The 5'UTR of ATXN1 is alternatively spliced and post-transcriptionally regulates Ataxin-1 expression
Rachna Manek, Tiffany Nelson, Edgardo Rodríguez-Lebrón PhD Department of Pharmacology and Therapeutics and the Center for Translational Research in Neurodegenerative Diseases. University of Florida, Gainesville, Florida
Spinocerebellar ataxia type-1 (SCA-1) is an autosomal dominantly inherited, progressive neurodegenerative disease caused by the abnormal expansion of a 'CAG' tri-nucleotide repeat within the coding region of the ATXN1 gene. Molecular strategies aimed at suppressing Ataxin-1 protein levels in mouse models of SCA1 have shown therapeutic promise in preclinical studies. Thus, understanding the endogenous cellular mechanisms that regulate Ataxin-1 expression, and how this regulation may be disrupted in SCA1, is key to the development of novel, clinically relevant therapeutics. Here we seek to better define how Ataxin-1 levels are regulated post-transcriptionally by evaluating the role that the 5'-untranslated region (5'UTR) of ATXN1 plays on the expression of Ataxin-1, in the context of normal and CAG-expanded transcripts. The 5'UTR of Ataxin-1 is unusually long (> 900bp), extending through at least 7 of the 9 exons that normally comprise the ATXN1 transcript. RT-PCR, quantitative PCR and single-allele analyses of the 5'UTR region in control and SCA1 human brain tissue revealed some novel findings. First, a previously unidentified 5'UTR alternatively spliced variant lacking exons -2 and -3 appears to be the major ATXN1 transcript expressed in the brain. Second, there are alterations to 5'UTR ATXN1 splicing patterns in SCA1 human cerebellum when compared to 5'UTR ATXN1 splicing in control brains. EGFP-based reporter assay showed that the 5'UTR of ATXN1 acts to repress Ataxin-1 expression, post-transcriptionally. Importantly, alternative splicing of the 5'UTR impacts the extent of this repression, suggesting a role for altered 5'UTR ATXN1 splicing in disease pathogenesis. Studies aimed at identifying the mechanisms by which the 5'UTR sequence Ataxin-1 expression revealed a potential role for uORFs and uAUGs, as deletion of all ATG codons in the 5'UTR ATXN1 sequence leads to greater than 2-fold rescue of EGFP expression in our reporter assay. Our data reveals a previously unknown role for the 5'UTR of ATXN1 in the regulation of Ataxin-1 levels and strongly implicates mis-regulation of ATXN1 5'UTR alternative splicing in the pathogenesis of SCA1.