**4.2. Atlantic cod mitochondrial structural RNAs**

The 22 mitochondrial tRNAs were found to be highly conserved in Atlantic cod, both in structure and sequence [5, 18], and some tRNAs (tRNAIle, tRNASer(UCN), tRNASer(AGY), and tRNACys) were invariant in the 200 specimens investigated. SOLiD deep sequencing confirmed a nontemplate CCA addition at the 3′ ends of tRNAs (our unpublished results). Thus, mitochondrial tRNA processing and probably modification are highly similar in human and Atlantic cod [47].

**Figure 3.** The mitochondrial transcriptome in Atlantic cod. (A) Schematic map of mitochondrial ribosomal RNA,

rRNA, mitochondrial small- and large-subunit ribosomal RNA (yellow boxes); ND1, ND2, ND3, ND4L/ND4, ND5, and ND6, NADH dehydrogenase subunit mRNAs; COI, COII, and COIII, cytochrome c oxidase subunit mRNAs; A8/ A6, ATPase subunit bicistronic mRNA; Cyt B, cytochrome b mRNA (all mRNAs indicated by blue boxes); lncCR-H and lncCR-L, long noncoding RNAs (orange boxes). (B) 3′ untranslated region (UTR) and 5' UTR in COI and COII mRNAs, respectively. Translation initiation codons (GUG and AUG) and termination codons (UAA) are indicated by green and red circles, respectively. The 3' UTR of COI mRNA contains a mirror tRNASer motif, and the 5' UTR of COII mRNA

, HSP2

Expanding the Coding Potential of Vertebrate Mitochondrial Genomes: Lesson Learned…

http://dx.doi.org/10.5772/intechopen.75883

101

, and LSP transcripts. mtSSU rRNA and mtLSU

messenger RNA, and long noncoding RNA generated from HSP1

contains a GAAA tetra-loop hairpin motif.

**4. Mitochondrial transcriptome**

100 Mitochondrial DNA - New Insights

identified in porcine and bovine [45].

**4.1. Atlantic cod mitochondrial mRNAs**

interactions [48].

addition [47].

**4.2. Atlantic cod mitochondrial structural RNAs**

initiated within CR from two H-strand promoters (HSP1

promoter (LSP) (**Figure 3A**) [36, 38–40]. The HSP1

Vertebrate mitochondrial transcriptomes have mainly been studied in human cells and tissues [36, 37]. Mature mitochondrial RNAs are generated from three polycistronic transcripts

tRNAs have recently been proposed to perform a second role as a mitochondrial rRNA, substituting the lacking 5S rRNA in vertebrate mitochondrial ribosomes [43, 44]. While tRNAVal appears associated with the mitochondrial ribosomes in human and rat, tRNAPhe has been

tronic, but two of the mRNAs are bicistronic (ND4/4 L mRNA and ATPase8/6 mRNA). Finally, the L-strand-specific transcript gives rise to the ND6 mRNA and eight tRNAs (**Figure 3A**).

Similar to that of human cells, 11 mature mRNAs were readily expressed from the Atlantic cod mitogenome [47]. There are, however, some minor differences in mitochondrial mRNA maturation and modification between human and Atlantic cod. Mapping of the 5′ ends in mitochondrial mRNAs by pyrosequencing revealed that 10 of the 11 mRNAs contain no, or very short (1–2 nt), 5′ untranslated regions (UTRs) [47]. The only exception is the 5' UTR of the COII mRNA, which contained a short hairpin structure. In Atlantic cod and all other Gadidae species, this hairpin structure is capped by a GAAA tetra-loop (**Figure 3B**) [47]. GAAA tetra-loops are known to frequently participate in long-range RNA:RNA tertiary

Most Atlantic cod mRNAs lack 3' UTRs, but the COI mRNA has a 3' UTR of 76 nt corresponding to the complete mirror sequence of tRNASer(UCN) (**Figure 3B**) [47]. A very similar 3' UTR (72 nt) has been reported in the human COI mRNA [49], indicating a conserved role in vertebrates. The 3' UTR of the ND5 mRNA is highly variable in length in vertebrates but is lacking completely in Atlantic cod [40, 47]. However, the closely related Gadidae species *Pollachius virens* (Saithe) contains an ND5 mRNA 3' UTR of 16 nucleotides [47]. In humans, mitochondrial mRNAs contain short polyA tails of 40–50 adenosines at their 3′ ends [40, 45]. PolyA tails were identified in all mRNAs, except for ND6 mRNA [40], and seven UAA termination codons were created in the human mitochondria by polyA posttranscriptional editing [50]. Similarly, all mitochondrial mRNAs (except the ND6 mRNA) were found to be polyadenylated in Atlantic cod, and six UAA termination codons were generated by polyA

The 22 mitochondrial tRNAs were found to be highly conserved in Atlantic cod, both in structure and sequence [5, 18], and some tRNAs (tRNAIle, tRNASer(UCN), tRNASer(AGY), and tRNACys)

generates mtSSU rRNA, mtLSU rRNA, as well as tRNAVal and tRNAPhe [41, 42]. HSP1

Ten H-strand-specific mRNAs are posttranscriptionally processed from the HSP<sup>2</sup>

together with 13 tRNAs and the two rRNAs (**Figure 3A**) [46]. Most HSP2

and HSP2


) and a single L-strand

mRNAs are monocis-


transcript,

**Figure 3.** The mitochondrial transcriptome in Atlantic cod. (A) Schematic map of mitochondrial ribosomal RNA, messenger RNA, and long noncoding RNA generated from HSP1 , HSP2 , and LSP transcripts. mtSSU rRNA and mtLSU rRNA, mitochondrial small- and large-subunit ribosomal RNA (yellow boxes); ND1, ND2, ND3, ND4L/ND4, ND5, and ND6, NADH dehydrogenase subunit mRNAs; COI, COII, and COIII, cytochrome c oxidase subunit mRNAs; A8/ A6, ATPase subunit bicistronic mRNA; Cyt B, cytochrome b mRNA (all mRNAs indicated by blue boxes); lncCR-H and lncCR-L, long noncoding RNAs (orange boxes). (B) 3′ untranslated region (UTR) and 5' UTR in COI and COII mRNAs, respectively. Translation initiation codons (GUG and AUG) and termination codons (UAA) are indicated by green and red circles, respectively. The 3' UTR of COI mRNA contains a mirror tRNASer motif, and the 5' UTR of COII mRNA contains a GAAA tetra-loop hairpin motif.

were invariant in the 200 specimens investigated. SOLiD deep sequencing confirmed a nontemplate CCA addition at the 3′ ends of tRNAs (our unpublished results). Thus, mitochondrial tRNA processing and probably modification are highly similar in human and Atlantic cod [47].

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 processed from two different precursor RNAs, the HSP<sup>1</sup> and HSP2 primary transcripts (**Figure 3A**).
