PCID2 Impacts Centrosome Duplication by Regulating the Nuclear Export of BARD1 mRNA in Breast Cancer Cells

Abstract

The nuclear export of proteins and other macromolecules out of the nucleus to different intracellular locations is a significant and crucial function of the cell that affects cellular processes such as DNA synthesis, RNA transcription, protein processing, the cell cycle, and apoptosis. Chromosome region maintenance (CRM1) is the major export receptor within the cell, regulating the localization of most proteins. One nuclear export cargo of CRM1 is BARD1, which dimerizes with BRCA1 to regulate DNA repair in the nucleus, while the pair also function in the cytoplasm as negative regulators of centrosome amplification during cell cycle. Despite cooperation at both of these locations BARD1 and BRCA1 are independently exported from the nucleus to the cytoplasm. Our laboratory is interested characterizing the protein PCID2, which is involved in CRM1-mediated protein export and localizes to the centrosome, where it potentially regulates centrosome duplication. We previously demonstrated that PCID2 helps mediate CRM1 dependent export of BRCA1 from the nucleus to the centrosome in Hs578T Breast Cancer cells. The present work aims to determine the role of PCID2 in BARD1 nuclear export and localization in these same cells. PCID2 siRNA knockdowns were used and confirmed via western blot, and BARD1 localization was viewed via immunofluorescence. PCID2 knockdown caused a twenty percent decrease in BARD1 protein in the nucleus and a thirty percent decrease in BARD1 protein at the centrosome. The loss of BARD1 at centrosomes was tied to a fifteen percent increase in Hs578T cells demonstrating excess centrosomes duplication. The loss of BARD1 at both locations in the absence of PCID2 suggests this protein is not involved in the CRM1 mediated export of BARD1 protein from nucleus to centrosomes, and instead changes in cellular localization of BARD1 may be due to an observed 50% decrease in total BARD1 protein with PCID2 knockdown. Interestingly, PCID2 is also a part of the TREX-2 mRNA nuclear export complex; therefore loss of PCID2 may have instead impacted the nuclear export, and translation of BARD1 mRNA. Indeed, PCID2 siRNA knockdown led to a two-fold increase in nuclear BARD1 mRNA as observed by in situ hybridization. Nuclear accumulation of BARD1 mRNA likely contributed the overall decrease in BARD1 expression. The correlation between loss of PCID2 and centrosome amplification suggests PCID2 plays impacts centrosome duplication by regulating both BARD1 mRNA export and BRCA1 protein export; with the loss of PCID2 creating an overall decrease in these negative regulators at the centrosome. Understanding PCID2’s effect on centrosome duplication helps to determine it's impact on cell cycle regulation and define it's potential role as tumor suppressor in Breast Cancer.