SELF-SPLICING OF A PODOSPORA-ANSERINA GROUP-IIA INTRON INVITRO - EFFECTS OF 3'-TERMINAL INTRON ALTERATIONS ON CLEAVAGE AT THE 5' AND 3' SPLICE-SITE

A shortened derivative of the group IIA intron from the mitochondrial cytochrome-c-oxidase subunit I gene (COI I1) of the ascomycete Podospora anserina can undergo self-splicing in vitro. When compared to self-splicing group IIB introns from yeast mitochondria (aI5c, BI1) the autocatalytic reaction shows a lower efficiency and 5’ cleavage takes place predominantly by hydrolysis. In order to test the influence on reaction efficiency and mode of 5’ cleavage of the long peripheral structure of domain VI (dVI) we generated mutant Podospora introns that have different structural forms of shortened dVI. Our results show that: (1) in general the size and structure of dVI distal from the branch site is essential for 5' transesterification and influences the efficiency of the second splicing step; (2) 5'; transesterification as well as the complete self-splicing reaction is more efficient when the structure of dVI is adapted to that of yeast group IIB introns. Moreover, our data indicate that the postulated γ-γ’ tertiary interaction is also functional for group IIA introns. A weakening or disruption of this interaction in the Podospora intron leads to a greatly reduced cleavage at the 3' splice site and to a selection of cryptic sites downstream in the 3’ exon that almost exclusively restore the strong wild-type γ-γ’ pairing. The so-called guide’ interaction seems to support the selection of 3’ cleavage sites but is of secondary importance in relation to the γ-γ’ interaction. © 1993 Academic Press Limited.