Fig. 2
From: Development of an in vitro pre-mRNA splicing assay using plant nuclear extract
In vitro splicing assays. a In vitro splicing assay with the Arabidopsis LHCB3 pre-mRNA substrate. Radioactive LHCB3 pre-mRNA substrate was synthesized in vitro with a DNA template using SP6 RNA polymerase (see Fig. 1b) as described in materials and methods. [32P]-labeled LHCB3 pre-mRNA substrate (25,000 cpm) was incubated with nuclear extract from Arabidopsis etiolated seedlings at 30 °C as described in materials and methods. Samples were withdrawn at intervals (0, 90 and 180 min), [32P]-RNA was extracted and analyzed by electrophoresis on a 6% polyacrylamide gel containing 7 M urea. The gel was dried and exposed to a phosphor-imaging screen. b Heat-inactivation of Arabidopsis NE abolished the production of a spliced product. NE from Arabidopsis etiolated seedlings was incubated at 90 °C for 3 min or kept on ice (as a control) were used for splicing assays at 30 °C with the LHCB3 [32P]-pre-mRNA. Samples were withdrawn at different time points (0, 90, and 180 min), [32P]-RNA was extracted and analyzed as described above. c The spliced product is increased with increasing nuclear extract concentration. In vitro splicing of [32P]-labeled LHCB3 pre-mRNA substrate (25,000 cpm) was carried out at 30 °C in 25 μl reaction volume containing different concentrations 0–50% (v/v) of nuclear extract as described in materials and methods. All reactions were stopped after three hours; [32P]-RNA was extracted and analyzed as described above. M indicates [32P]-labeled RNA markers synthesized in vitro using RNA Century™-Plus Marker Templates (Applied Biosystems, AM7782). M* lane contains [32P]-labeled LHCB3 pre-mRNA, spliced mRNA, and exon1. Schematic diagrams on the right show pre-mRNA, spliced mRNA and exon 1 and their sizes. One of the [32P]-RNA products formed in in vitro splicing assay corresponds to the size of spliced [32P]-mRNA marker, suggesting that it could be a spliced product. The asterisks indicate the potential splicing intermediates. Other [32P]-RNA products could be another pre-mRNA splicing intermediates and/or degradation products