Fig. 4

Elevated STAU1 levels induce aberrant processing of RNA targets. a, b In qRT-PCR analyses, SCA2- FBs (a) or LBCs (b) revealed significantly reduced PCP2 mRNA abundance compared to normal controls; ATXN2 mRNA levels are not changed. c, d Exogenous STAU1 reduces levels of PCP2 and CALB1 mRNAs, two mRNAs greatly reduced in SCA2 mouse models^{8, 9}. c HEK-293 cells overexpressing Flag-tagged STAU1 were analyzed 48 h post-transfection by western blotting and had reduced PCP2 levels compared to controls. d Matching cultures were analyzed by qRT-PCR demonstrating decreased mRNAs for PCP2 and CALB1 but not ATXN2 compared with controls. Gene expression levels were normalized to GAPDH. e–i Increased STAU1 expression results in reduced PCP2 protein levels in a manner requiring the PCP2-3′UTR. Schematics of MYC-tagged PCP2 cDNA constructs without (e) or with (f) the 3′UTR. g PCP2 constructs (e, f) were transfected into short-term hygromycin selected HEK-293 cells expressing Flag-STAU1. Forty-eight hours post-transfection, western blotting of cellular extracts showed significantly reduced PCP2 levels only from the constructs retaining the 3′UTR (f). Transfection equalities were monitored by anti-neomycin phosphotransferase (NPTII), expressed independently by the PCP2 plasmids. h Quantification of MYC-tagged PCP2 expression on western blots. β-Actin was used as loading control and representative blots of three independent experiments are shown. i In the presence of exogenous STAU1, the 3′UTR is required to reduce luciferase expression. Luciferase expression was reported relative to Renilla luciferase (RLU). Data are mean ± SD, **P < 0.01, ***P < 0.001, Student t-test