Figure 7.

RNA Lassos can redirect splicing of Fas pre-mRNA. (a) Target sequences (red) of Lassos Fas1, Fas2 and Fas3 that span the intron 5/exon 6 splice junction. (b) Schematic of intron/exon structure of Fas receptor primary transcript (not to scale). The Lasso target site (intron 5/exon 6 junction) chosen to prevent inclusion of exon 6 in the final mRNA product is shown. The PCR primer site locations for analysis are shown as horizontal arrows. (c) RT–PCR amplification (using the exon 4 and exon 9 primers, black arrows) of products of Fas splicing in the presence of Lassos Fas1, Fas2 or Fas3. No shorter DNA fragment corresponding to Fas lacking exon 6 was seen in the absence of Fas Lassos (ct). β-actin serves as a loading control. Stds is a DNA ladder (100–1000 bp). (d) Confirmation that Fas Lassos lead to exon 6 skipping revealed by PCR analysis using a downstream primer spanning the exon 5 and exon 7 junction (red arrow) along with the exon 4 upstream primer, which are unable to amplify a product that includes exon 6. Lanes 2–4 and 5–7 correspond to independent experiments with anti-Fas Lassos 1, 2 and 3. Control lane 8 corresponds to the RT–PCR products obtained with cells that were not transfected by Lassos (ct), and lane 9 contains no PCR template (n/a).