**3.7. Methylation of** *trn* **genes and tRNAs and their possible roles in transcription and translation**

Methylation is much rarer in mt- than nuclear-DNA [60]. However, these might occur at *trn* genes (particularly around TAR10 and ATR49) and might have deleterious consequences especially because differential mtDNA methylations are linked to aging and diseases (including diabetes and cancers) [60]. Methylation of nts of UAR10 and AUR49 is known as those of A9 and G10 which can be important for correct tRNA foldings [61]. We are unaware whether posttranscriptional modifications occur on bicistronic mt-transcripts containing complete or partial tRNAs. This would be worth investigating including possible consequences on maturation and translation.

#### **3.8. Reassignments of codons and ss-tRNA**

Several codon-amino acid reassignments are known, mainly from mitochondria [62, 63]. In 11 different mt-genetic codes, UGA stops code for tryptophan and AUA codes for methionine instead of isoleucine in 8 and 5 mt-genetic codes, respectively [63]. Both reassignments avoid potential errors along traditional wobble rules. Reassigning UGA-stop to UGA-Trp fits the "capture" hypothesis, and UGA codons mutate first to synonymous UAA codon in AT-rich mtgenomes. Then, UGA reappears occasionally by mutations, free for "capture" by an amino acid, like Trp [64]. AUA is frequently used as alternative initiation codon. Its reassignment to internal sense Met codon could also have evolved in AT-rich genomes. Moreover, the standard genetic code assigns six codons to arginine, whereas two would fit arginine's relatively low frequency in current proteins [65]. In 8 out of 11 mt-codes, different strategies reduce Arg codons to four, AGR reassignments to other amino acids (in six genetic codes), lack of two Arg codons (CGA and CGC yeast mt-code), and AGR as terminators in vertebrates. These AGR codons were believed mt-stop codons since early vertebrate evolution [66]. However, at least in humans, AGRs are not recognized terminators [67], suggesting that AGRs have no assignment. Hence, the vertebrate mt-genetic code could be the most optimized known genetic code (that of yeast was not retained because four Leu codons were reassigned to Thr). Characteristics of the nt triplets at the position 8–10 and ending at position 49 should be analyzed for each mt-genetic code.

The study of ss-tRNAs suggests a model partially explaining canonical tRNA origins (**Figure 3**). The DNA region specifying the "bottom half" would be integrated in a sequence that can specify the "top half" but at the junction between the parts corresponding to the 3′-end of the

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

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

17

On the other hand, the "bottom half"/"cherry bob" structure could also be integrated at RNA level, either in the RNA world by intermolecular RNA-RNA recombination or template switches or later with retrotranscription events. Fujishima and Kanai [70] also proposed an equivalent model where a long hairpin corresponding to about the "top half" region merged with a viral RNA element corresponding to the "bottom half" to give the TLS found in modern viral genomes (who however possessed a pseudoknotted acceptor-stem). Besides, rare pretRNA molecules from the three domains of life exhibit an intron. The intron's origin is debated. The "introns-early" scenario assumes most of them were lost during evolution, and the opposite scenario theorizes that introns were inserted into some *trn* genes after their emergence [75]. To date, our hypothesis would rather favor the second scenario, even though it could be considered that the "cherry bob" structure could be an ancestral intron becoming unspliceable. In tRNAs, the two first nts of both UAR10 and AUR49 belong to connector 1 and 2, respectively. They are thus at the junction between the top and bottom halves and are very close physically in the 3D structure (**Figure 1**). The belonging of some of the nts of the TAR10 and ATR49 triplets to either of the two parts is not discussed here because the theoretical model of **Figure 3** is applicable independently of "bottom half" extremities. However, as the V-R is important for aminoacylation [76], ATR49 triplets could rather integrally belong to the "top half." The tRNA L-shape is stabilized by various tertiary interactions of the V-R with the D-arm and between the D- and T-loops. Nucleotides of the connectors form contacts with the D-arm, and in some

**Figure 3.** Proposed model for the origin of genes specifying tRNAs with canonical cloverleaf structure. It can be summarized by insertion (follow the arrow) of the region specifying the "bottom half" into those specifying the "top half". Here, the entire TAG10 triplet presumably belonged to the "bottom half" region as well as the first 2 nts of ATG49. Colors as **Figure 1**.

5′-acceptor stem and the 5′-end of the 5′-T-stem.

#### **3.9. Origin of the cloverleaf structure of tRNA and ss-tRNA**

Various models could explain tRNA origins (see reviews [68–70]). The modern tRNA cloverleaf structure might result from direct duplication of primordial RNA hairpins (e.g., [68]). However, studies lend strong support to the "two halves" hypothesis [43], in which tRNAs consist of two coaxially stacked helices with presumed independent structural and functional domains. These correspond to the "top half" containing the acceptor-stem and the T-arm and the "bottom half" with the D-arm and anticodon-arm (**Figure 1**). The 2D representation of the latter corresponds to the "cherry-bob" structure (**Figure 2**). The "top half" of modern tRNA embeds the "operational code" in the identity elements of the acceptor-stem that interacts with the catalytic domain of specific aaRSs and is recognized by RNases P and Z and the CCA-adding enzyme (therefore mainly RNA end processing reactions) [70, 71]. This domain also interacts with translation elongation factor Tu and one rRNA subunit [71]. The importance of this domain in most macromolecular interactions involving tRNAs (including *in vitro* even when it is detached from the "bottom half") suggests that these half's specificities were established before the tRNA's "bottom half," presumably incorporated later [72]. Growing evidence for tRNA elements involved in both RNA and DNA replication with the 3′-end playing a determinant role has led to the idea that the "top half" initially evolved for replication in the RNA world before the advent of protein synthesis [73]. The supposed evolutionarily recent tRNA "bottom half" provides genetic code specificity. This suggests late implementation of the standard genetic code and late appearance of interactions between the tRNA "bottom half" and ribosomes [74]. Whether the "bottom half" derived from a loop or extra loop belonging to the "top half" or was an independent structural and functional domain that was subsequently incorporated into the "top half" remains unresolved [71]. Some authors suggest independent evolutionary origins [71, 72].

The study of ss-tRNAs suggests a model partially explaining canonical tRNA origins (**Figure 3**). The DNA region specifying the "bottom half" would be integrated in a sequence that can specify the "top half" but at the junction between the parts corresponding to the 3′-end of the 5′-acceptor stem and the 5′-end of the 5′-T-stem.

**3.8. Reassignments of codons and ss-tRNA**

16 Mitochondrial DNA - New Insights

**3.9. Origin of the cloverleaf structure of tRNA and ss-tRNA**

Several codon-amino acid reassignments are known, mainly from mitochondria [62, 63]. In 11 different mt-genetic codes, UGA stops code for tryptophan and AUA codes for methionine instead of isoleucine in 8 and 5 mt-genetic codes, respectively [63]. Both reassignments avoid potential errors along traditional wobble rules. Reassigning UGA-stop to UGA-Trp fits the "capture" hypothesis, and UGA codons mutate first to synonymous UAA codon in AT-rich mtgenomes. Then, UGA reappears occasionally by mutations, free for "capture" by an amino acid, like Trp [64]. AUA is frequently used as alternative initiation codon. Its reassignment to internal sense Met codon could also have evolved in AT-rich genomes. Moreover, the standard genetic code assigns six codons to arginine, whereas two would fit arginine's relatively low frequency in current proteins [65]. In 8 out of 11 mt-codes, different strategies reduce Arg codons to four, AGR reassignments to other amino acids (in six genetic codes), lack of two Arg codons (CGA and CGC yeast mt-code), and AGR as terminators in vertebrates. These AGR codons were believed mt-stop codons since early vertebrate evolution [66]. However, at least in humans, AGRs are not recognized terminators [67], suggesting that AGRs have no assignment. Hence, the vertebrate mt-genetic code could be the most optimized known genetic code (that of yeast was not retained because four Leu codons were reassigned to Thr). Characteristics of the nt triplets at the position 8–10 and ending at position 49 should be analyzed for each mt-genetic code.

Various models could explain tRNA origins (see reviews [68–70]). The modern tRNA cloverleaf structure might result from direct duplication of primordial RNA hairpins (e.g., [68]). However, studies lend strong support to the "two halves" hypothesis [43], in which tRNAs consist of two coaxially stacked helices with presumed independent structural and functional domains. These correspond to the "top half" containing the acceptor-stem and the T-arm and the "bottom half" with the D-arm and anticodon-arm (**Figure 1**). The 2D representation of the latter corresponds to the "cherry-bob" structure (**Figure 2**). The "top half" of modern tRNA embeds the "operational code" in the identity elements of the acceptor-stem that interacts with the catalytic domain of specific aaRSs and is recognized by RNases P and Z and the CCA-adding enzyme (therefore mainly RNA end processing reactions) [70, 71]. This domain also interacts with translation elongation factor Tu and one rRNA subunit [71]. The importance of this domain in most macromolecular interactions involving tRNAs (including *in vitro* even when it is detached from the "bottom half") suggests that these half's specificities were established before the tRNA's "bottom half," presumably incorporated later [72]. Growing evidence for tRNA elements involved in both RNA and DNA replication with the 3′-end playing a determinant role has led to the idea that the "top half" initially evolved for replication in the RNA world before the advent of protein synthesis [73]. The supposed evolutionarily recent tRNA "bottom half" provides genetic code specificity. This suggests late implementation of the standard genetic code and late appearance of interactions between the tRNA "bottom half" and ribosomes [74]. Whether the "bottom half" derived from a loop or extra loop belonging to the "top half" or was an independent structural and functional domain that was subsequently incorporated into the "top half" remains unresolved [71]. Some authors suggest independent evolutionary origins [71, 72].

On the other hand, the "bottom half"/"cherry bob" structure could also be integrated at RNA level, either in the RNA world by intermolecular RNA-RNA recombination or template switches or later with retrotranscription events. Fujishima and Kanai [70] also proposed an equivalent model where a long hairpin corresponding to about the "top half" region merged with a viral RNA element corresponding to the "bottom half" to give the TLS found in modern viral genomes (who however possessed a pseudoknotted acceptor-stem). Besides, rare pretRNA molecules from the three domains of life exhibit an intron. The intron's origin is debated. The "introns-early" scenario assumes most of them were lost during evolution, and the opposite scenario theorizes that introns were inserted into some *trn* genes after their emergence [75]. To date, our hypothesis would rather favor the second scenario, even though it could be considered that the "cherry bob" structure could be an ancestral intron becoming unspliceable.

In tRNAs, the two first nts of both UAR10 and AUR49 belong to connector 1 and 2, respectively. They are thus at the junction between the top and bottom halves and are very close physically in the 3D structure (**Figure 1**). The belonging of some of the nts of the TAR10 and ATR49 triplets to either of the two parts is not discussed here because the theoretical model of **Figure 3** is applicable independently of "bottom half" extremities. However, as the V-R is important for aminoacylation [76], ATR49 triplets could rather integrally belong to the "top half." The tRNA L-shape is stabilized by various tertiary interactions of the V-R with the D-arm and between the D- and T-loops. Nucleotides of the connectors form contacts with the D-arm, and in some

**Figure 3.** Proposed model for the origin of genes specifying tRNAs with canonical cloverleaf structure. It can be summarized by insertion (follow the arrow) of the region specifying the "bottom half" into those specifying the "top half". Here, the entire TAG10 triplet presumably belonged to the "bottom half" region as well as the first 2 nts of ATG49. Colors as **Figure 1**.

tRNAs, the G10 can establish potential tertiary interactions with a nt of the V-R upstream the putative start codon [77]. At least in cytosolic tRNAs, frequently U8 and sometimes U48 form noncanonical pairs. Moreover, generally, base pair 15–48 is more conserved in mt-tRNAs than 8–14, and this is probably due to the fundamental role played by the first in maintaining the tRNA L-shape [5]. UAR10 and AUR49 had to play first only a role in the L-shaped tertiary structure of tRNAs, and their implication as codons, if it exists, would be only a derived character. It was hypothesized that DNA punctuation evolved from 2D structures signaling polymerization initiation, termination, and/or processing to linear sequence motifs, which further evolved to translational signals [78]. In ss-tRNA, UAR10 triplet probably already plays a structural role in proto-tRNAs, whereas AUR49 would have appeared only during the evolution of organelle tRNAs and was related to L-shaped tertiary structures of organelle tRNAs and due to severe genome reduction and extreme base compositions. The opposite hypothesis would imply that the AUR49 triplet would have been a plesiomorphic character counterselected in large genomes but kept in certain bacterial genomes up to mt-ancestors.

the RNA/protein world. Structures with both start and stop codons partially in a stem-loop (as ss-tRNA), constituting basic signals for translation, could be a missing link of the RNA world hypothesis. Furthermore, in these proto-tRNAs, 3D structures could act as initiation and termination signals before the emergence of standard codons. Moreover, mRNAs in the form of ss-tRNA or a combination of several of these molecules would have been relatively stable. The cloverleaf structure could facilitate its entry into the PTC, and then interactions with other factors could allow a short region to be in linear form and thus could be read. Upstream and downstream of the linear region, the arrangement in hairpins protected the proto-mRNA from degradation during its reading, and as soon as a long enough region was read, it could take again its original 3D structure. Otherwise, circular proto-mRNAs derived from ss-tRNA-like molecules could not be excluded, although the hypothesis of circular tRNA-like ancestor ("proto-tRNA") was first proposed by Ohnishi in 1990 [89]. Furthermore, nuclear-encoded mt-tRNAs of Kinetoplastid protists are imported into the mitochondrion, and circularized mature tRNA molecules are produced probably by mt-endogenous RNA ligase activity (*in vivo* or during mt-isolation) [90]. Moreover, in red and green algae and possibly in one Archaea, the maturation of permuted *trn* genes, in which the sequences encoding the 5′-half and 3′-half of the specific tRNA are separated and inverted on the genome, needs the formation of a characteristic circular RNA intermediate which after cleavage at the acceptor-stem generates the typical cloverleaf structure with functional termini [91]. If in a ss-tRNA with a T-loop of 7 nts, the nt72 is ligated to the nt1; this creates a small ORF starting with a start codon (AUR49), which potentially codes for a peptide of 12 amino acids if UAR10 is used as stop codon. However, the circularization could be done elsewhere than at levels of nts 72 and 1. Thus, UAR10 would not be in frame, and therefore, this could allow the synthesis of smaller or longer peptides. To date, the formation of this type of structure and its translation remains hypothetical; however, experimental data shown that circular RNAs can be translated in prokaryotic and eukaryotic systems in the absence of any particular element for internal ribosome entry as SD sequence, poly-A tail, or cap structure [92]. Therefore, the evolutionary advantage of a circular proto-mRNA is also posited to be the simplicity of its replication mechanism and not be able to be degraded by the extremities that do not have one.

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

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

19

Besides, the fusion of tRNA-like mRNA and a classical tRNA could be at the origin of the ancestors of tmRNAs, and it can be mentioned just for guidance that the size of the tag peptide encoded by bacteria is of the same order of magnitude as those corresponding to putative translation of a ss-tRNA from the ATR49 triplet. Moreover, evolution of self-charging prototRNAs may also be selected [93], it has even been proposed that the activity of the juxtaposed 2′/3′-OHs of the tRNA A76 ribose qualifies tRNA as a ribozyme [94] and some RNAs (the early tRNA adaptor) must have had the ability to undergo 3′-aminoacylation. It has also been previously shown that many hairpin-structured RNAs bear ribozyme activity. These catalyze self-cleavage and ligation reactions [95]. In addition, it remains possible that circular ss-tRNAs with amino acid-anchored structure could be at the origins of tmRNAs. Indeed, two-piece bacterial tmRNAs (e.g., in α-proteobacteria) are encoded by a circularly permuted gene sequence implying that pre-tmRNA is processed, and that the two pieces are held together by noncovalent interactions. Moreover, in line with an α-proteobacterial origin of mitochondria, probable mt-encoded circular permuted *tmRNA* genes have been found in the oomycete (water mold) *Phytophthora sojae* and in the jakobid *Reclinomonas americana* [96]. A proto-*trnA* gene could be

#### **3.10. tRNAs at the origin of all the nucleic members of the RNA/protein world**

Some authors have hypothesized that tRNAs may be the precursors of mRNAs, rRNAs (and therefore proto-ribosomes), and also of the first genomes. Several suggested similar origins for tRNA and rRNA [79]. Analyzes of sequences and secondary structures of ribosome suggested that the ribosomal peptidyl transferase center (PTC) which forms peptide bonds between adjacent amino acids originates from fused proto-tRNAs [80]. Strikingly, the ribosome is a ribozyme, since only RNA catalyzes peptide bond formation [81]. Otherwise, current eubacterial rRNAs themselves could encode several tRNAs [82] and chaetognath 16*S rRNA* genes appear as tRNA nurseries [12] (or the opposite). Eubacterial 5S rRNAs contain TLSs similar to alanine and arginine tRNAs [82], exhibiting tRNA-like 2D structures [83]. Some suggest that rRNAs are fused tRNA molecules [80].

Molecular biology dogmatically assumes that "tRNA genes are of course entirely noncoding" [84]. But in 1981, Eigen and Winkler-Oswatitsch suggested that in the RNA world to the RNA/ protein world transition, ancestral tRNAs were mRNAs [85]. Assuming that the first mRNAs had been recruited from proto-tRNAs, it follows that TLSs were inside viral and cellular mRNAs [86]. Self-recognition between tRNA-like mRNAs and canonical cloverleaf tRNAs could stabilize these molecules and produce proto-proteins [87]. The first proteins potentially emerged from junctions of ancestral tRNAs, and among the modern proteins, the only polymerase which matched with tRNAs translated like a mRNA was the RNA-dependent RNA polymerase [87]. Otherwise, eubacterial rRNAs could also encode several active sites of key proteins involved in the translation machinery [82]. Then, analyzes of sequences and secondary structures of ribosomes suggested that these derived from tRNAs also functioned as a protogenome [82]. The very parsimonious syncretic model "tRNA core hypothesis" assumes that some proto-tRNAs were classical tRNAs and also functioned as rRNAs and mRNAs, a self-recognition between these molecules allowed to obtain proto-proteins [88].

Assuming that the ATR49 triplets are a primitive character lost during the first genome expansions and that they could already act as an initiation codon seems too speculative, but RNA structures having characteristics of ss-tRNAs could have accumulated many advantages in the RNA/protein world. Structures with both start and stop codons partially in a stem-loop (as ss-tRNA), constituting basic signals for translation, could be a missing link of the RNA world hypothesis. Furthermore, in these proto-tRNAs, 3D structures could act as initiation and termination signals before the emergence of standard codons. Moreover, mRNAs in the form of ss-tRNA or a combination of several of these molecules would have been relatively stable. The cloverleaf structure could facilitate its entry into the PTC, and then interactions with other factors could allow a short region to be in linear form and thus could be read. Upstream and downstream of the linear region, the arrangement in hairpins protected the proto-mRNA from degradation during its reading, and as soon as a long enough region was read, it could take again its original 3D structure. Otherwise, circular proto-mRNAs derived from ss-tRNA-like molecules could not be excluded, although the hypothesis of circular tRNA-like ancestor ("proto-tRNA") was first proposed by Ohnishi in 1990 [89]. Furthermore, nuclear-encoded mt-tRNAs of Kinetoplastid protists are imported into the mitochondrion, and circularized mature tRNA molecules are produced probably by mt-endogenous RNA ligase activity (*in vivo* or during mt-isolation) [90]. Moreover, in red and green algae and possibly in one Archaea, the maturation of permuted *trn* genes, in which the sequences encoding the 5′-half and 3′-half of the specific tRNA are separated and inverted on the genome, needs the formation of a characteristic circular RNA intermediate which after cleavage at the acceptor-stem generates the typical cloverleaf structure with functional termini [91]. If in a ss-tRNA with a T-loop of 7 nts, the nt72 is ligated to the nt1; this creates a small ORF starting with a start codon (AUR49), which potentially codes for a peptide of 12 amino acids if UAR10 is used as stop codon. However, the circularization could be done elsewhere than at levels of nts 72 and 1. Thus, UAR10 would not be in frame, and therefore, this could allow the synthesis of smaller or longer peptides. To date, the formation of this type of structure and its translation remains hypothetical; however, experimental data shown that circular RNAs can be translated in prokaryotic and eukaryotic systems in the absence of any particular element for internal ribosome entry as SD sequence, poly-A tail, or cap structure [92]. Therefore, the evolutionary advantage of a circular proto-mRNA is also posited to be the simplicity of its replication mechanism and not be able to be degraded by the extremities that do not have one.

tRNAs, the G10 can establish potential tertiary interactions with a nt of the V-R upstream the putative start codon [77]. At least in cytosolic tRNAs, frequently U8 and sometimes U48 form noncanonical pairs. Moreover, generally, base pair 15–48 is more conserved in mt-tRNAs than 8–14, and this is probably due to the fundamental role played by the first in maintaining the tRNA L-shape [5]. UAR10 and AUR49 had to play first only a role in the L-shaped tertiary structure of tRNAs, and their implication as codons, if it exists, would be only a derived character. It was hypothesized that DNA punctuation evolved from 2D structures signaling polymerization initiation, termination, and/or processing to linear sequence motifs, which further evolved to translational signals [78]. In ss-tRNA, UAR10 triplet probably already plays a structural role in proto-tRNAs, whereas AUR49 would have appeared only during the evolution of organelle tRNAs and was related to L-shaped tertiary structures of organelle tRNAs and due to severe genome reduction and extreme base compositions. The opposite hypothesis would imply that the AUR49 triplet would have been a plesiomorphic character counter-

selected in large genomes but kept in certain bacterial genomes up to mt-ancestors.

**3.10. tRNAs at the origin of all the nucleic members of the RNA/protein world**

rRNAs are fused tRNA molecules [80].

18 Mitochondrial DNA - New Insights

Some authors have hypothesized that tRNAs may be the precursors of mRNAs, rRNAs (and therefore proto-ribosomes), and also of the first genomes. Several suggested similar origins for tRNA and rRNA [79]. Analyzes of sequences and secondary structures of ribosome suggested that the ribosomal peptidyl transferase center (PTC) which forms peptide bonds between adjacent amino acids originates from fused proto-tRNAs [80]. Strikingly, the ribosome is a ribozyme, since only RNA catalyzes peptide bond formation [81]. Otherwise, current eubacterial rRNAs themselves could encode several tRNAs [82] and chaetognath 16*S rRNA* genes appear as tRNA nurseries [12] (or the opposite). Eubacterial 5S rRNAs contain TLSs similar to alanine and arginine tRNAs [82], exhibiting tRNA-like 2D structures [83]. Some suggest that

Molecular biology dogmatically assumes that "tRNA genes are of course entirely noncoding" [84]. But in 1981, Eigen and Winkler-Oswatitsch suggested that in the RNA world to the RNA/ protein world transition, ancestral tRNAs were mRNAs [85]. Assuming that the first mRNAs had been recruited from proto-tRNAs, it follows that TLSs were inside viral and cellular mRNAs [86]. Self-recognition between tRNA-like mRNAs and canonical cloverleaf tRNAs could stabilize these molecules and produce proto-proteins [87]. The first proteins potentially emerged from junctions of ancestral tRNAs, and among the modern proteins, the only polymerase which matched with tRNAs translated like a mRNA was the RNA-dependent RNA polymerase [87]. Otherwise, eubacterial rRNAs could also encode several active sites of key proteins involved in the translation machinery [82]. Then, analyzes of sequences and secondary structures of ribosomes suggested that these derived from tRNAs also functioned as a protogenome [82]. The very parsimonious syncretic model "tRNA core hypothesis" assumes that some proto-tRNAs were classical tRNAs and also functioned as rRNAs and mRNAs, a

self-recognition between these molecules allowed to obtain proto-proteins [88].

Assuming that the ATR49 triplets are a primitive character lost during the first genome expansions and that they could already act as an initiation codon seems too speculative, but RNA structures having characteristics of ss-tRNAs could have accumulated many advantages in Besides, the fusion of tRNA-like mRNA and a classical tRNA could be at the origin of the ancestors of tmRNAs, and it can be mentioned just for guidance that the size of the tag peptide encoded by bacteria is of the same order of magnitude as those corresponding to putative translation of a ss-tRNA from the ATR49 triplet. Moreover, evolution of self-charging prototRNAs may also be selected [93], it has even been proposed that the activity of the juxtaposed 2′/3′-OHs of the tRNA A76 ribose qualifies tRNA as a ribozyme [94] and some RNAs (the early tRNA adaptor) must have had the ability to undergo 3′-aminoacylation. It has also been previously shown that many hairpin-structured RNAs bear ribozyme activity. These catalyze self-cleavage and ligation reactions [95]. In addition, it remains possible that circular ss-tRNAs with amino acid-anchored structure could be at the origins of tmRNAs. Indeed, two-piece bacterial tmRNAs (e.g., in α-proteobacteria) are encoded by a circularly permuted gene sequence implying that pre-tmRNA is processed, and that the two pieces are held together by noncovalent interactions. Moreover, in line with an α-proteobacterial origin of mitochondria, probable mt-encoded circular permuted *tmRNA* genes have been found in the oomycete (water mold) *Phytophthora sojae* and in the jakobid *Reclinomonas americana* [96]. A proto-*trnA* gene could be at the origin of modern tmRNAs [41]. Metazoan mt-*trnA* genes combine the highest levels of TAR10 and ATR49 triplets (>95% for each), but in the prokaryotic world, if the rate of TAG10 is always higher than 91%, only one ATR49 occurs in Eubacteria and none in Archaea.

mt-genomes. These overlaps can have a variable (sometimes large) number of nts; however, when annotating their genomes, several authors voluntarily underestimated the number and the size of overlaps, speculating that there would be upstream abbreviated stop codons or downstream alternative start codons but most often without any direct demonstration so far. However, the high number of possible overlaps on the same strand in which the first in-frame complete stop codon or standard start codon are located at specific positions in the sequences of *trn* genes (TAR10 and ATR49, respectively) strongly suggest an exclusive relationship between obtaining tRNAs and translation of mRNAs and/or the development of repair system to keep the two genes functional due in some cases to co-evolution during several hundred MY. We can therefore speculate that ss-*trn* genes could allow true tRNA punctuation and initiation. Noted that ss-tRNAs seem to be hybrid molecules which would contain three essential coding or decoding informations in the form of nt triplets (i.e., anticodon and stop/ start codons) which are all at least in part integrated into stem or loop; moreover, after the ATR49, nt triplets play the role of internal sense codons. To date, it is unclear what biochemical mechanism would allow to choose between different alternate cleavage sites, leading to the complete tRNA rather than to the mRNA or *vice versa*, but reduced/expanded proteins can be functional, and various processes including editing suggest this also for incomplete tRNAs. Hence, despite lacking experimental evidence, TAR10 and ATR49 triplets have probable roles, including regulation. Future analyzes of the processed bicistronic transcripts (tRNA/ protein-encoding or the contrary) are required. Moreover, even if mt-*trn* genes are most often expressed at very low levels [53], only direct sequencing of tRNAs can validate transcription, epitranscriptomic maturation and can pinpoint nt modifications including post-transcriptionally edited positions. Purified native, or even synthetic, tRNAs should also be tested for their *in vitro* activity to confirm the functionality of aberrant transcripts. Similar experiments must be made on the flanking mRNAs and their products. If as we think, ss-tRNAs could play regu-

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

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

21

latory roles, initially experiments should compare stress and nonstress conditions.

mini while accounting tRNA species, taxa, and genomic systems.

The authors declare no potential commercial or financial conflicts of interest.

should be more thoroughly investigated.

**Conflict of interest**

Here, the bias for metazoan mtDNA does not allow for a complete picture of variation in the entire eukaryotic world, and protist mt-genomes should also be considered. Special attention should also be paid to noncanonical base pairings potentially formed by UAR10 and AUR49 nts, in perspective with tRNA structure and V-R length. Accounting for TAR10 and ATR49 triplet presences in the algorithms predicting tRNAs could improve mt-genome annotations, reducing numbers of false positives and negatives, and more accurately determine tRNA ter-

MtDNA plays a central role in apoptosis, aging, and cancer [13]. Moreover, mt-diseases are among the most common inherited metabolic and neurological disorders [101]. In addition, as new functions and new mechanisms of action of tRNAs are continuously discovered [1] and as ss-*trn* genes could affect the cellular dynamic during normal and stress conditions leading to pathologies, potential subtleties of action and regulation of these genes and products
