A somatic mutation in the 5′UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency
A somatic mutation in the 5′UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency
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DOI：
10.1038/sj.onc.1204620
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发表时间：
2001-07-27
期刊：
影响因子：
8
通讯作者：
Fazio, VM
中科院分区：
文献类型：
--
作者：
Signori, E;Bagni, C;Fazio, VM
引起翻译效率增加/减少的5' UTR中的突变最近被描述为疾病的新分子机制。翻译起始的共有序列的改变可以促进核糖体的上下文依赖性泄漏扫描和/或从下游AUG密码子的起始。最近在正常细胞和可能在乳腺癌中发现了BRCA 1基因下游符合读框的AUG密码子的翻译起始。在这里，我们提出了进一步深入了解BRCA 1的翻译病理生理学调查的BRCA 1的启动一致序列的典型结构的作用。我们分析了BRCA 1基因AUG位点-3位点的体细胞点突变（117 G > C）的影响，该突变在高度侵袭性散发性乳腺癌中被发现。我们构建了编码野生型或突变的BRCA 1 5 'UTR下游的荧光素酶报告序列的嵌合基因。测试这些转录物在体外和体内系统中的活性。在体外转录/翻译测定中，具有突变BRCA 1 5 'UTR的构建体的估计翻译效率比具有野生型BRCA 1 5' UTR的构建体低30-50%。通过在人细胞中瞬时转染，分析相同嵌合基因的体内表达。虽然两种构建体被同等地转录，但携带突变序列的质粒产生的荧光素酶活性比野生型序列低70%。最后，为了获得对体内翻译效率的直接评估，我们分析了从转染细胞分离的转录活性和非活性核糖体上的mRNA翻译。突变体mRNA部分定位于亚多核糖体颗粒分析确认多核糖体募集缺陷。因此，BRCA 1 5 'UTR和翻译效率的表征似乎为BRCA 1在乳腺癌和卵巢癌发病机制中的作用提供了新的见解。
Mutations in the 5' UTR which cause increment/ decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G > C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly, aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/ translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.