Figure 2.

Stau1⁵⁵-HA₃ increases translation of TAR-Rluc transcripts in HEK293T cells. (A) HEK293T cells were co-transfected with plasmids expressing either Rluc or TAR-Rluc transcripts and different concentrations of a plasmid coding for Stau1⁵⁵-HA₃. Resulting luciferase activity was quantified 24 h post-transfection. In the absence of Stau1⁵⁵-HA₃, a 2-fold repression of translation of the TAR-Rluc RNA was observed as compared with translation of Rluc RNA. Results are expressed as fold induction in luciferase activity versus the concentration of the Stau1⁵⁵-HA₃ coding plasmid. To facilitate comparison, fold induction of the luciferase activity in the absence of Stau1⁵⁵-HA₃ was defined as 1. Black bars, TAR-Rluc RNA; hatched bars, Rluc RNA. ^**P ≤ 0.01, n = 3. (B) HEK293T cells were transfected with different concentrations of a plasmid coding for Stau1⁵⁵-HA₃. Western blot analyses showed that, for most dilutions, Stau1⁵⁵-HA₃ (60 kDa) is not overexpressed as compared with endogenous Stau1⁵⁵ (55 kDa). (C) Schematic representation of Stau1⁵⁵Δsh1-HA₃ and position of the sh1 and sh2 RNAs. Symbols are described in the legend of Figure 1. (D) HEK293T cells were transfected with plasmids expressing sh1 or shsh2 RNAs (left panel) or co-transfected with plasmids expressing sh1 RNA and Stau1⁵⁵Δsh1-HA₃ (right panel). Proteins were analysed by SDS–PAGE and western blotting using anti-Stau1 and anti-calnexin (Cnx) antibodies. The percentage of down-regulation of endogenous Stau1⁵⁵ is indicated at the bottom of the figure. Position of endogenous Stau1⁶³ and of Stau1⁵⁵ is indicated by two and one asterisk, respectively. (E) HEK293T cells were transfected as described in (D) in the presence of either TAR-Rluc or Rluc expressors. Resulting luciferase activity was analysed as described in (B) (n = 3). Expression of sh1 decreased luciferase activity that is rescued by expression of Stau1⁵⁵Δsh1-HA₃. ^*P ≤ 0.05.