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

Abstract

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.

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

“…S14D). However, contrary to previous statements (Humphrey et al, 1997;Bennett et al, 2008;Couch et al, 2008), we prove here that the toxicity induced by the full-length BRCA1 is not mediated by the BRCT domain. This indicates that the toxicity resulting from the full-length BRCA1 protein and from small synthetic NLS-BRCT constructs (Humphrey et al, 1997;Coyne et al, 2004) have distinct origins.…”

“…However, the fact that deficiency in Nup49 alleviates the growth inhibition (Millot et al, 2011) might be an indication that the toxicity is not the consequence of importin titration by these NLSs. Bennett et al proposed that the toxicity results from a Spt4-dependent BRCA1 interaction with the yeast RNA polymerase II, inducing a protease activity that cleaves this polymerase (Bennett et al, 2008). We cannot exclude an effect of the nuclear Spt4 protein in the BRCA1 toxicity, which would corroborate the importance of the BRCA1 localization in the nucleus, but we were unable to confirm the RNA pol II cleavage in western blot experiments (Fig.…”

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

“…In this case, BRCA1-Ku80-dependent precise end-joining may provide a second chance for accurate repair during G 1 phase (49). DNA-PKcs has recently been shown to be required for arrest of RNA polymerase II during stalled transcription (50), and given that BRCA1 forms a complex with RNA polymerase II to repress transcription (51)(52)(53)(54), it is possible BRCA1 may also be involved in DSBs repair during stalled transcription (52,53,55,56). Strikingly, another recent report showed that BRCA1 maintained heterochromatin-mediated silencing, which involved in silencing satellite RNA transcripts (57).…”

CtIP: A DNA damage response protein at the intersection of DNA metabolism

“…BRCA1 also forms complexes with transcription factors and RNA polymerase [77][78][79], and has been shown to be important for the resolution of stalled replication forks [80,81]. Thus, the interaction of BRCA1 with CtIP is likely only one part of its complex set of roles in DNA damage repair.…”