Functional genomics and biochemical characterization of theC. elegansorthologue of the Machado‐Joseph disease protein ataxin‐3

Abstract

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.

Deubiquitylases From Genes to Organism

“…However, there is a general elevation in the amount of ubiquitylated protein (229). ATXN3 regulates transcription of multiple genes (65,215), a property which may allow for a coordinated response to proteotoxic stresses, which have been shown to promote nuclear accummulation of ATXN3 (206). In C. elegans, ATXN3 has been linked to the control of longevity by the IGF-I signaling axis (131).…”

Absence of Ataxin-3 Leads to Enhanced Stress Response in C. elegans

“…Although human, mouse and C. elegans ataxin-3 are highly conserved and ubiquitously expressed, the worm and mouse knockout animals do not display any major phenotype [19], [20]. The C. elegans atx-3 deletion strains are apparently normal, with similar lifespan and brood size when compared to controls in basal conditions [19].…”

Model Systems for Spinocerebellar Ataxias: Lessons Learned About the Pathogenesis

“…The knockdown of ataxin-2 by siRNA results in reduced numbers of eggs and developmental arrest whereas the knock-out of this gene was embryonically lethal (Kiehl et al, 2000). In comparison, the knock-out of ataxin-3 results in viable animals, which show no obvious morphological abnormalities as well as normal lifespan and behaviour (Rodrigues et al, 2007) but a significantly increased resistance to stress .…”

“…Except for ataxin-7, the worm contains orthologues for all SCA caused by polyQ expansion. Interestingly, for SCA C. elegans strains have only been generated and characterized for SCA2 and SCA3 (Ciosk et al, 2004;Khan et al, 2006;Kiehl et al, 2000;Rodrigues et al, 2007;Teixeira-Castro et al, 2011).…”

“…Likewise, the expression of the worm orthologue of the human ataxin-3 was strongly detected during the late embryogenesis and during all stages of postnatal development. Interestingly, ataxin-3 was not only detected in the central nervous system (in the neuronal dorsal and ventral cord as well as in neurons of the head and tail) but was also observed in the spermatheca, vulval muscle, hypoderm, coelomocytes and body muscles (Rodrigues et al, 2007).…”