Protein interaction analysis of senataxin and the ALS4 L389S mutant yields insights into senataxin post-translational modification and uncovers mutant-specific binding with a brain cytoplasmic RNA-encoded peptide

• Published in: PloS one Vol. 8; no. 11; p. e78837
• Main Authors: Bennett, Craig L, Chen, Yingzhang, Vignali, Marissa, Lo, Russell S, Mason, Amanda G, Unal, Asli, Huq Saifee, Nabiha P, Fields, Stanley, La Spada, Albert R
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11.11.2013; Public Library of Science (PLoS)
• Subjects: Adult; Amino Acid Substitution; Amyotrophic lateral sclerosis; Amyotrophic Lateral Sclerosis - genetics; Amyotrophic Lateral Sclerosis - metabolism; Amyotrophic Lateral Sclerosis - pathology; Analysis; Antisense RNA; Ataxia; Binding; Brain; Cell survival; Cloning; Coding; Cognitive ability; Deoxyribonucleic acid; Dimerization; DNA; DNA helicase; DNA Helicases; Female; Genes; Genetic aspects; Genomes; HEK293 Cells; Homology; Humans; Male; Medical screening; Middle Aged; Monomers; Motor neurone disease; Multifunctional Enzymes; Mutation; Mutation, Missense; Neurodegeneration; Non-coding RNA; Nucleotide sequence; Pathogenesis; Pediatrics; Peptides; Post-translation; Post-translational modifications; Protein interaction; Protein Processing, Post-Translational; Proteins; Ribonucleic acid; RNA; RNA helicase; RNA Helicases - genetics; RNA Helicases - metabolism; RNA, Small Cytoplasmic - genetics; RNA, Small Cytoplasmic - metabolism; Screens; Self-association; SUMO-1 Protein - genetics; SUMO-1 Protein - metabolism; Terminal protein; Translation; Ubiquitin; Ubiquitin - genetics; Ubiquitin - metabolism; Yeast; La Jolla California; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0078837

Senataxin is a large 303 kDa protein linked to neuron survival, as recessive mutations cause Ataxia with Oculomotor Apraxia type 2 (AOA2), and dominant mutations cause amyotrophic lateral sclerosis type 4 (ALS4). Senataxin contains an amino-terminal protein-interaction domain and a carboxy-terminal DNA/RNA helicase domain. In this study, we focused upon the common ALS4 mutation, L389S, by performing yeast two-hybrid screens of a human brain expression library with control senataxin or L389S senataxin as bait. Interacting clones identified from the two screens were collated, and redundant hits and false positives subtracted to yield a set of 13 protein interactors. Among these hits, we discovered a highly specific and reproducible interaction of L389S senataxin with a peptide encoded by the antisense sequence of a brain-specific non-coding RNA, known as BCYRN1. We further found that L389S senataxin interacts with other proteins containing regions of conserved homology with the BCYRN1 reverse complement-encoded peptide, suggesting that such aberrant protein interactions may contribute to L389S ALS4 disease pathogenesis. As the yeast two-hybrid screen also demonstrated senataxin self-association, we confirmed senataxin dimerization via its amino-terminal binding domain and determined that the L389S mutation does not abrogate senataxin self-association. Finally, based upon detection of interactions between senataxin and ubiquitin-SUMO pathway modification enzymes, we examined senataxin for the presence of ubiquitin and SUMO monomers, and observed this post-translational modification. Our senataxin protein interaction study reveals a number of features of senataxin biology that shed light on senataxin normal function and likely on senataxin molecular pathology in ALS4.