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 degrees 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 3' 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.

FIGURE 1.

(A) Sequence alignment showing the IS of group II introns encoding putative LHEases in the mt rns gene of Leptographium spp. The intron IS was delimited by comparison of intron-minus and intron-plus alleles of the mt rns gene. The corresponding sequence of the mt rns gene of C. parasitica, which contains a related intron (I3), is included. Both the 5′ exon and 3′ exon sequences that flank the intron IS and the intron sequences at the 5′ and 3′ termini, which follow the group II consensus GUGYG and AY, respectively, are shown. The rest of the intervening sequence is represented by dots. The 5′ and 3′ splice junctions are designated by black arrows. Heteroplasmy (intron-plus and intron-minus alleles) has been observed in L. truncatum strains CBS929.85 and NFRI1813/1. (B) Secondary structure model of the group IIB1 intron, Lt.SSU/1, of L. truncatum showing the insertion of the ORF in a looped-out region in DIII. The 5′ and 3′ splice sites are marked by black arrowheads and the bulged adenosine residue in DVI is indicated by the asterisk. EBS and IBS nucleotides are shown, as well as nucleotides/segments involved in tertiary interactions (α-α′, γ-γ′, δ-δ′, ɛ-ɛ′, ζ-ζ′, κ-κ′, and θ-θ′).

FIGURE 2.

Gel electrophoresis of in vitro splicing reactions by Lt.SSU/1 intron under various temperature and ionic conditions. Internally labeled precursor transcripts were incubated for the length of time indicated prior to loading onto a 4% polyacrylamide/8 M urea gel. (A) The LtrΔORF.3 version of the intron, from which the ORF sequence is absent, comprises 819 nt. Products from a self-splicing reaction of the Pl.LSU/2 intron from P. littoralis (Costa et al. 1997) were used to generate a molecular weight calibration. (B) The 19U/Ltr.2 version of the intron (1840 nt) contains the full-length ORF sequence. “19U/-Ltr.2 transcript” designates unincubated precursor transcript.

FIGURE 3.

(A) Gel electrophoresis of in vitro splicing reactions of the LtrΔORF.3 precursor transcript in the presence of N-terminal His₆-tagged I-LtrII. Internally labeled precursor transcripts were incubated with increasing concentrations of I-LtrII at 37°C for the length of time indicated prior to loading onto a 4% polyacrylamide/8 M urea gel. The composition of buffers C1 and C2 are described in Materials and Methods. (B) RNA-binding assays of N-terminal His₆-tagged I-LtrII. Values were corrected for binding of the eluate to an RNA-binding Hybond N+ filter, and also for the residual binding observed in the absence of protein.

FIGURE 4.

(A) Gel electrophoresis of in vitro splicing reactions with the 19U/Ltr.2 precursor transcript in the presence of near-native I-LtrII. Internally labeled precursor transcripts were incubated with near native I-LtrII at 37°C for the length of time indicated prior to loading onto a 4% polyacrylamide/8 M urea gel. Products from the self-splicing reaction of the Pl.LSU/2 intron from P. littoralis were used to generate a molecular weight calibration. (B) Quantitation of the progress of the splicing reaction based on the molar ratio of [ligated exons] to ([precursor] + [ligated exons]). The calculation did not take into account the relatively greater vulnerability of the lengthy precursor molecules to degradation over extended reaction times. Curves were fitted to the equation m1 * [1 − exp(-m2 * t)] using the KaleidaGraph software. Circles (m1 = 0.294 ± 0.025; m2 = 0.054 ± 0.012; Pearson's R = 0.986) and squares (m1 = 0.477 ± 0.043; m2 = 0.036 ± 0.007; Pearson's R = 0.993) correspond to reactions in the absence and presence, respectively, of the I-LtrII protein.

FIGURE 5.

Cleavage activity of near-native I-LtrII protein. The ability of I-LtrII to cleave linearized plasmid DNA containing exon sequences (pCR4mtrnsEX) and the intron sequences flanking the LHEase ORF (pSKmtrnsORFis) was tested. The cleavage assays were carried out using nonlabeled plasmid DNA that was first linearized with either NcoI (pCR4mtrnsEx) or Not1 (pSKmtrnsORFis). Negative controls were NcoI-linearized plasmid DNA containing the intron-plus allele of the mt rns gene (pCR4mtrnsExIn), in which both potential homing sites are disrupted, or the mt cytochrome b (cob) gene (construct pCR4mtcob), an unrelated gene, that was cloned into the pCR4-TOPO vector. The molecular weight marker, L, used was the 1Kb Plus DNA ladder (Life Technologies).

FIGURE 6.

Mapping of the cleavage site of the I-LtrII LHEase in the mt rns gene. Shown is a representative sequencing ladder generated for the top (A) and bottom (B) DNA strands that flank the intron IS. The uncleaved product represents the 248-bp PCR amplicon that served as the template for the sequencing reaction. The cleaved product was generated by incubating this amplicon with near-native I-LtrII. The asterisk marks the nt that is immediately 5′ to the cut site. Schematic of the region of the mt rns gene that flanks the intron IS and includes the cleavage site (CS) of I-LtrII (C).