A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions

Abstract

A group II intron encoding a protein belonging to the LAGLIDADG family of homing endonucleases was identified in the mitochondrial rns gene of the filamentous fungus Leptographium truncatum, and the catalytic activities of both the intron and its encoded protein were characterized. A model of the RNA secondary structure indicates that the intron is a member of the IIB1 subclass and the open reading frame is inserted in ribozyme domain III. In vitro assays carried out with two versions of the intron, one in which the open reading frame was removed and the other in which it was present, demonstrate that both versions of the intron readily self-splice at 37°C and at a concentration of MgCl 2 as low as 6 mM. The open reading frame encodes a functional LAGLIDADG homing endonuclease that cleaves 2 (top strand) and 6 (bottom strand) nucleotides (nt) upstream of the intron insertion site, generating 4 nt 39 OH overhangs. In vitro splicing assays carried out in the absence and presence of the intron-encoded protein indicate that the protein does not enhance intron splicing, and RNA-binding assays show that the protein does not appear to bind to the intron RNA precursor transcript. These findings raise intriguing questions concerning the functional and evolutionary relationships of the two components of this unique composite element.

Tapping natural reservoirs of homing endonucleases for targeted gene modification

“…In the subset of LHEs that included the previously characterized enzymes I-OnuI, I-LtrI, and I-LtrII (13,15), many additional putative endonucleases reside within a wide variety of different host genes (see the I-OnuI subfamily in Fig. 1 A and B and protein alignment in Fig.…”

“…(B) Schematic of homing sites recognized by the I-OnuI subfamily. (C) Target sequences for I-OnuI, I-LtrI, and I-LtrII were identified in previous studies (13,15). Recognition sequences for the other LHEs were predicted through comparative sequence alignments of each host gene to related species lacking an embedded endonuclease.…”

Recurrent insertion of 5′-terminal nucleotides and loss of the branchpoint motif in lineages of group II introns inserted in mitochondrial preribosomal RNAs

“…The following facts provide further evidence that mitochondrial subgroup IIB1 introns acquired ORFs for proteins other than reverse transcriptases through independent insertion events: (1) in introns inserted at the SSU952 site, the ORF is inserted in ribozyme domain III, rather than in domain IV (Table 1; Mullineux et al 2010); (2) the ORF in the A. parasiticum SSU788 intron encodes a protein belonging to the GIY-YIG family of homing endonucleases, rather than a member of the LAGLIDADG family; (3) the proteins possibly encoded by the LSU2059 introns are closely related (Supplemental Fig. S2) to the protein specified by a group I intron inserted at position 2066 of the mitochondrial LSU gene of Tuber melanosporum, just 7 nt 39 of position 2059.…”

“…All known examples of group II introns encoding proteins completely unrelated to reverse transcriptases also come from mitochondrial genes encoding ribosomal RNA precursor transcripts (Toor and Zimerly 2002;Monteiro-Vitorello et al 2009;Mullineux et al 2010); moreover, these introns belong again to subgroup IIB1, and multiple events of the insertion of an ORF (at least six of them) ( Fig. 5; Supplemental Fig.…”

“…More generally, we propose that the loss of the ability to form a branched structure should be regarded as an ultimate consequence of the recently documented (Mullineux et al 2010) evolutionary conversion of some mitochondrial group II introns into DNA transposons (the class II mobile elements of Wicker et al ½2007 that move at the DNA level, contrary to retrotransposons that change location as RNA).…”