Primer extension analysis of the Rz cleavage sites. (A) Transcripts corresponding to the central domain bearing the wild-type sequence or two different nucleotide substitutions (GUAG in the GNRA tetraloop and CGCCC in the RAAA loop) were subjected to reverse transcriptase (RT) extension with a 5′-end-labeled primer. cDNA products were analyzed on denaturing 6% acrylamide gels parallel to a DNA sequence prepared with the same oligonucleotide. IRES residues are indicated on the left side of the gel every 10 nt. Arrows depict the position of cleavages specifically detected in the Rz digestion samples. Synthesis of the full-length cDNA product is detected at the top of the gel. (B) Position of the cleavage sites indicated on the RNA structure previously determined by RNA probing (Fernandez-Miragall and Martinez-Salas 2003; Fernandez-Miragall et al. 2006) obtained for wt sequence, GUAG mutant sequence (C), and CGCCC mutant sequence (D).
