Structure and expression of the trans-split gene for NADH dehydrogenase subunit I in wheat mitochondria.

Abstract

Plant mitochondrial genomes are known to undergo frequent DNA rearrangements during evolution. Such DNA rearrangements have been observed within the extreme 5' or 3' termini of plant mitochondrial genes. The present studies of the gene for NADH dehydrogenase subunit I (nad1) in wheat demonstrate that DNA rearrangements can even occur at internal sites. This work shows that the nad1 gene is comprised of five single-copy exons in wheat mitochondria. The predicted NAD1 amino acid sequence is closely related to that from non-plant and chloroplast counterparts. Somewhat surprisingly, these nad1 sequences are scattered at four distantly-separated sites in the wheat mitochondrial genome. The analysis of regions flanking nad1 coding segments revealed the presence of sequence motifs and helical structures that are hallmarks of group II introns. A model is proposed for nad1 gene expression in which one cis- and three trans-splicing events are necessary for the production of nad1 mRNAs. To investigate nad1 gene expression at the RNA level, transcripts arising from the four nad1 coding regions were analyzed. Northern blot hybridizations and cDNA sequence analysis show that stable transcripts contain all five correctly-linked nad1 exons. S1 nuclease analysis in the regions flanking the nad1 coding segments also revealed the presence of stable transcripts which would be large enough to contain group II intron structures of normal sizes (<3 kb). As has been observed for virtually all plant mitochondrial mRNAs, the maturation of nad1 transcripts also involves RNA editing events. Interestingly, one of these RNA editing sites converts an ACG codon to AUG to create an initiation codon, and this suggests that RNA editing at this site is obligatory for translation of nad1 mRNAs to proceed. RNA editing is also observed within the discontinuous nad1 a/b intron in wheat (which is of particular interest because it lacks several key features of group II introns) but any contribution to intron structure or splicing is as yet uncertain. The wheat nad1 gene organization seems to have resulted from DNA rearrangements that occurred within previously continuous introns. This organization of the nad1 gene in wheat, a monocot, differs from that of dicot nad1 genes and this suggests that some DNA rearrangements have occurred relatively recently during plant evolution. The unusual nad1 gene structure shows that DNA rearrangements can alter not only gene order but also gene structure, provided that scattered gene pieces are properly transcribed and spliced.