**5.2. Atlantic cod mitochondrial small RNAs**

of CR and is the precursor of three enriched small RNAs (below CR map).

The annotated mtSSU rRNA and mtLSU rRNA genes in Atlantic cod are 952 and 1664 bp, respectively [7]. The corresponding rRNAs are highly conserved within the species [18] and well conserved between different fish species [7, 51]. The 5′ and 3′ ends of Atlantic cod mitochondrial rRNAs have been precisely mapped using different approaches. Primer extension and pyrosequencing confirmed the 5′ ends to correspond to the annotated features based on comparative sequence alignments [47, 51]. The 3′ ends were mapped by pyrosequencing and by RNA ligation sequencing [51]. Interestingly, non-template adenosines were added at both rRNAs. Whereas the 3′ end of mtSSU rRNA was found to be homogenous and mono-adenylated, the corresponding end of mtLSU rRNA was heterogeneous and oligo-adenylated [51]. The observed mtLSU rRNA heterogeneity is consistent with the notion that mitochondrial rRNAs are transcribed and pro-

In addition to the canonical mitochondrial genes and the newly proposed MDPs, vertebrate mitogenomes encode several noncoding RNAs [36]. The first discovered mitochondrial long noncoding RNA (lncRNA) was the human L-strand-specific 7S RNA (lncCR-L) [52, 53].

At least eight vertebrate mitochondrial lncRNAs have now been proposed and characterized [54]. Two lncRNAs correspond to the H-strand and L-strand of CR (lncCR-H and lncCR-L) [10, 18, 47, 52, 55, 56], one is an antisense chimer to partial regions of the CytB and COI mRNAs (LIPCAR) [57–59], three are mRNA antisense RNAs (lncND5, lncND6, and lncCytB) [60], and two are chimeric RNAs that involve sense and antisense mtLSUrRNAs (SncmtRNA and ASncmtRNA) [61–63]. So far, LIPCAR, rRNA chimers, and lncCR-H have been associated with human diseases [56, 57, 61, 63–66]. There are apparently a large number of small noncoding RNAs (mitosRNAs) generated from vertebrate mitochondrial transcripts [36, 67–69]. None of these mitosRNAs have been assigned to a specific function funded on experimental evidence. However, in a recent study by Riggs and Podrabsky [70], mitosRNAs were associ-

Two lncRNAs (lncCR-H and lncCR-L) have been identified and investigated in Atlantic cod mitochondria (**Figure 4**) [10, 18, 47]. Both lncRNAs were found to be polyadenylated but transcribed from opposite strands within the CR [10]. We showed that the Atlantic cod lncCR-L has a mutation rate and an expression level corresponding to that of Complex I mRNAs [10, 18, 47]. The lncCR-L apparently corresponds to the 7S RNA in human mitochondria [52], and recently we showed that lncCR-L is differentially expressed in a human cancer-matched cell line pair [56].

The lncCR-H was found to be highly variable in sequence and structure, both between and within Atlantic cod specimens [10, 18]. A schematic overview of the lncCR-H RNA is presented in **Figure 4**. Here, the noncoding T–P spacer is present at the 5′ end and includes two potential RNA hairpin structures. The T–P spacer domain is followed by a mirror tRNAPro, before entering the HTR array motifs. The HTR copy numbers vary between 2 (80 bp)

and HSP2

primary transcripts (**Figure 3A**).

cessed from two different precursor RNAs, the HSP<sup>1</sup>

ated to a hypoxia stress response in killifish embryos.

**5.1. Atlantic cod mitochondrial long noncoding RNAs**

**5. Mitochondrial noncoding RNAs**

102 Mitochondrial DNA - New Insights

The Atlantic cod mitogenomes express a number of small RNAs, revealed by SOLiD small RNA sequencing experiments (our unpublished results). Here, the majority of mitosRNA was identified as mitochondrial tRNA-derived fragments (tRFs; see [69, 70]). Interestingly, most Atlantic cod mitochondrial tRFs correspond to H-strand tRNAs, and some tRFs were differentially expressed during early developmental stages (our unpublished results). Many of the same tRF species detected in Atlantic cod have recently been noted in rainbow trout egg cells [69] and in killifish embryos [70], suggesting a conserved feature at least among some bony fishes.

The SOLiD experiments also detected several abundant small RNAs mapping to the mitochondrial CR [17]. We found three small RNA candidates generated from lncCR-L, suggesting this lncRNA to be a precursor for mitosRNA (**Figure 4**). Similarly, two mitosRNA were generated from lncCR-H, one corresponded to a pyrimidine-rich motif and the other to tRF-1 derived from tRNAThr (**Figure 4**). What functions these small RNAs may serve in the mitochondria are not currently known, but we speculate that regulatory roles related to transcription elongation, mtDNA replication, or ribosome functions are likely.

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