Quantifying domain-ligand affinities and specificities by high-throughput holdup assay.

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.

Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing.

Multiple sequence variants of BRCA2 exon 7 alter splicing regulation.

BACKGROUND: Exonic variants of unknown biological significance (VUS) identified in patients can affect mRNA splicing, either by changing 5' or 3' splice sites or by modifying splicing regulatory elements. Bioinformatic predictions of these elements are still inaccurate and only few such elements have been functionally mapped in BRCA2. We studied the effect on splicing of eight exon 7 VUS, selected from the French UMD-BRCA2 mutation database. METHODS: We performed splicing minigene assays and analyses of patient RNA. We also developed a pyrosequencing-based quantitative assay, to measure, in patient RNA, the relative contribution of each allele to the production of exon 7-containing transcripts. Moreover, an exonic splicing enhancer (ESE)-dependent minigene assay was used to evaluate the splicing regulatory properties of wild-type and mutant segments. RESULTS: Six out of the eight variants induced splicing defects. In the minigene assay, c.517G>T and c.631G>A altered the natural splice sites, c.572A>G created a new 5' splice site, and c.520C>T, c.587G>A and c.617C>G induced exon 7 skipping (66%, 25% and 46%, respectively). Pyrosequencing of patient RNA confirmed these levels of exon skipping for c.520C>T and c.617C>G. Results from the ESE-dependent minigene assay indicated that c.520C>T and c.587G>A disturb splicing regulatory elements. CONCLUSIONS: BRCA2 exon 7 splicing is regulated by multiple exonic elements and is sensitive to disease-associated sequence variations. Measurements of allelic imbalance in patient-derived RNA and/or quantitative analyses using minigene assays provide valuable estimates of the extent of partial splicing defects. Assessment of pathogenicity of variants with partial splicing effect awaits additional evidence and especially the completion of segregation analyses.