Accurate in vitro splicing of two pre-mRNA plant introns in a HeLa cell nuclear extract

Abstract

Two plant introns along with flanking exon sequences have been isolated from an amylase gene of wheat and a legumin gene of pea and cloned behind the phage SP6 promoter. Pre-mRNAs produced by in vitro transcription with SP6 RNA polymerase were tested for their ability to be spliced in a HeLa cell nuclear extract. The plant introns were accurately spliced and the predicted splice junctions were used. Lariat RNAs were observed as both intermediates and final products during the splicing reaction. The branch points were mapped to adenosine residues lying within sequences that showed good homology to the animal branch point consensus. Consensus sequences for the 5' and 3' splice junctions and for putative branch point sequences of plants were derived from an analysis of 168 plant intron sequences.