Fig. 1

Preparation of LHCB3 [³²P]-labeled pre-mRNA substrate used for in vitro splicing assay. a Top, a schematic representation of a region of Arabidopsis LHCB3 (AT5G54270) gene used to prepare DNA template to synthesize pre-mRNA substrate. A portion of the third and fourth exons (Orange and green boxes, respectively, labeled as exon 1 and exon2) and second intron (black line, labeled as intron) was used. F primer, forward primer with SP6 promoter sequence (red line), R primer reverse primer with an adaptor sequence (red line). Bottom, PCR fragment amplified with F and R primers using Arabidopsis genomic DNA. The PCR product was gel purified and used for in vitro transcription reaction. b Top, schematic of DNA template that was used to synthesize [³²P]-labeled LHCB3 pre-mRNA substrate. Bottom, A representative autoradiogram of in vitro [³²P]-labeled LHCB3 pre-mRNA substrate. c Description of in vitro splicing assay. Top, Schematic representation of labeled LHCB3 pre-mRNA substrate used for in vitro splicing assay. Bottom, predicted mRNA after in vitro splicing of pre-mRNA substrate. Sizes of intron, exons, pre-mRNA, and predicted mRNA are indicated. Red asterisks indicate [³²P]-nucleotides in RNA