Enrichment of G4DNA and a Large Inverted Repeat Coincide in the Mitochondrial Genomes of Termitomyces

Abstract

Mitochondria retain their own genome, a hallmark of their bacterial ancestry. Mitochondrial genomes (mtDNA) are highly diverse in size, shape, and structure, despite their conserved function across most eukaryotes. Exploring extreme cases of mtDNA architecture can yield important information on fundamental aspects of genome biology. We discovered that the mitochondrial genomes of a basidiomycete fungus (Termitomyces spp.) contain an inverted repeat (IR), a duplicated region half the size of the complete genome. In addition, we found an abundance of sequences capable of forming G-quadruplexes (G4DNA); structures that can disrupt the double helical formation of DNA. G4DNA is implicated in replication fork stalling, double-stranded breaks, altered gene expression, recombination, and other effects. To determine whether this occurrence of IR and G4DNA was correlated within the genus Termitomyces, we reconstructed the mitochondrial genomes of 11 additional species including representatives of several closely related genera. We show that the mtDNA of all sampled species of Termitomyces and its sister group, represented by the species Tephrocybe rancida and Blastosporella zonata, are characterized by a large IR and enrichment of G4DNA. To determine whether high mitochondrial G4DNA content is common in fungi, we conducted the first broad survey of G4DNA content in fungal mtDNA, revealing it to be a highly variable trait. The results of this study provide important direction for future research on the function and evolution of G4DNA and organellar IRs.

Keywords: G-quadruplex; Lyophyllaceae; fungi; inverted repeat; mtDNA.

Fig. 1.

—Mitochondrial genomes of 12 Lyophyllaceae species. The phylogeny is rooted with Tricholoma matsutake, but the genome of this species is not included in the figure. Images of genomes created with OGDRAW (Lohse et al. 2013).

Fig. 2.

—(A) Mauve alignment of four Termitomyces mitochondrial genomes: sp. T32 (used as reference), T159, T132, and T123. Colored blocks indicate predicted homologies, which are connected through vertical lines. Genes associated with homologous regions are indicated in text for each block. If a block is shown underneath the line it indicates an inversion with respect to the reference. The inverted repeat is represented by a bold line for each genome (only one copy is shown). Several rearrangements are likely due to contraction/expansion of the inverted repeat: 1) The movement of nad4 from an upstream position of cox1 in SC1 of sp. T32 to the IR in the other species is probably a result of either contraction or expansion; 2) the repositioning of a large tRNA island from an upstream position of cox1 in sp. T32 and T159 to a downstream position in sp. T132 and T123 most likely involved both contraction and expansion; 3) the large ribosomal subunit (rnl) is only partially included in the IR in sp. T132 and T123, showing a potential expulsion or enveloping in progress. (B) Example of gene translocation through successive inclusion and expulsion by the IR (Goulding et al. 1996). When the IR expands, for instance through illegitimate recombination, it can overlap a flanking ORF. This creates a copy of the overlapping part of the ORF on the opposite IR. The ORF may even end up completely within the IR, in which case two complete copies of the ORF are created. When the IR shrinks and expunges the ORF, one of the copies will disappear, resulting in an ∼50% chance of the ORF translocating to a new position.

Fig. 3.

—Barplot showing average G4DNA motif content per 1-kb mtDNA for 62 species. Values were estimated with strict settings in G4Hunter (w = 25, s = 1.7). The phylogenetic tree was reconstructed using Maximum Likelihood with IQtree. Nodes with <70 bootstrap support were collapsed into polytomies. The tree was rooted with Phytophthora infestans. Ascomycetes are shaded in green and Basidiomycetes in red. Species with mitochondrial IRs are shaded in a darker hue.

Fig. 4.

—Observed and expected G4DNA motif content for exons, introns, and intergenic regions in human and fungal mtDNA. Exons were only considered for the conserved protein-coding genes (cox1-3, nad1-6, atp6, atp8, atp9, cob, and rps3) and the ribosomal subunits (rns and rnl, while these are not protein coding they produce large, functional transcripts). Human mtDNA has no introns. For Termitomyces sp. DKA19, T13, and T123, no G4 motifs were observed in coding regions. Asterisks indicate significant deviation of observed values from the expected distribution (χ² test, P < 0.05).

Fig. 5.

—Average G4DNA motif content per 1-kb mtDNA for Lyophyllaceae species with IRs, and Agaricus bisporus as outgroup. Dark gray denotes the G4DNA motif content for the SC region. Light gray indicates the G4DNA motif content of the IR. The expected 50/50 divide if the G4DNA motif content was equal for IR and SC is shown by a vertical line halfway along each bar. The phylogenetic tree is the same as that of figure 1, but rooted with A. bisporus rather than Tricholoma matsutake as it is the closest relative to the other species that has a mitochondrial IR.