(A) Switch from IRES-dependent to 5′-end-dependent translation in defective IRES constructs in response to coexpression of the rnpB ribozyme with the Lb protease. BHK-21 cells were transfected with the ribozyme-GUAG IRES constructs in the presence or absence of the Lb-expression plasmid. Lysates collected 4 hpt were used to measure CAT (IRES construct) or luciferase (all constructs). Levels of luciferase activity measured in the ribozyme-IRES constructs were made relative to those detected in the extract corresponding to the control GUAG IRES vector (591200 RLU 4 hpt), set to 100%. (B) Primer extension analysis of the ribozyme cleavage sites. Total RNA isolated from cells transfected with Rz-IRES, RzP4-IRES, Rz-GUAG, or asRs-GUAG was subjected to RT extension with a 5′-end-labeled primer complementary to the IRES sequence. cDNA products were analyzed on denaturing 6% acrylamide gels in parallel with a DNA sequence prepared with the same primer. IRES nucleotides (numbered as in Serrano et al. 2007) are indicated on the left. Arrows denote the position of RT stops specifically detected in the Rz-IRES and Rz-GUAG RNA. Full-length products are show at the top. The relative intensity of RT stops observed in lanes with Rz (measured as the % of the full-length product) is indicated at the bottom.
