In-vitro splicing of IR intron 10 templates. Panel A: Schematic of RNA templates used for in-vitro splicing. Left panel shows the percentage of skipping of exon 11 for four minigenes pIRA, pIRC, pIRK, and pIRL in HepG2 and HeLa cells in-vivo. Right panel shows the four templates generated by PCR amplification across exons 10 and 11 of the respective minigenes. The templates lacked the downstream intron and exon 12 that are present in the minigenes. The presence of the purine-rich enhancer (GA) and inhibitor (INH) are indicated, dashed lines represent deleted sequence. The size of the intron is shown. Panel B: Splicing of RNA templates either lacking the first seven nucleotides of intron 11 (left lanes), or containing the natural 5' splice site (center lanes) or a consensus 5' splice site (right lanes). All templates were incubated with 40% HeLa nuclear extract under splicing conditions for 60 min, the RNA extracted and separated on 5% sequencing gels. The RNA templates, spliced RNA (153 nt), and 5' exon (110 nt) are indicated by symbols on the right. Lariat intermediates were identified by differential mobility on 8% acrylamide gels and are indicated by asterisks. Panel C: Splicing efficiency was quantified by densitometric scanning of autoradiographs. The natural 5' splice site is represented by nat, the consensus 5' splice site by con. Second step efficiency was calculated from the ratio of spliced product to total RNA (spliced, 5' exon, and template, adjusted for labeling), first step efficiency was calculated from the ratio of spliced product plus 5' exon to total RNA. Values below the gel show the mean of three determinations and show the percent efficiency of each step for each template.