title: Requirement of heterogeneous nuclear ribonucleoprotein C for BRCA gene expression and homologous recombination.
abstract: BACKGROUND
  Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR), DNA double strand break (DSB) repair and cell cycle regulation following DNA damage.
  
  
  METHODS
  PALB2 nucleoprotein complexes were isolated using tandem affinity purification from nuclease-solubilized nuclear fraction. Immunofluorescence was used for localization studies of proteins. siRNA-mediated gene silencing and flow cytometry were used for studying DNA repair efficiency and cell cycle distribution/checkpoints. The effect of hnRNP C on mRNA abundance was assayed using quantitative reverse transcriptase PCR.
  
  
  CONCLUSIONS
  We identified hnRNP C as a component of a nucleoprotein complex containing breast cancer suppressor proteins PALB2, BRCA2 and BRCA1. Notably, other components of the 40S ribonucleoprotein particle were not present in the complex. hnRNP C was found to undergo significant changes of sub-nuclear localization after ionizing radiation (IR) and to partially localize to DNA damage sites. Depletion of hnRNP C substantially altered the normal balance of repair mechanisms following DSB induction, reducing HR usage in particular, and impaired S phase progression after IR. Moreover, loss of hnRNP C strongly reduced the abundance of key HR proteins BRCA1, BRCA2, RAD51 and BRIP1, which can be attributed, at least in part, to the downregulation of their mRNAs due to aberrant splicing. Our results establish hnRNP C as a key regulator of BRCA gene expression and HR-based DNA repair. They also suggest the existence of an RNA regulatory program at sites of DNA damage, which involves a unique function of hnRNP C that is independent of the 40S ribonucleoprotein particles and most other hnRNP proteins.
authors.affiliation: Department of Radiation Oncology, The Cancer Institute of New Jersey and Robert Wood Johnson Medical School, New Brunswick, New Jersey, United States of America.
authors.forename: Rachel W
authors.initials: RW
authors.lastname: Anantha
authors.forename: Allen L
authors.initials: AL
authors.lastname: Alcivar
authors.forename: Jianglin
authors.initials: J
authors.lastname: Ma
authors.forename: Hong
authors.initials: H
authors.lastname: Cai
authors.forename: Srilatha
authors.initials: S
authors.lastname: Simhadri
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.forename: Julian
authors.initials: J
authors.lastname: König
authors.forename: Bing
authors.initials: B
authors.lastname: Xia
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics* / Q000378:metabolism; D024682:BRCA2 Protein / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D000076164:Fanconi Anemia Complementation Group N Protein; D005720:Gamma Rays; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects*; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D059765:Homologous Recombination / Q000528:radiation effects; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D000076164:Fanconi Anemia Complementation Group N Protein; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D009687:Nuclear Proteins; C512790:PALB2 protein, human; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins