Factor-Dependent Splicing of Chloroplast Group II Introns

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9630213 Barken Group II introns, found in bacterial and organelle genomes, are ribozymes that catalyze their own excision via a mechanism that is closely related to that of nuclear pre-mRNA splicing. In analogy to other enzymes containing catalytic RNA moieties, proteins are required for efficient group II splicing in vivo. However the identities of only two facilitators of group II splicing have been reported and their biochemical roles are not understood. Chloroplast genomes of higher plants contain numerous group II introns, providing potential targets for nuclear-encoded splicing factors, Previous work from this laboratory has identified two nuclear mutations in maize, crs1 and crs2, that block the splicing of group II introns in the chloroplast. Preliminary results suggest that the crs1 mutation blocks the splicing of just one group II intron and of no group I introns. In contrast, crs2 blocks the splicing of most group II introns in the chloroplast. These are the first described mutations that block group II cis-splicing in chloroplasts. Four aims of this work are,(1) Further characterization of the mutant phenotypes, including a complete analysis of all 5' and 3'-splice junctions in the maize chloroplast genome, the expression of the single putative chloroplast "maturase", identification of additional alleles, double-mutant analysis and a search for RNA-binding activities that are modulated by crs1 and crs2 gene function, (2) Cloning of the crs1 and crs2 genes, (3) Analysis of the developmental and genetic control of crs1 and crs2 expression correlated with the splicing of their target introns, and (4) Identification of new nuclear genes that modulate the splicing of group II introns in the chloroplast. %%% The results of the study may have implications for the understanding of the mechanisms and evolution of spliceosomal splicing and for RNA-catalyzed, protein-facilitated reactions in general. In addition, this project will improve our understanding of the role and mechanisms of regu lated RNA splicing in chloroplast development. ***