Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro.

Abstract

The first intron of the mitochondrial gene coding for cytochrome oxidase subunit I (COI I1) of Podospora anserina can undergo self-splicing in vitro at high concentrations of NH 4 Cl or KCl. Under these conditions cleavage at the 5' splice junction takes place without branch formation probably via hydrolysis by water or OH - and the intron is released in a linear form. In vitro transcripts that contain mutated introns with large deletions in nonconserved domain IV comprising > 50% of the intronic sequence display a more efficient splicing reaction and, surprisingly, 5' cleavage via transesterification and lariat formation is re-established to a low degree under NH 4 Cl. In contrast to the self-splicing group II introns aI5γ and bI1 from yeast mitochondria cleavage at the 3' splice site of the Podospora intron is reduced and cleavage by hydrolysis in trans (i.e. exon reopening) is almost completely suppressed. Both observations could be interpreted as a result of unfavourable spatial conformations of the intron that (i) lead to a steric hindrance of the 5' exon to attack the 3' splice site in cis and (ii) block intron-dependent cleavage reaction of the ligated exons in trans. Alternatively, the possibility that a weak overall interaction of the postulated exon- with the corresponding intron-binding site (EBS-IBS pairings) is responsible for the remarkable differences to the self-splicing reaction of other group II introns is discussed.