A homing endonuclease with a switch: Characterization of a twintron encoded homing endonuclease

• The Chaetomium thermophilum mtDNA rns gene is interrupted by a twintron.
• The external intron encoded homing endonuclease gene is interrupted by a group II intron.
• In vitro RNA splicing assays shows that the group II intron can self-splice.
• The mS1247 twintron encoded homing endonuclease is active and cuts within the rns gene.
• This twintron has the potential to be developed into a genome editing tool.

The small ribosomal subunit gene residing in the mitochondrial DNA of the thermophilic fungus Chaetomium thermophilum var. thermophilum La Touche DSM 1495 is interrupted by a twintron at position mS1247. The mS1247 twintron represents the first mixed twintron found in fungal mtDNA, composed of an external group I intron encoding a LAGLIDADG open reading frame that is interrupted by an internal group II intron. Splicing of the internal group II intron reconstitutes the open reading frame and thus facilitates the expression of the encoded homing endonuclease. The cleavage assays suggest that the twintron encodes an active homing endonuclease that could potentially mobilize the twintron to rns genes that have not yet been invaded by this mobile composite element.