Cleavage efficiency mediated by the N117 hammerhead ribozyme affects EDA alternative splicing. (A) Diagram of the pEDAL and pEDs minigenes. The position of the N117 ribozyme is indicated. To distinguish amplification products in cotransfection experiments, two active or mutant N117 hammerhead ribozymes were inserted in tandem between the EDA exon and the DRE. The DRE is indicated, and the thick line represents the 25-nt-long spacer (see text). The primers used in RT-PCR analysis to amplify nascent transcripts are indicated. (B) Analysis of mature spliced transcripts. RT-PCR analysis of total RNA from Hep3B cells expressing different minigenes was performed, and the resulting amplification products were resolved on 2% agarose gel. The two splicing products are schematically indicated. (M) The 1-kb molecular weight marker. (Histogram on the right) The percentage of EDA exon inclusion (±SD) based on at least three independent duplicate experiments. (C) Analysis of nascent transcripts. Amplification of nascent mRNA with (+) and without (−) RT resolved on 2% agarose gel. The identity of the amplified fragments (a, b, and c) is reported on the right of each panel. (D) Analysis of nascent b transcript across the Rzs. The indicated minigenes (0.45 μg) were cotransfected with pEDAL (0.15 μg) (lanes 1–3) or with pEDs (0.15 μg) (lanes 4–6) in Hep3B cells and the b fragment amplified from total RNA. The identity of b bands originating from pEDAL or pEDS (n1) and other minigenes (n2) is reported. (Lower panel) The PCR control, without reverse transcriptase (−RT). (E) Ratio of the intensity of the n1/n2 bands in D expressed as the mean ±SD of three independent experiments done in duplicate. Quantification of pre-mRNA was done using a fluorescent primer.
