Characterization of the I-Spom I Endonuclease from Fission Yeast: Insights into the Evolution of a Group I Intron-Encoded Homing Endonuclease

Abstract

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.

Frequent Gain and Loss of Introns in Fungal Cytochrome b Genes

“…Mitochondrial introns are classified into two main groups, group I and group II, based on their distinct RNA structures that facilitate their self-splicing activity [6]–[7]. Mitochondrial introns are usually group I introns and contain internal open reading frames (ORFs) which facilitate intron removal from RNA transcripts and intron propagation to new sites in genes [8]–[10].…”

The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi

“…Mitochondrial introns mostly belong to the group I, based on a conserved secondary structure with 10 stem-loop domains and a tertiary organization involved in the autocatalytic ribozyme activity (Cech et al, 1994). About 30% of the group I introns possesses a long ORF which encodes a protein described as a maturase required for the splicing, but actually considered as an homing endonuclease (HE) involved in the mobility and site-speciWc integration (homing) of the intron (Pellenz et al, 2002). Most of these HE genes (HEGs) are considered as mobile genetic elements independent of the introns and able to invade various loops of the intron secondary structure (Lambowitz and Belfort, 1993).…”

“…In fungi, functional studies about these mitochondrial intron-encoded proteins and their role in the splicing and transfer of introns have been carried out mainly on species belonging to the Ascomycota phylum such as Schizosaccharomyces pombe or Saccharomyces cerevisiae (Dujon, 1989;Henke et al, 1995;Pellenz et al, 2002). In the Basidiomycota phylum, data on mitochondrial introns occurrence and transfer are scarce and highly conXicting.…”

Characterization and Phylogenetic Analysis of the Mitochondrial Genome of Glarea lozoyensis Indicates High Diversity within the Order Helotiales

“…Group I introns are considered to be mobile genetic elements interrupting protein-coding and structural RNA genes [45]. Most of them carry a “homing endonuclease gene” (heg) encoding a DNA endonuclease (HE), which catalyzes in the transfer and site-specific integration (“homing”) of the intron [46], [47], [48]. There are four families of DNA endonucleases (HEs) [49] denoted by the presence of conserved amino acid sequence motifs: GIY-YIG, HC-box, HNH and LAGLIDADG [50], [51].…”