I-OmiI and I-OmiII: two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

Area of Science:

• Molecular Biology
• Mycology
• Genomics

Background:

• Mitochondrial genomes of fungi contain introns with embedded open reading frames.
• Homing endonucleases are enzymes encoded by introns that facilitate intron mobility.
• The Ophiostoma minus fungus serves as a model organism for studying fungal genetics.

Purpose of the Study:

• To identify and characterize homing endonucleases within the mitochondrial small subunit ribosomal RNA (rns) gene of Ophiostoma minus.
• To investigate the biochemical activity of the identified homing endonucleases.
• To explore the potential for novel homing endonuclease discovery in fungal mitochondrial genomes.

Main Methods:

• Identification of group IC2 and group IIB1 introns in the Ophiostoma minus rns gene.
• Synthesis and overexpression of codon-optimized homing endonuclease genes (I-OmiI and I-OmiII) in Escherichia coli.
• In vitro characterization of I-OmiII endonuclease activity using various substrates and temperatures.
• In vivo assays to evaluate the endonuclease activity of I-OmiI.

Main Results:

• Two introns, at positions mS569 and mS952, were found in the Ophiostoma minus rns gene, each encoding a LAGLIDADG homing endonuclease (I-OmiI and I-OmiII).
• I-OmiII was confirmed as a functional homing endonuclease, cleaving the rns target site with specific overhangs.
• I-OmiI showed endonuclease activity through in vivo assays, despite purification challenges.

Conclusions:

• Ophiostoma minus harbors functional homing endonucleases encoded by its mitochondrial introns.
• These findings contribute to understanding intron evolution and endonuclease diversity in fungi.
• The study suggests a vast, undiscovered reservoir of functional homing endonucleases in fungal mitochondrial DNA.