**3.5. Multifunctionality of tRNAs**

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.

**3.4. Some known structures in the living world bearing a start or stop codon at least** 

In the living world, many nucleic sequences with secondary structures playing a physiological role involving stop and/or start codons have been discovered. Some representative examples are briefly presented here. (1) The tropism switching of the bacteriophage BPP-1 is mediated by a phage-encoded diversity-generating retroelement, which introduces nt substitutions in a gene that specifies a host cell-binding protein (*Mtd*) [37]. The nt substitutions are introduced in a variable repeat located at the 3′-end of this gene. Two nts after this region, the UAG stop codon is present, and its last nt is situated at the 5′ beginning of the 5′-stem of a hairpin. Both the UAG codon and hairpin are required for phage tropism switching. (2) Programmed translational bypassing is a process, whereby ribosomes "ignore" a substantial interval of mRNA sequence. In a bacteriophage T4 gene, bypassing requires translational blockage at a "takeoff codon" immediately upstream of the UAG stop codon, and both codons are in the 5′-stem of a hairpin; moreover, this region is mobile [38]. (3) The operon *flgFG* of the bacterium *Campylobacter jejuni* can encode two genes (*flgF* and *flgG*). Its expression in *E. coli* produces a fusion protein probably due to ribosomal frameshifting (translational hopping) [39]. The putative hop region contains, among others, a hairpin beginning by the last nt of the UAA stop codon of the first mRNA. The AUG start codon of the second gene is in the loop of the following hairpin. (4) In Eubacteria, riboswitches are regulatory segments of DNA or mRNA that can bind a small molecule (the effector), which repress or activate their cognate genes at transcriptional and/or translational levels. In the riboflavin and *cob* operons, conformational changes can form a stem loop which sequesters the translational start site, consisting of the Shine-Dalgarno (SD) sequence plus start codon thus preventing gene translation [40]. (5) Bacterial transfer-messenger RNAs (tmRNAs) have dual TLS and mRNA-like properties. They rescue stalled ribosomes on mRNAs lacking proper translational stop signal; the tRNA-like

**in part of a stem-loop structure**

12 Mitochondrial DNA - New Insights

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).

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

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

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.

on positions of upstream stop codons completed by polyadenylation and/or on downstream (alternative) initiator codons. Not only complete proteins may be functional. Depending on cleavage positions in polycistronic transcripts, consequences may be neutral, disadvantageous, or favorable in specific contexts. In yeast, extended proteins can increase fitness under stress conditions [58]. In addition, in bacteria and in organelles, alternative initiation codons decrease efficiency [5], and it must be noted that ATR49 triplets are "canonical" start codons. In other conditions, incomplete mRNAs could be favored. Mitosolic mRNA accumulations can be due to lack of translation because of tRNA paucity. Thus, high mRNA levels might indirectly promote cleavage of entire tRNA transcripts while reducing the synthesis of new functional mRNAs and favoring translation of those which are already present into proteins. Presence/absence of hairpins involving stop or start codons might regulate translation. This regulation could involve proteins that stabilize the hairpins or posttranscriptional modifications. Moreover, translational products of "incomplete" mRNAs might have housekeeping

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

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

15

Regulation of alternative processing producing either complete tRNAs or complete mRNAs requires elucidation. Factors, probably proteins, need characterization. Note that metazoan mt-*atp8* and *atp6* genes overlap (mainly by 10 bp in vertebrates) and are transcribed as joint bicistronic transcript [59]. This proven overlap is inherent to mt-metabolism. Hence, similar

Overlap conservation might reflect the need to produce bicistronic transcripts (5′-tRNAmRNA-3′ or 5′-mRNA-tRNA-3′) or functional constraints at protein level (i.e., preserving specific amino acid patterns upstream or downstream the ORF). When overlap regions have conserved, amino acid sequences at the protein N- or C-terminal functional constraints at protein level for overlaps are probable [19]. In viruses, mutation rates are low in DNA regions coding for multiple protein products in separate reading frames (called overprinted genes) because point mutations compatible with functional products from all frames are rare. In these regions, the frame is said "close off." Partial overlap between protein-encoding genes and *ss-trn* genes would present similar situations explaining greater conservation of extremities of protein and tRNA sequences when the corresponding genes overlap. This lock almost only concerns the ss-tRNA's "top half," limiting changes in the region interacting with many processing enzymes. The *ss-trn* genes could also regulate translation upstream, bicistronic mRNA/ss-tRNA transcripts could be more stable, and likewise, ss-*trn* genes could also play

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

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.

functions.

overlaps assumed for TAR10 triplets are plausible.

roles in replication and transcription.

**and translation**

**Figure 2.** 2D "cherry-bob"/"bottom half" structure. AUG/UAG triplets are discussed in text. Colors as **Figure 1**.

Overlaps involve numerous constraints for genes including sequence bias. Constraints are probably less stringent for *trn* genes, which can evolve rapidly because relatively standard secondary structure coupled with a specific anticodon might suffice for tRNA function [52]. Incomplete cloverleaf structures may also be repaired post-transcriptionally [53].

Alternative processing might be possible for the production of either a supposed complete mRNA or a complete tRNA. In the first case, the synthesis of new complete mRNAs could be promoted by high mitosolic tRNA numbers. Moreover, amino acid starvation can regulate mt-tRNA levels [54]. However, if mt-tRNAs already present are not destroyed, translation would not immediately stop because mt-tRNA half-life which is lower than that of their cytosolic counterparts can nevertheless exceed 10 h [54]. Moreover, aberrant mt-tRNAs can be corrected by RNA editing during or after transcription, and this process appeared independently several times in a wide variety of eukaryotes [5]. As an extreme example, due to large overlaps between *trn* genes, up to 34 nts are added post-transcriptionally during the editing process to the mt-tRNA sequences encoded in an onychophora species, rebuilding the acceptor-stem, the T-arm, and in some extreme cases, the V-R and even a part of the anticodon-stem [55]. In that species, several edition types must be combined, including template-dependent editing [55]. This last example suggests that complete tRNA could be restored after a cleavage just upstream of ATR49. However, edition of parts of the 5′-end of tRNAs seems more problematic. Besides, mRNAs with upstream or downstream ss-tRNA can form a partially double strand region with a homologous ss-tRNA at the level of the acceptor-stem. This might induce mRNA degradation via antisense mechanisms. In bacteria, uncharged tRNAs cause antisense RNA inhibition [56], and small interfering cytosolic tRNA-derived RNAs exist [57]. Modifications (methylation, edition, etc.) of incomplete tRNAs generated after cleavages of polycistronic transcripts at TAR10 or ATR49 triplets would indicate regulatory functions.

Putative use of TAR10 or ATR49 triplets affects protein length. When in frame, this could generate a protein at least 3 or 9 amino acids longer, respectively. Extension length depends on positions of upstream stop codons completed by polyadenylation and/or on downstream (alternative) initiator codons. Not only complete proteins may be functional. Depending on cleavage positions in polycistronic transcripts, consequences may be neutral, disadvantageous, or favorable in specific contexts. In yeast, extended proteins can increase fitness under stress conditions [58]. In addition, in bacteria and in organelles, alternative initiation codons decrease efficiency [5], and it must be noted that ATR49 triplets are "canonical" start codons.

In other conditions, incomplete mRNAs could be favored. Mitosolic mRNA accumulations can be due to lack of translation because of tRNA paucity. Thus, high mRNA levels might indirectly promote cleavage of entire tRNA transcripts while reducing the synthesis of new functional mRNAs and favoring translation of those which are already present into proteins. Presence/absence of hairpins involving stop or start codons might regulate translation. This regulation could involve proteins that stabilize the hairpins or posttranscriptional modifications. Moreover, translational products of "incomplete" mRNAs might have housekeeping functions.

Regulation of alternative processing producing either complete tRNAs or complete mRNAs requires elucidation. Factors, probably proteins, need characterization. Note that metazoan mt-*atp8* and *atp6* genes overlap (mainly by 10 bp in vertebrates) and are transcribed as joint bicistronic transcript [59]. This proven overlap is inherent to mt-metabolism. Hence, similar overlaps assumed for TAR10 triplets are plausible.

Overlaps involve numerous constraints for genes including sequence bias. Constraints are probably less stringent for *trn* genes, which can evolve rapidly because relatively standard secondary structure coupled with a specific anticodon might suffice for tRNA function [52].

**Figure 2.** 2D "cherry-bob"/"bottom half" structure. AUG/UAG triplets are discussed in text. Colors as **Figure 1**.

14 Mitochondrial DNA - New Insights

Alternative processing might be possible for the production of either a supposed complete mRNA or a complete tRNA. In the first case, the synthesis of new complete mRNAs could be promoted by high mitosolic tRNA numbers. Moreover, amino acid starvation can regulate mt-tRNA levels [54]. However, if mt-tRNAs already present are not destroyed, translation would not immediately stop because mt-tRNA half-life which is lower than that of their cytosolic counterparts can nevertheless exceed 10 h [54]. Moreover, aberrant mt-tRNAs can be corrected by RNA editing during or after transcription, and this process appeared independently several times in a wide variety of eukaryotes [5]. As an extreme example, due to large overlaps between *trn* genes, up to 34 nts are added post-transcriptionally during the editing process to the mt-tRNA sequences encoded in an onychophora species, rebuilding the acceptor-stem, the T-arm, and in some extreme cases, the V-R and even a part of the anticodon-stem [55]. In that species, several edition types must be combined, including template-dependent editing [55]. This last example suggests that complete tRNA could be restored after a cleavage just upstream of ATR49. However, edition of parts of the 5′-end of tRNAs seems more problematic. Besides, mRNAs with upstream or downstream ss-tRNA can form a partially double strand region with a homologous ss-tRNA at the level of the acceptor-stem. This might induce mRNA degradation via antisense mechanisms. In bacteria, uncharged tRNAs cause antisense RNA inhibition [56], and small interfering cytosolic tRNA-derived RNAs exist [57]. Modifications (methylation, edition, etc.) of incomplete tRNAs generated after cleavages of polycistronic transcripts at TAR10 or ATR49 triplets would indicate regulatory functions.

Putative use of TAR10 or ATR49 triplets affects protein length. When in frame, this could generate a protein at least 3 or 9 amino acids longer, respectively. Extension length depends

Incomplete cloverleaf structures may also be repaired post-transcriptionally [53].

Overlap conservation might reflect the need to produce bicistronic transcripts (5′-tRNAmRNA-3′ or 5′-mRNA-tRNA-3′) or functional constraints at protein level (i.e., preserving specific amino acid patterns upstream or downstream the ORF). When overlap regions have conserved, amino acid sequences at the protein N- or C-terminal functional constraints at protein level for overlaps are probable [19]. In viruses, mutation rates are low in DNA regions coding for multiple protein products in separate reading frames (called overprinted genes) because point mutations compatible with functional products from all frames are rare. In these regions, the frame is said "close off." Partial overlap between protein-encoding genes and *ss-trn* genes would present similar situations explaining greater conservation of extremities of protein and tRNA sequences when the corresponding genes overlap. This lock almost only concerns the ss-tRNA's "top half," limiting changes in the region interacting with many processing enzymes. The *ss-trn* genes could also regulate translation upstream, bicistronic mRNA/ss-tRNA transcripts could be more stable, and likewise, ss-*trn* genes could also play roles in replication and transcription.
