Spliceozymes: Ribozymes that Remove Introns from Pre-mRNAs in Trans
Figure 2
Influence of the spliceozyme 5′-terminus design on the reaction in vitro.
The ribozyme 5′-terminus ends in (A) a P1 duplex, (B) a P1 extension, or (C) a 5′-duplex (see Figure 1). The corresponding constructs are labeled as 5′ P1, 5′ P1ex, and 5′ EGS, respectively. The top panels show autoradiograms of internally radiolabeled splicing products after separation by denaturing polyacrylamide gel electrophoresis. The marker (M) shows the position of pre-mRNA (778 nt), mRNA (678 nt), and 5′-exon (278 nt). The 3′-exon and the intron had a length of 400 nt and 100 nt, respectively. For each spliceozyme construct, two splicing reactions are analyzed with spliceozyme concentrations of 100 nM and 1 µM. Samples were taken at reaction times between 0 and 60 minutes. A schematic of the substrates and reaction products is shown to the right of the image, with exons in red and the intron in blue. Bottom panels show the quantitation of the disappearance of the CAT pre-mRNA (triangles) and the appearance of the CAT mRNA (squares). Empty symbols correspond to 100 nM spliceozyme, while filled symbols correspond to 1 µM spliceozyme concentration. Grey lines show single-exponential curve fits to the products and double-exponential curve fits to the reaction products. Error bars are standard deviations from three experiments.