Figure 9.

Binding of Stau1⁵⁵ to the 5′ end increases translation of structure-repressed transcripts. (A) Schematic representation of 5′-structure-repressed transcripts. RNAs coding for the Rluc reporter protein are shown with one copy of the SBS or two copies of the MS2-binding site (MS2bs) at the 5′ end. (B) HEK293T cells were co-transfected with plasmids expressing either Rluc or SBS-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 100-fold repression of translation of the SBS-Rluc RNA was observed as compared with translation of Rluc RNA. Results are expressed as luciferase activity versus concentration of the Stau1⁵⁵-HA₃ coding plasmid. To facilitate comparison, the luciferase activity in the absence of Stau1⁵⁵-HA₃ was defined as 1. P ≤ 0.01, n = 3. Black bars, SBS-Rluc RNA; hatched bars, Rluc RNA. (C) HEK293T cells were co-transfected with plasmids expressing the SBS-Rluc transcript and different concentrations of a plasmid coding for Stau1⁵⁵-HA₃. Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (D) HEK293T cells were co-transfected with plasmids expressing the MS2bs-Rluc transcript and different concentrations of plasmids coding for either MS2-Stau1⁵⁵-HA₃, MS2-HA or Stau1⁵⁵-HA₃. Resulting luciferase activity was quantified 24 h post-transfection. In the absence of MS2-Stau1⁵⁵-HA₃, a 100-fold repression of translation of the MS2bs-Rluc RNA was observed as compared with translation of Rluc RNA. To facilitate comparison, the luciferase activity in the absence of expressor plasmids was defined as 1, n = 3.