Assessment of human nter and cterBRCA1mutations using growth and localization assays in yeast

Abstract

A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.

Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein

“…This indicates that at least one of the two endogenous NLSs of BRCA1 is functional in yeast. It also confirms our previous results using the deficient nucleoporin Nup49-GFP construct, which revealed that an active transport of BRCA1-mCherry from the cytoplasm towards the nucleus is necessary for nuclear aggregation (Millot et al, 2011). NLS inactivation completely impaired this mechanism, resulting in cytoplasmic aggregation.…”

“…5A). The Y1853X nonsense mutation results in the loss of the ten C-terminal amino acids of BRCA1, which destabilizes the BRCT structure (Williams et al, 2001) and delocalizes the full-length BRCA1 aggregation into the cytoplasm in yeast cells (Millot et al, 2011). Strikingly, both W1837C and Y1853X mutations strongly impaired the nuclear localization of the Nter-NLS-Cter peptide (Fig.…”

“…In a previous study, we speculated that the full-length BRCA1-mCherry fluorescent spot is a protein aggregate, excluded from the nucleolus, and presumably amorphous rather than amyloid-like, as it is sensitive to sodium dodecyl sulfate (SDS) and to sarkosyl (Millot et al, 2011). To confirm that the fluorescent spot visualized in microscopy is an aggregate, we analyzed the organization of large nuclear structures.…”

“…It has been known for a long time that expression of the full-length human BRCA1 protein in yeast cells compromises cell growth (Humphrey et al, 1997;Caligo et al, 2009;Millot et al, 2011). However, the origin of this growth inhibition is poorly understood.…”

“…Recently, we proposed evaluating the pathogenic relevance of BRCA1 missense variants by using the yeast Saccharomyces cerevisiae model organism, through four different assays: the colony size, liquid medium, spot formation and yeast localization assays (Millot et al, 2011;Thouvenot et al, 2016). The colony size and liquid medium assays monitor the growth inhibition of yeast cells expressing the full-length wild-type (WT) BRCA1 protein, either by plating the cells on agarose medium and measuring the number of cells in the formed colonies (colony size assay) or by monitoring the concentration of cells grown in liquid medium (liquid medium assay).…”