miR-19, miR-101, and miR-130 regulate ATXN1 levels. (a and b) miR-19a, miR-101 and miR-130a decrease ATXN1 level in HEK293T cells. A representative western blot image (a) and mean relative levels of ATXN1 (negative control =1) and standard deviation (s.d.) (b) are presented. (c and d) 2´-O-methyl inhibitors specific for each miRNA increase ATXN1 levels. (e) Luciferase assays using portions of the ATXN1 3´UTR depicted by nucleotide number within the 3´UTR identify the regions regulated by three different miRNAs. (f) Schematic of human ATXN1 3´UTR shows location of the authentic miR-19, miR-101, and miR-130 target sites conserved in vertebrates and verified by mutagenesis in Supplementary Figures 6 and 7. * P≤0.05 and ** P<0.01.

Purkinje cell expression of miR-19, miR-101 and miR-130. In situ hybridization using LNA probes for (a) miR-19b, (b) miR-130a, (c) scrambled (control for miR-19b and miR-130a), (d) miR-101a, (e) miR-101b, (f) scrambled (control for miR-101a and miR-101b), (g) miR-130b, and (e) scrambled (control for miR-130b). Upper panels show miR-19b, miR-101 and miR-130 expression in anterior cerebellar lobules and lower panels are enlarged images of boxed regions to show expression in Purkinje cells.

Inhibition of miRNA-mediated posttranscriptional regulation of polyQ expanded ATXN1 causes more severe cytotoxicity in HEK293T cells. (a) Schematic diagrams of three FLAG-hATXN1[86Q] constructs that either lack the 3´UTR (F86Q), contain a wild type 3´UTR (F86Q-3UTR WT), or contain a 3´UTR harboring mutated miRNA target sites depicted as “x” (F86Q-3UTR Mut). (b and c) A mixture of 2´-O-methyl inhibitors specific for each miRNA (19a+19b+101+130a) increases the level of FLAG-hATXN1[86Q] expressed from F86Q-3UTR WT. A representative western blot image (b) and mean relative levels of FLAG-hATXN1[86Q] (negative control =1) and s.d. (c) are presented. EGFP is a normalization control for transfection efficiency. (d) Lethality is significantly enhanced in cells transfected with the mixture of 2´-O-methyl inhibitors specific for each miRNA and F86Q-3UTR WT compared to cells transfected with 2´-O-methyl inhibitor control and F86Q-3UTR WT. (e and f) Disruption of miRNA target sites in the ATXN1 3´UTR increases FLAG-hATXN1[86Q] level. A representative western blot image (e) and mean relative levels of FLAG-hATXN1[86Q] (F86Q of lane 3 in the western image = 1) and s.d. (f) are presented. (g) Assays of cell viability 48h or 72h after transfection with each vector demonstrate significantly more lethality in cells expressing either F86Q or F86Q-3UTR Mut in comparison to cells expressing F86Q-3UTR WT. * P≤0.05 and ** P<0.01.