journal homepage: www.elsevier.com/locate/yexcr Available online at www.sciencedirect.com Research Article Human Homolog of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies Fatima Elmehdawi a , Gabrielle Wheway a , Katarzyna Szymanska a , Matthew Adams b , Alec S. High c , Colin A. Johnson a,n , Philip A. Robinson a,y a Division of Clinical Sciences, Leeds Institute of Molecular Medicine, Level 8, Wellcome Trust Brenner Building, University of Leeds, Leeds, LS9 7TF West Yorkshire, UK b BioScreening Technology Group, Biomedical Health Research Center, Wellcome Trust Brenner Building, University of Leeds, Leeds, LS9 7TF West Yorkshire, UK c Department of Histopathology, Bexley Wing, St. James’s University Hospital, Beckett Street, Leeds, LS9 7TF West Yorkshire, UK articleinformation Article Chronology: Received 25 July 2012 Received in revised form 18 September 2012 Accepted 2 October 2012 Available online 8 October 2012 Keywords: HHARI ARIH1 UbcH7 Ki67 HNSCC Nuclear bodies P80 coilin SC35 NOPP140 Cell proliferation Reverse genetics screen abstract HHARI (also known as ARIH1) is an ubiquitin-protein ligase and is the cognate of the E2, UbcH7 (UBE2L3). To establish a functional role for HHARI in cellular proliferation processes, we performed a reverse genetics screen that identified n ¼86/522 (16.5%) ubiquitin conjugation components that have a statistically significant effect on cell proliferation, which included HHARI as a strong hit. We then produced and validated a panel of specific antibodies that establish HHARI as both a nuclear and cytoplasmic protein that is expressed in all cell types studied. HHARI was expressed at higher levels in nuclei, and co-localized with nuclear bodies including Cajal bodies (p80 coilin, NOPP140), PML and SC35 bodies. We confirmed reduced cellular proliferation after ARIH1 knockdown with individual siRNA duplexes, in addition to significantly increased levels of apoptosis, an increased proportion of cells in G2 phase of the cell cycle, and significant reductions in total cellular RNA levels. In head and neck squamous cell carcinoma biopsies, there are higher levels of HHARI expression associated with increased levels of proliferation, compared to healthy control tissues. We demonstrate that HHARI is associated with cellular proliferation, which may be mediated through its interaction with UbcH7 and modification of proteins in nuclear bodies. & 2012 Elsevier Inc. All rights reserved. Introduction The human homolog of Drosophila Ariadne protein, HHARI (also known as ARIH1), is a cognate [1] of the ubiquitin conjugation enzyme UbcH7 [2,3]. HHARI consists of a 557 amino acid residue, ‘RING/HECT’ hybrid ubiquitin-protein ligase (E3), of molecular weight 64 kDa encoded by the ARIH1 gene [1,4]. It is evolutionary highly conserved, sharing 72% and 98% amino acid sequence 0014-4827/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.10.002 n Corresponding author. Fax: þ44 113 343 8603. E-mail address: c.johnson@leeds.ac.uk (C.A. Johnson). y Deceased. EXPERIMENTAL CELL RESEARCH 319 (2013) 161 –172