Ophiostoma minus

Characterization of a double motif LAGLIDADG homing endonuclease from the blue stain fungus Ophiostoma minus. Mohamed Hafez and Georg Hausner 

Department of Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada 

ABSTRACT         The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus was found to be invaded with several group I and group II introns. In the O. minus strain WIN(M)371 rns gene, group IC2 and group IIB1 introns are inserted at positions mS569 and mS952 respectively. The mS952 intron encodes a double motif LAGLIDADG homing endonuclease (I-OmiII) from an open reading frame (ORF) located in domain III. A codon-optimized version of the I-OmiII sequence was synthesized and cloned into the pET200/D/TOPO vector and transformed into Escherichia coli BL21 star (DE3). Thereafter, the 6xHisTagged I-OmiII protein was purified using Ni-NTA resin. A second purification step was carried out using a HiTrap heparin HP column. The results showed that: I-OmiII is a functional homing endonuclease that cleaves the target site two nucleotides upstream (top strand) of the intron insertion site generating 4 nucleotides 3’overhangs. The endonuclease activity of I-OmiII was tested using a variety of possible substrates. The optimum temperature of I-OmiII was also determined.

RESULTS Fig. 4: Construction of the expression plasmid

Fig. 1: A schematic overview of the O. minus WIN(M)371 rns gene

Two introns were found at positions S569 and S952 with reference to the E. coli SSU rRNA sequence. The O.mimS569 is a group IC2 intron, while the O.mi-S952 intron is a group IIB1 intron and they both encode a double motif LAGLIDADG ORF. Fig. 2: Secondary structure of the O.mi-mS952 group IIB1 intron RNA.

The optimized I-OmiII ORF was amplified from the pBluescript plasmid with the primers I-OmiII-F and I-OmiII-R. The I-OmiII-F primer was designed with CACC 5’ tail to enable the directional cloning of the I-OmiII ORF into the pET200/D/TOPO plasmid and thus generating the expression plasmid pET200/D/I-OmiII.

[a]

INTRODUCTION

Fig. 5: Purification of I-OmiII. [b]

•Ophiostoma minus is an economically important agent of blue-stain (sap-stain) of various pine species. Blue-stain is a discoloration in the wood of infected trees [1]. •Fungal mitochondrial DNA (mtDNA) genomes are frequently encountered with group I and group II introns which are potentially self-splicing introns [2]. •Group II introns have conserved secondary structures at the RNA level, that can be visualized as six stem-loop domains (D I to D VI) emerging from a central wheel [3]. Typically group II introns encode multifunctional reverse transcriptase-like (RT) proteins, but recently a family of group II introns was found to encode LAGLIDADG Homing Endonucleases (LHEases) [4]. When RT -like ORFs are present they tend to be embedded within domain IV. However in some bacterial and mitochondrial group II introns RT type ORFs have been observed in domain II. LAGLIDADG type ORFs are inserted in domains III or IV [4]. •Homing endonucleases (HEs) are encoded by homing endonuclease genes (HEGs) which are embedded within group I introns, group II introns and archael introns, as well as inteins. HEs are named based on conserved amino acid motifs. At least four homing endonuclease families have been identified on the basis of conserved sequence motifs that provide residues critical for enzyme folding and catalysis: the LAGLIDADG, HNH, His-Cys Box and GIY-YIG [5].

The O.mi-mS952 intron is a typical group II intron containing the characteristic features of class B1. The ORF finder program identified an ORF within the O.mimS952 intron, this ORF encodes a double motif LHEase rather than the RT-ORFs that typically are associated with group II introns. Folding the intron RNA indicates that the location of the LAGLIDADG ORF is within domain III. Fig. 3: RT-PCR analysis for demonstrating the in vivo splicing of the O.mi-mS569 and O.mi-mS952 introns. The rns transcripts for the O. minus WIN(M)371 were analyzed by RT-PCR. The standard PCR reaction using genomic DNA as a template is shown in lane 1 and it generated a 4.4 kb fragment. The amplicon length for the cDNA was 1.2 kb (Lane 2) indicating that both O.mimS969 and O.mi-952 introns were spliced out in vivo.

DISCUSSION •Here we describe a group II intron that has been invaded by LHEase, and together they could represent the evolution of a new type of composite mobile elements. •O. minus is a sexual species that can be used as a model organism in genetic experiments involving transmission of mobile intron from an intron containing to an intronless strain. •HEs require long DNA recognition sites and therefore cut infrequently within a genome; this makes them useful for DNA engineering and genomics. HEs can be engineered to cleave at desired locations and therefore HEs can become site specific tools that can be used to target specific genes. Strategies are also being developed that would allow for therapeutic applications of HEs that target genes associated with human diseases [6].

Fig. 6: In vitro cleavage assay with I-OmiII

[a] The ability of I-OmiII (purple pac-man symbol) to cleave/linearize the circular pCR4-1574 plasmid, which contains the O. minus strain WIN(M)1574 rns gene (orange box) with the recognition site of I-OmiII (black box). The circular pCR4-1574 plasmid incubated with IOmiII for 0, 30 and 60 minutes. I-OmiII completely linearized the plasmid after 1 hour generating a 5167 bp fragment. [b] The ability of I-OmiII to cleave the pCR41574 plasmid that was first linearized with NcoI. The linearized plasmid also was incubated with I-OmiII for 0, 30 and 60 minutes. I-OmiII was cleaving the template into two fragments (2194 and 2973 bp). Fig. 7: Cleavage site mapping of I-OmiII

[a] SDS-PAGE (10%) gel of I-OmiII purification by NiNTA resin (Qiagen). Lanes are indicated as follows: M, Marker; CL, Clear Lysate; FT, Flow Through; W, wash and E, Elution. Non His-Tagged proteins were washed out with a narrow range of imidazole 20, 30 and 40 mM (W1, W2 & W3 respectively) and the I-OmiII was eluted in three fractions E1 (125 mM imidazole), E2 and E3 (250 mM imidazole). The red arrow marks the 37 kDa IOmiII protein (the expected size of I-OmiII is 34 kDa, but the size increases due to the N-terminus fusion tag). [b] 10% SDS-PAGE gel for the second purification step of I-OmiII. The elution fractions (E1-E3) from the first purification step were combined and applied to a HiTrap heparin HP column (GE Healthcare) and then washed with one column volume of binding buffer over a range of 200 mM to 900 mM NaCl as indicated by the red leftfacing triangle. The I-OmiII eluted in three fractions (400, 500 and 600 mM NaCl).

Cleavage site mapping for I-OmiII by DNA sequencing of the pCR4-1574 and pCR4-1574-T4 plasmids. The pCR4-1574-T4 plasmid was obtained from pCR4-1574 which was cleaved with I-OmiII (purple pac-man symbol) generating 4 nucleotides 3’ overhangs and then blunted with T4 DNA polymerase and religated with T4 DNA ligase.

SUMMARY Here we describe a double motif LAGLIDADG-type ORF (I-OmiII) encoded within a group IIB1 intron in the rns gene of the blue stain fungus O. minus. The I-OmiII ORF was over expressed in E. coli BL21 (DE3) and its ability to recognize, bind and cleave the target site was evaluated in vitro. The cleavage pattern of I-OmiII was tested and it is found to cleave the target site generating 4 nucleotides 3’ overhangs.

REFERENCES [1] Zink P. and Fengel D., 1990. Holzforschung, 44(3): 163–168.

ACKNOWLEDGMENTS

[2] Belfort M. et al., 2002. Mobile DNA II, American Society for Microbiology Press, Washington DC, pp. 761–783. [3] Michel F. and Ferat J.L., 1995. Annual Review of Biochemistry 64: 435–461. [4] Toor N. and Zimmerly S., 2002. RNA 8: 1373–1377. [5] Stoddard B.L., 2006. Quarterly Review of Biophysics 38: 49–95. [6] Marcaida M.J. et al., 2010. Cellular and Molecular Life Sciences 67: 727-748.

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