Using multiplex single-base extension typing to screen for mutants defective in RNA editing

Abstract

RNA editing is an RNA maturation process that changes the nucleotide present at particular positions (editing sites) in specific RNAs; in plant organelles, the most common nucleotide change is from cytidine (C) to uridine (U). In a mutant suspected of affecting RNA editing, all known editing sites have to be analyzed. Therefore, to screen a population of mutants, all individuals must be analyzed at every editing site. We describe a multiplex single-nucleotide polymorphism (SNP)-typing procedure to economically screen a mutant individual or population for differences at hundreds of nucleotide positions in RNA or DNA. By using this protocol, we have previously identified mutants defective in RNA editing in a randomly mutated population of Arabidopsis thaliana. The procedure requires 2-3 weeks to identify the individual plant in the mutant population. The time required to locate the mutated gene is between 3 and 24 months in Arabidopsis. Although this procedure has been developed to study RNA editing in plants, it could also be used to investigate other RNA modification processes. It could also be adapted to investigate RNA editing in other organisms.