Fig. 5

STAU1 directly interacts with human PCP2 mRNA. a Non-RNase A treated HEK-293 whole-cell extracts expressing Flag or Flag-STAU1 were subjected to immunoprecipitation with Flag mAb beads. Flag-STAU1 pulled down ATXN2 protein, and PCP2 and CALB1 mRNAs on western blot and RT-PCR analysis. b–e Northwestern blotting showing that STAU1 binds directly to human PCP2 RNA in a manner requiring the 3′UTR. Construction of DIG-labeled human PCP2 RNA probes with both UTRs (b), 3′ UTR only (c), and 5′UTR only (d). e Bacterial expressed His-STAU1, His-GFP, or control bacterial lysate (vector alone) were run on SDS–PAGE and stained with Coomassie brilliant blue (CBB) or western blotted with anti-His antibody (blot 1). Protein blots were hybridized with DIG-labeled PCP2 RNA probes followed by anti-DIG antibody staining. STAU1 directly interacts with PCP2 RNAs: (5′ + 3′)UTRs (blot 2) or 3′UTR (blot 3), but not with PCP2(5′UTR) RNA (blot 4). His-GFP shows no interactions. The blot represents one of three independent experiments. f STAU1-PCP2 RNA granules in SCA2 cells. Normal and SCA2 FBs were immunostained with STAU1 antibodies and STAU1 granules (green) are visualized in SCA2 FBs but not in normal FBs. PCP2 RNA granules (red) in SCA2 FBs were detected by FISH using a PCP2-Cy3 probe. Merged images demonstrate positive STAU1-PCP2 RNA granules (white arrows) for SCA2 FBs. Scale bar, 30 µM