Journal Article

Repeat Associated Non-AUG Translation (RAN Translation) Dependent on Sequence Downstream of the ATXN2 CAG Repeat

Ho, Mi H. T., Dansithong, Warunee, Scoles, Daniel R., Pflieger, Lance T., Petersen, Lance W., Thai, Khanh K., Pulst, Stefan M.

2015

Overview

Spinocerebellar ataxia type 2 (SCA2) is a progressive autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 disease phenotype is characterized by cerebellar atrophy, gait ataxia, and slow saccades. ATXN2 mutation causes gains of toxic and normal functions of the ATXN2 gene product, ataxin-2, and abnormally slow Purkinje cell firing frequency. Previously we investigated features of ATXN2 controlling expression and noted expression differences for ATXN2 constructs with varying CAG lengths, suggestive of repeat associated non-AUG translation (RAN translation). To determine whether RAN translation occurs for ATXN2 we assembled various ATXN2 constructs with ATXN2 tagged by luciferase, HA or FLAG tags, driven by the CMV promoter or the ATXN2 promoter. Luciferase expression from ATXN2-luciferase constructs lacking the ATXN2 start codon was weak vs AUG translation, regardless of promoter type, and did not increase with longer CAG repeat lengths. RAN translation was detected on western blots by the anti-polyglutamine antibody 1C2 for constructs driven by the CMV promoter but not the ATXN2 promoter, and was weaker than AUG translation. Strong RAN translation was also observed when driving the ATXN2 sequence with the CMV promoter with ATXN2 sequence downstream of the CAG repeat truncated to 18 bp in the polyglutamine frame but not in the polyserine or polyalanine frames. Our data demonstrate that ATXN2 RAN translation is weak compared to AUG translation and is dependent on ATXN2 sequences flanking the CAG repeat.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Ataxia / Ataxin / Ataxin-2 - genetics / Ataxin-2 - physiology / Atrophy / Binding sites / Cerebellum / Cloning / Disease / Firing rate / Gait / Genetic aspects / HEK293 Cells / Hereditary diseases / Humans / Luciferase / Luciferases - metabolism / Mutation / Neurology / Phenotypes / Plasmids / Polyalanine / Polyglutamine / Promoter Regions, Genetic - genetics / Promoter Regions, Genetic - physiology / Proteins / Real-Time Polymerase Chain Reaction / Saccades (Eye movements) / Spinocerebellar ataxia / Spinocerebellar Ataxias - genetics / Translation / Trinucleotide repeat diseases / Trinucleotide Repeat Expansion - genetics / Trinucleotide Repeat Expansion - physiology / Trinucleotide repeats / Western blotting