Yeast Screens Identify the RNA Polymerase II CTD and SPT5 as Relevant Targets of BRCA1 Interaction

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression

Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation

G.M. and A.G. were funded by the FCT-Harvard Medical School Program Portugal grant (HMSP-CT/SAU-ICT/0075/2009) and individual FCT post-doctoral fellowships (SFRH/BPD/98439/2013 and SFRH/BPD/82420/2011, respectively). The M.B-D. laboratory is supported by IGC, an EMBO installation grant, ERC grant ERC-2010-StG-261344, FCT grants (FCT Investigator to M.B-D., POCI-01-0145-FEDER-016390 and PTDC/BIM-ONC/6858/2014) and a FCT-Harvard Medical School Program Portugal grant (HMSP-CT/SAU-ICT/0075/2009)

DiMSum: an error model and pipeline for analyzing deep mutational scanning data and diagnosing common experimental pathologies

Deep mutational scanning (DMS) enables multiplexed measurement of the effects of thousands of variants of proteins, RNAs, and regulatory elements. Here, we present a customizable pipeline, DiMSum, that represents an end-to-end solution for obtaining variant fitness and error estimates from raw sequencing data. A key innovation of DiMSum is the use of an interpretable error model that captures the main sources of variability arising in DMS workflows, outperforming previous methods. DiMSum is available as an R/Bioconda package and provides summary reports to help researchers diagnose common DMS pathologies and take remedial steps in their analyses.This work was supported by a European Research Council (ERC) Consolidator grant (616434), the Spanish Ministry of Economy and Competitiveness (BFU2017-89488-P and SEV-2012-0208), the Bettencourt Schueller Foundation, Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 1322.), and the CERCA Program/Generalitat de Catalunya. We also acknowledge the support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and the Centro de Excelencia Severo Ochoa. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement 752809 (J.M.S.)