Translation of Mutant Repetitive Genomic Sequences in Hirsutella sinensis and Changes in the Secondary Structures and Functional Specifications of the Encoded Proteins

Multiple repetitive sequences of authentic genes commonly exist in fungal genomes. AT-biased genotypes of <i>Ophiocordyceps sinensis</i> have been hypothesized as repetitive pseudogenes in the genome of <i>Hirsutella sinensis</i> (GC-biased Genotype #1 of <i>O. sinensis</i>) and are generated through repeat-induced point mutation (RIP), which is charactered by cytosine-to-thymine and guanine-to-adenine transitions, concurrent epigenetic methylation, and dysfunctionality. This multilocus study examined repetitive sequences in the <i>H. sinensis</i> genome and transcriptome using a bioinformatic approach and revealed that 8.2% of the authentic genes had repetitive copies, including various allelic insertions/deletions, transversions, and transitions. The transcripts for the repetitive sequences, regardless of the decreases, increases, or bidirectional changes in the AT content, were identified in the <i>H. sinensis</i> transcriptome, resulting in changes in the secondary protein structure and functional specification. Multiple repetitive internal transcribed spacer (ITS) copies containing multiple insertion/deletion and transversion alleles in the genome of <i>H. sinensis</i> were GC-biased and were theoretically not generated through RIP mutagenesis. The repetitive ITS copies were genetically and phylogenetically distinct from the AT-biased <i>O. sinensis</i> genotypes that possess multiple transition alleles. The sequences of Genotypes #2-17 of <i>O. sinensis</i>, both GC- and AT-biased, were absent from the <i>H. sinensis</i> genome, belong to the interindividual fungi, and differentially occur in different compartments of the natural <i>Cordyceps sinensis</i> insect-fungi complex, which contains >90 fungal species from >37 genera. Metatranscriptomic analyses of natural <i>C. sinensis</i> revealed the transcriptional silencing of 5.8S genes in all <i>C. sinensis</i>-colonizing fungi in natural settings, including <i>H. sinensis</i> and other genotypes of <i>O. sinensis</i>. Thus, AT-biased genotypes of <i>O. sinensis</i> might have evolved through advanced evolutionary mechanisms, not through RIP mutagenesis, in parallel with GC-biased Genotype #1 of <i>H. sinensis</i> from a common genetic ancestor over the long course of evolution.