Figure 8.

In the presence of Stau1⁵⁵-HA₃, TAR-Rluc transcripts are shifted to fractions containing heavy polysomes. HEK293T cells were transfected with plasmids expressing either TAR-Rluc (A) or Rluc (B) transcripts (top panels) or co-transfected with the same plasmids and plasmids coding for Stau1⁵⁵-HA₃ (bottom panels). Twenty-four hours post-transfection, cell extracts were centrifuged onto 15–45% sucrose gradients and the position of ribosomal subunits, ribosomes and polysomes determined by OD₂₅₄ (data not shown). Position of polysomes is indicated. RNA was isolated from each fraction and analysed by slot-blotting and hybridization using [³²P]labelled luciferase cDNA. As control, the probe recognized a single band on a northern blot made with RNA isolated from Rluc-transfected HEK293T cells (data not shown). The percentage of RNA in each fraction in relation to the amount of total RNA is plotted above the hybridization blots. The percentage of RNA found in heavy polysomes is indicated below the blots. Similar results were obtained three times. The increase in the amounts of TAR-Rluc transcripts associated with heavy polysomes in the presence of Stau1⁵⁵-HA₃ is statistically significant (P ≤ 0.05), whereas the small decrease in the amounts of Rluc transcripts associated with heavy polysomes in the presence of Stau1⁵⁵-HA₃ is not statistically significant.