Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

Abstract

AbstractBackgroundPre-mRNA splicing is an essential post-transcriptional process in all eukaryotes. In vitro splicing systems using nuclear or cytoplasmic extracts from mammalian cells, yeast, and Drosophila have provided a wealth of mechanistic insights into assembly and composition of the spliceosome, splicing regulatory proteins and mechanisms of pre-mRNA splicing in non-plant systems. The lack of an in vitro splicing system prepared from plant cells has been a major limitation in splicing research in plants.ResultsHere we report an in vitro splicing assay system using plant nuclear extract. Several lines of evidence indicate that nuclear extract (NE) derived from Arabidopsis seedlings can convert pre-mRNA substrate (LHCB3) into a spliced product. These include: i) generation of an RNA product that corresponds to the size of expected mRNA, ii) a junction-mapping assay using S1 nuclease revealed that the two exons are spliced together, iii) the reaction conditions are similar to those found with non-plant extracts and iv) finally mutations in conserved donor and acceptor sites abolished the production of the spliced product.ConclusionsThis first report on the plant in vitro splicing assay opens new avenues to investigate plant spliceosome assembly and composition, and splicing regulatory mechanisms specific to plants.