RNA splicing and editing of group II introns in flowering plant mitochondria.

Abstract

The focus of this study is on examining the features and splicing of unusual group II introns observed in plant mitochondria. Plant mitochondrial group II introns contain a number of mispairs within the helical regions of D5 and D6 which are normally among the most highly conserved structures of classical group II introns. In introns within the wheat gene encoding subunit 7 of the NADH dehydrogenase (nad7), six AC mispairs; in these domains were predicted to be corrected to Watson-Crick AU pairs by RNA editing to restore helical structures. The editing status of these regions was determined by direct sequencing of RT-PCR products from partially-spliced and excised intron molecules. Editing was observed at only two of the six predicted sites and it was not conserved among angiosperms. Editing of AC mispairs within intron sequences was unexpectedly low and did not restore the normally highly conserved D5 helical structures. The 3' regions of several group II introns within the mitochondrial nad1, nad4 and nad7 genes show unexpected sequence divergence among flowering plants. The D6 structures of nad1 intron 1 and nad4 intron 2 are highly variable in sequence among plants and, for nad1 intron 1, editing actually reduces similarity among plants. These observations suggest that the core structures and sequences of certain mitochondrial introns in flowering plants are under reduced or different evolutionary constraint compared to conventional group II introns. Steady state levels of nad7 intron-containing precursors and excised intron RNAs were higher in 24 hr germinating embryos than in 6 day etiolated seedlings, suggesting different RNA processing pathways or RNA stabilities. Moreover, excised intron molecules have differing electrophoretic mobilities, indicating the presence of several forms of this intron RNA. Nevertheless, RT-PCF, northern and S1 nuclease protection analyses demonstrate the presence of an abundant lariat form of excised nad7 introns 3 and 4 in 24 hr wheat mt RNA whereas linear forms of excised intron were not detected by these methods. (Abstract shortened by UMI.)