**3.3. Why mt-***ss-trn* **genes with TAG10 and ATR49 triplets as putative stop and start codons only occur in Metazoa?**

similar secondary structures, including a D-arm. In addition*,* the high level of nt conservation in the 5′-end of the *trnT* genes of cestoda (i.e., G1, G2, T7, T8, A9, G10, T11, T12 and A14) suggests strongly that the 5′-acceptor-stem and the D-stem are under positive selection. All this implies

Species names are followed by their accession number(s). \*: sequences for which the authors of these latter considered that there was an abbreviated stop codon and this latter was upstream the *trn* sequence. Symbols: <sup>X</sup> TAR10 was the first

or downstream TAR10 or nts 8–10, downstream the *tg*, respectively. *Abbreviations*: *P. c*., *Pseudanoplocephala crawfordi*;

**Table 3.** Position of the first complete in-frame stop codon of the *cox1* gene versus the following *trnT* gene in Cestoda

Proteoce., Proteocephalidea; stop cod., putative stop codon according to the authors of the sequences.

, & and μ, the putative stop codon was upstream the *trn* gene (*t*g), in the *t*g but upstream

Concerning the putative ATR49 start codon, in GenBank, the number of complete mt-genomes found using the keywords previously mentioned was relatively low; moreover, in some cases, the upstream gene encoded a protein, specified a rRNA and/or there was only one mention for a given taxon. A significant example within Deuterostomia (frogs) is presented in **Table 4**. In the superfamily Hyloidea, the ATA49 triplet is frequently the first potential complete start codon at the level of the gene pair encoding and specifying NAD1 and tRNA-Leu2, respectively. In two families (Bufonidae, Hylidae), for all the sequences (16 belonging to 14 different species), the first ATR triplet found in frame in the ORF of the *nd1* gene is ATA49. For four sequences belonging to three other frog families, the ATR49 triplet is missing from the *trnL2*

that the hypothesis of D-armless tRNAs is, according to us, improbable.

in-frame putative stop codon; §

10 Mitochondrial DNA - New Insights

(Platyhelminthes).

, \$

Foremost, biases in the search strategy cannot be excluded, but the important point to note is that mt-genomes of animals, fungi, protists, and plants differ drastically in all major characteristics including gene content and large size variation. Generally, metazoans have ultra-compact mtDNAs (from c.10,000 to c.50,000 bp); usually, nonfunctional sequences are rapidly eliminated, and there are short intergenic regions and frequent overlaps [13]. However, nonbilaterian mtgenomes have higher variation in size, gene content, shape, and genetic code [32]. The mtDNA size range is from 30,000 to 90,000 bp in fungi, and generally, intergenic regions are relatively long. A broader range of mtDNA size is found in higher plants (from 0.2 × 10<sup>6</sup> to about 11.3 × 10<sup>6</sup> bp [33]), and the largest known mt-genome in this lineage exceeds sizes of reduced bacterial and nuclear genomes [34]. The increased sizes of plant mtDNAs are mostly due to noncoding DNA sequences, large inserted nuclear regions, and many introns and not to a large increase in gene numbers. The nuclear-derived sequences amount to up nearly half of their size as in melon [35], and so presence of mt-*trn* genes with nuclear origin cannot be excluded. Although not directly correlated, intergenic distances are generally much higher in larger genomes, reducing the number of overlaps. In addition, the situation of plant mt-tRNAs is very complex. Indeed, they contain few "native" tRNAs expressed from true mt-*trn* genes. They possess "chloroplastlike" *trn* genes inserted into the mtDNA. They compensate the loss of mt-*trn* genes by importing several nucleus-encoded tRNAs [36]. In addition, most often in plants, the standard code applies to the reading of organelle genomes, even if ATA is frequently used as start codon. Metazoan mtgenomes are generally small, very constrained and exhibit several gene overlaps between *trn* and protein-encoding genes or between *trn* genes. Their tRNAs have sequence and structural peculiarities and tend to shortening [19]. Our exploration is not exhaustive, but this might explain the presence of putative stop or start codons specifically within mt-*ss-trn* genes of this taxon.

structure acts first as an alanine-tRNA, and then the short mRNA reading frame is translated and the product is released [41]. This *trans*-translation terminates at the stop codon terminating the tmRNA reading frame. This stop can be in a little loop or totally or partially integrated in the stem of a hairpin-like structure. In eukaryotes, structurally reduced tmRNAs (no mRNA-like domain) rarely occur in chloroplasts [42] and in mt-genomes (in Jakobids, presumably close to the most ancient living eukaryotes with bacterial-like mt-genome) [41]. Moreover, tmRNA TLSs function even without any canonical initiation factors. These examples show that start or stop codons

True Mitochondrial tRNA Punctuation and Initiation Using Overlapping Stop and Start Codons…

http://dx.doi.org/10.5772/intechopen.75555

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Ancient tRNAs probably had diverse functions in replication and proto-metabolism before protein translation [43] and modern tRNAs have also various functions in all the living organisms [1]. These functions include cell wall synthesis, protein N-terminal modification, nutritional stress management, porphyrin biosynthesis (heme and chlorophyll), lipid remodeling, and initiation of retrovirus reverse transcription. Accumulating experimental evidence suggests also that they have important regulatory roles in translation, viral infections, and tumor development (reviewed in [44]). Mt-tRNAs interfere with a cytochrome *c*-mediated apoptotic pathway and promote cell survival [45] and function as replication origins [46]. Moreover, nuclear-tRNA abundance and modifications are dynamically regulated, and tRNAs and their tRNA-derived RNA fragments (tRFs) are centrally involved in stress signaling and adaptive translation [47, 48]. This suggests that the choice of cleavage sites of mRNA transcripts with or not part of the neighboring ss-tRNA could be dynamic and also respond to environmental changes. Some of the noncanonical translation functions of tRNAs can also be driven or enhanced by their ability to adopt different complex three-dimensional structures, and these conformational changes can be linked to functional states [49]. Moreover, the tRNA multifunctionality has also been considered to be, at least in part, random due to the high amount of tRNA species within the cell [1]. In addition, the mt-*trn* genes represent natural pause sites for replication forks and could also prone double-strand breaks [50], and their role, as "punctuation signals," for processing of mtDNA polycistronic transcripts has already been mentioned. Enormous numbers of tRFs in all domains of life were found in the last decade [44]. In the plant *Arabidopsis thaliana*, nucleus-, plastid-, and mt-encoded tRNAs can produce tRFs [36]. The tRFs are not randomly degraded tRNAs. Experiments showed several functions including regulation of tumor development and viral infections [44]. Degradations resulting from cleavages at TAR10 and ATR49 triplets could produce a conformation exhibiting two loops linked by a forked-stem structure, roughly resembling a pair of cherries, so called "cherry-bob" (**Figure 2**).

located in hairpin may have various functions, as we suggest for TAR10 and ATR49.

Our hypothesis predicts this structure which however has never been observed [51].

Metazoan mt-genomes are believed optimized for rapid replication and transcription. Potentially, TAG10 and ATR49 make transcription/translation more complex but perhaps more efficient. Examples in the Section 3.2 (i.e., Eucestoda) suggest mt-overlaps appeared 100s millions of years (MY) ago, enabling co-evolution between protein-encoding genes and those specifying tRNA.

**3.6. Other putative roles of TAR10 and ATR49**

**3.5. Multifunctionality of tRNAs**

One may wonder why the hypotheses concerning the TAR10 and ATR49 triplets were not proposed before? At least, the presence of these characteristic triplets could have been observed by some authors but considered as having no connection with the translation of neighboring protein genes. Among the first sequenced and the most studied were mt-genomes of *Homo sapiens* (J01415) and *Mus musculus* (J01420). In these latter, no putative start or stop codon occurs at these positions within *trn* sequences adjacent to protein genes (data not shown).
