Using high-resolution annotation of insect mitochondrial DNA to decipher tandem repeats in the control region

In this study, we used a small RNA sequencing (sRNA-seq) based method to annotate the mitochondrial genome of the insect Erthesina fullo Thunberg at 1 bp resolution. Most of the new annotations were consistent with the previous annotations which were obtained using PacBio full-length transcripts. Two important findings are that animals transcribe both entire strands of mitochondrial genomes and the tandem repeat in the control region of the E. fullo mitochondrial genome contains the repeated Transcription Initiation Sites (TISs) of the H-strand. In addition, we found that the copy numbers of tandem repeats showed a great diversity within an individual, enriching the fundamental knowledge of mitochondrial biology. This sRNA-seq based method uses 5′ and 3′ end small RNAs to annotate nuclear non-coding and mitochondrial genes at 1 bp resolution and can also be used to identify new steady-state RNAs, particularly long non-coding RNAs (lncRNAs). Animal mitochondrial genomes containing one control region only encode two steady-state lncRNAs, which are the Mitochondrial D-loop 1 (MDL1) and its antisense gene (MDL1AS), while all other reported mitochondrial lncRNAs could be degraded fragments of transient RNAs or random breaks during experimental processing. The high-resolution annotations of mitochondrial genomes can be used to study the phylogenetics and molecular evolution of animals or to investigate mitochondrial gene transcription, RNA processing, RNA maturation and several other related topics.