive option to evaluate the efficiency of NMD in multiple mammalian tissues and organs, given that it is much easier to obtain these from a mouse than from a single individual carrying a germline truncating mutation. In this study, we have uncovered in the thirteen different murine tissues that we examined up to a two-fold difference in NMD efficiency.
Assuntos
Códon sem Sentido/genética , Especificidade de Órgãos/genética , Estabilidade de RNA/genética , Animais , Regulação da Expressão Gênica , Camundongos , Modelos Animais , Modelos Genéticos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The 185delAG mutation (c.68_69delAG; ter39) in the BRCA1 gene is a founder Jewish Ashkenazi mutation that is carried by 1% of this population and has been identified in thousands of breast or ovarian cancer patients. We have previously described that transcripts bearing this mutation, as well as transcripts bearing the 188del11 mutation (c.71_81del; ter36), are not degraded by nonsense-mediated mRNA decay (NMD), contrary to our observations of other truncating mutations that introduce premature termination codons (PTCs) farther downstream in the coding sequence [Perrin-Vidoz et al., 2002]. To test the hypothesis that these two mutations fail to trigger NMD because of translation reinitiation, we have constructed BRCA1 minigenes and studied their protein expression after transient expression in HeLa cells. We show here that in the presence of a (PTC) at position 36 or 39, translation reinitiation occurs in the BRCA1 minigenes at position 128.