Jorgensen, T. E.; Wardale, A.; Wolf Profant, S.; Amundsen, C.; Emblem, A.; Joakimsen, I. S.; Brekke, O.-L.; Karlsen, B. O.; Babiak, I.; Johansen, S. D.

Even though teleost fish and mammals share the same mitochondrial gene content and organization, the teleost mitochondrial transcriptome is still poorly understood. We characterized the mitochondrial transcriptome during zebrafish (Danio rerio) early development by long-read direct RNA sequencing. All heavy-strand specific mRNAs were found to carry 3 poly-A tails of approximately 50-60 residues, and the transcriptome profile was distinctive but practically invariant between stages. Three unusual transcripts were however noted. These included two mRNAs (COI and ND5 mRNAs), with significant 3 untranslated regions corresponding to antisense gene sequences, and a previously not described noncoding RNA named here lncOriL. The ND5 mRNA was found to carry one third of all detected m 6A methylation sites in the zebrafish mitochondrial transcriptome. The 313 nt-long lncOriL transcript had an abundance comparable to that of ND5 mRNA and it mapped to mitochondrial genome region covering the origin of light strand replication and four flanking antisense tRNAs. A mitochondrial tRNA-derived fragment (tiRNA5-Asn), with a 35 nt perfect pairing-potential to lncOriL, was present at all stages. Additional analyses including adult zebrafish, scissortail (Rasbora rasbora), and monkfish (Lophius piscatorius) strongly corroborate the results of COI mRNA, ND5 mRNA, and lncOriL transcript prevalence among teleost fish. Surprisingly, our findings in zebrafish were further supported by mitochondrial transcriptome analyses in domestic pig (Sus scrofa) and human (Homo sapiens), including tiRNA5-Asn commonly present in human tissues, suggesting that lncOriL is ubiquitously expressed and regulated in vertebrates.