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Functional genomics and biochemical characterization of the C. elegans orthologue of the Machado-Joseph disease protein ataxin-3

Ana-João Rodrigues^*,, Giovanni Coppola, Cláudia Santos, Maria do Carmo Costa^*, Michael Ailion, Jorge Sequeiros^*, Daniel H. Geschwind and Patrícia Maciel^*,1

^* Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal;

Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine-UCLA, Los Angeles, USA;

UnIGENe, Institute for Molecular and Cell Biology, Porto, Portugal; and

Department of Biology, University of Utah, Salt Lake City, Utah, USA

¹Correspondence: Life and Health Sciences Research Institute, Health Sciences School, University of Minho, Campus de Gualtar, 4710–057 Braga, Portugal. E-mail: pmaciel{at}ecsaude.uminho.pt

Machado-Joseph disease (MJD) is the most common dominant spinocerebellar ataxia. MJD is caused by a CAG trinucleotide expansion in the ATXN3 gene, which encodes a protein named ataxin-3. Ataxin-3 has been proposed to act as a deubiquitinating enzyme in the ubiquitin-proteasome pathway and to be involved in transcriptional repression; nevertheless, its precise biological function(s) remains unknown. To gain further insight into the function of ataxin-3, we have identified the Caenorhabditis elegans orthologue of the ATXN3 gene and characterized its pattern of expression, developmental regulation, and subcellular localization. We demonstrate that, analogous to its human orthologue, C. elegans ataxin-3 has deubiquitinating activity in vitro against polyubiquitin chains with four or more ubiquitins, the minimum ubiquitin length for proteasomal targeting. To further evaluate C. elegans ataxin-3, we characterized the first known knockout animal models both phenotypically and biochemically, and found that the two C. elegans strains were viable and displayed no gross phenotype. To identify a molecular phenotype, we performed a large-scale microarray analysis of gene expression in both knockout strains. The data revealed a significant deregulation of core sets of genes involved in the ubiquitin-proteasome pathway, structure/motility, and signal transduction. This gene identification provides important clues that can help elucidate the specific biological role of ataxin-3 and unveil some of the physiological effects caused by its absence or diminished function.—Rodrigues, A-J., Coppola, G., Santos, C., do Carmo Costa, M., Ailion, M., Sequeiros, J., Geschwind, D. H., Maciel, P. Functional genomics and biochemical characterization of the C. elegans orthologue of the Machado-Joseph disease protein ataxin-3.

Key Words: polyglutamine disorders • ubiquitin-proteasome pathway • microarray • ataxia • knockout