A somatic mutation in the 5 ' UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency

Mutations in the 5' UTR which cause increment/ decrement of translation eff iciency have been recently described as a novel molecular mechanism of dise ase. Alterations in the consensus sequence for the translation initiation m ay 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 trans lational 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 c ancer. We constructed chimeric genes encoding the luciferase reporter seque nce downstream of the wild type or the mutated BRCA1 5'UTR. These transcrip ts were tested for their activity in in vitro and in vivo systems. In in vi tro transcription/ translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that wi th the wild type BRCA1 5'UTR. The same chimeric genes were analysed for the ir expression in vivo by transient transfection in human cells. While the t wo constructs were equally transcribed, the plasmid carrying the mutated se quence produced 70% less luciferase activity compared to the wild type sequ ence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-activ e ribosomes separated from transfected cells. Mutant mRNA was partially loc alized in subpolysomal particles analytically confirming a polysome recruit ment defect. Thus, characterization of BRCA1 5'UTR and translation efficien cy seems to provide new insight into BRCA1 role in breast and ovarian cance r pathogenesis.