Fig. 5

Analysis of optimum conditions for splicing assay. a The amount of spliced product at different temperatures. In vitro splicing of LHCB3 [³²P]-pre-mRNA substrate (25,000 cpm) was carried out as described earlier at different temperatures (24, 30, 37, and 40 °C). b Addition of ATP to in vitro splicing assay increased the amount of spliced product from LHCB3 pre-mRNA. c Effect of various concentrations of Mg²⁺ on the production of the spliced product. In vitro splicing reaction of LHCB3 [³²P]-pre-mRNA substrate (25,000 cpm) was performed as described previously with different concentrations of Mg²⁺ (2.5 and 5 mM), or in the presence of different concentration (2,5 and 5 mM) of EDTA, a divalent cation chelator (EDTA). In vitro splicing reaction of LHCB3 [³²P]-pre-mRNA substrate (25,000 cpm) was carried out as described above without (0 mM) with increasing concentrations ATP (1, 2 and 3 mM). All reactions were stopped after 3 h. [³²P]-RNA was recovered and analyzed by electrophoresis on a 6% polyacrylamide-7 M urea gel, followed by autoradiography. RNA markers (M and M*) and schematic diagrams on the right were described in Fig. 2. The asterisks indicate the potential splicing intermediates. Other [³²P]-RNA products could be another pre-mRNA splicing intermediates and/or degradation products