Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plantmitochondrialgroup II intronsdonotallpossess hallmark ribozymic features such as the bulged aden-osine involved in lariat formation. To gain insight into their splicing pathways, we have examined the phys-ical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step tran-sesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns,which possess non-encoded 30terminal adenosines, as well as heterogeneous circular introns, which lack 30 nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo