Figure 4.

Tau expression and phosphorylation regulates nuclear deadenylase activity. (A) Tau depletion inhibits nuclear deadenylation under non-stress and UV-induced conditions in HCT116 cells. NEs were prepared from HCT116 cells treated with either MAPT or control siRNAs for 48 h, and/or treated with UV (40 Jm⁻²) and allowed to recover for 2 h. NEs and radiolabeled capped L3(A₃₀) RNA substrate were used in deadenylation assays. Deadenylation reactions were incubated for 90 min (Cevher et al., 2010). RNAs were purified and analyzed by denaturing PAGE. Representative deadenylation reactions from three independent biological assays are shown. Positions of the polyadenylated RNA L3(A₃₀) and the L3 deadenylated product are indicated. On the right, relative deadenylation (RD) levels, calculated as {L3 fragment/[L3 fragment + L3(A₃₀)]} × 100, were plotted for each condition. The RD level for samples from control siRNA treated cells was arbitrarily set at 1.0. The means ± standard deviation of RD values are indicated. Quantifications were done using ImageJ software (http://rsb.info.nih.gov/ij/). The P-values are indicated as *(≤0.05) or **(≤0.005). Lower panels show expression levels of indicated proteins from NEs tested in deadenylation assays. (B,C) Expression of WT-Tau but not of its phospho-derivative PH-Tau induces nuclear deadenylase activity in (B) HCT116 and (C) chinese hamster ovary (CHO) cells. NEs from cells transfected with pAc-GFP expression vectors containing either WT-Tau or PH-Tau (described in Figure 3; Alonso et al., 2010) were used in deadenylation assays and analyzed as described in (A). Additionally, HCT116 cells were treated with UV (40 Jm⁻²) and allowed to recover for 2 h. The RD level for samples treated with the only vector was arbitrarily set at 1.0. (D) Nuclear deadenylation is induced in SH-SY5Y cells treated with hydroxyurea (HU). NEs from untreated SH-SY5Y cells or cells treated with 1 μM HU for 2 h were used in deadenylation assays and analyzed as described in (A). The RD level for untreated samples was arbitrarily set at 1.0.