(A) Diagram of ribozyme-IRES constructs. Three versions of the RNase P ribozyme (PCC6803 rnpB wild type, the GG to CC mutation in helix P4, and the antisense orientation of the rnpB gene) were combined with three different sequences of the IRES element (the wild type, the GUAG mutation in the GNRA motif, and the CGCCC mutation in the RAAA motif). A schematic representation of the FMDV IRES structure with indication of the different domains, the location of the GNRA and RAAA motifs, and the RNase P cleavage site (depicted by an asterisk) mapped in vitro is represented at the top. CAT and luciferase indicate the reporter genes. (B) Determination of reporter RNA amount. Real-time RT-PCR analysis of reporter RNAs in cells transfected with the different versions of the ribozyme constructs. The amount of RNA corresponding to domain 3 (nucleotides 880–1022, relative to the +1 residue of the reporter RNA) or domains 4–5 (nucleotides 1007–1101) of the FMDV IRES was determined in duplicate assays of three independent experiments by RT-qPCR and made relative to the amounts of the 5′ sequence (nucleotides 4–147) present in the same samples. Error bars indicate the standard deviation. The Student's t-test was used for statistical calculation, and P values are indicated.
