3.6 to operate. The overlapping involves numerous constraints

3.6 Other putative roles of TAR10 and ATR49

Metazoan mt-genomes are generally thought of as being
economic and optimized for rapid replication and transcription. The potential
use of TAG10 and ATR49 apparently makes the system more complex but it could be
hypothezed that this should be compensated by large benefits that they could
provide. Moreover, examples given in the chapter 3.2, as those of Eucestoda, suggest
that some overlaps appeared hundreds of millions of years ago; so, a strong co-evolution between gene sequences had
time to operate.

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The overlapping
involves numerous constraints for the genes including sequences bias. However, in
contrast to protein-encoding genes, such constraints are more likely to be less
stringent for trn genes overlaps. The trn gene
sequences can evolve rapidly, a relatively standard secondary structure coupled
with a specific anticodon may be sufficient to generate a functional tRNA (Yona et al., 2013)  ; moreover, incomplete cloverleaf structure may be
repaired post-transcriptionally (Doublet et al., 2015) .

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 is favored
to the detriment of those of tRNAs, e.g., this could be promoted by a too large
number of tRNAs in
mitosol. Moreover, it is known that the mt-tRNA levels can be regulated in response amino acid starvation
(Schild et al., 2014) . However, if tRNAs
already present are not destroyed, the stop of the translation would not be immediate as although the half-life of mt-tRNAs is lower than
that of their cytosolic counterparts, it can exceed ten hours (Schild et al., 2014) . Moreover, aberrant mt-tRNAs can be corrected by RNA editing during or after
transcription, this process has been invented independently several
times, in a wide variety of eukaryotes (Lang et al., 2012) . 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 (Segovia et al.
2011) . In this species, a combination of several types of editing must be
involved including a template-dependent editing (Segovia et al.
2011) . This last example,
suggests that complete tRNA sequences could be restored after a cleavage just
upstream of the ATR49. However, to date, edition of parts of the 5′-end of
tRNAs seems more problematic. Besides, a mRNA
which contains an upstream or downstream sequence of a 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
an antisens mechanism. By the way, in bacteria, uncharged tRNAs can play an antisense RNA inhibition (Camps, 2010) and small
interfering RNAs derived from cytosolic tRNAs (Honda et al. 2017 . Furthermore, it will be useful to investigate whether incomplete
tRNAs generated after cleavages of polycistronic transcripts at TAR10 or ATR49
triplets would not be modified (edition, methylation, etc.), suggesting that
they could have regulatory functions.

Moreover, the putative use of TAR10 or ATR49 triplets affects
inevitably the protein length. Indeed, if they are in frame, this could generate a protein at least 3 or 9 amino acids longer respectively. Variations in the extension
length depending principally of the position of the upstream stop codons
which are complete by polyadenylation or of the
first downstream (alternative) initiator
codon respectively. Nonetheless, we should not be inevitably thinking in
Manichean terms of deleted versus complete proteins, the latter supposed to be
the only functional. Depending of cleavage positions in the
polycistronic transcripts, the consequences may be only neutral or slightly disadvantageous but can also favorable in a specific
context. This is supported by experiments showing that
extended proteins can increase fitness under stress conditions in yeast (Halfmann et al., 2012) . In addition, in bacteria and in organelles use of
alternative initiation codons induces a loss of efficiency, all the more due to
the alternative initiation does not depend on extra tRNA even if
post-transcriptional modifications could occur (Lang et al., 2012) . So, in this context, it may therefore be advantageous to
use the ATR49 triplet.

At the opposite, production of supposed incomplete
mRNAs could be favored, e.g, because accumulations of mRNAs in the mitosol,
this may be due to an absence of or slowdown in the translation due, among others, to
tRNA paucity. Thus, high levels of mRNAs might, indirectly,
promote cleavage of entire tRNA transcripts
while reducing the synthesis of new functional mRNAs and favors the
reading of those which are already present in
mitochondria into proteins. The control
of the translation could also depend on the presence or
not of hairpins involving stop or start cordons. This
regulation could involve proteins
that stabilize the hairpins or post-transcriptional modifications as methylamine.
Moreover, the products of “incomplete” mRNAs could ensure the
housekeeping functions.

The mechanism regulating alternative processing for the production of either a complete
tRNA or a complete mRNA would need to be elucidated. Factors, probably proteins,
are likely implicated and need to be characterized in the future. Besides, it is a common feature of metazoan mt-genomes that atp8 and atp6 genes overlap (of mainly 10 bp in length in vertebrates) and that they are transcribed as
a joint bicistronic transcript (Stewart and
Beckenbach 2009) . Therefore, overlaps
whose the size is equal to those observed to reach to the TAR10 triplet does
not seem to pose insuperable problems.

The conservation of the overlaps
could be due to the need to produce a bicistronic tRNA/mRNA or mRNA/tRNA, or to
a functional constraint at protein level, such as the need to preserve a
specific amino acid pattern in the regions corresponding to the upstream or
downstream ORF. When analyses of the
overlap regions show well conserved amino acid sequences at the N- or
C-terminal of proteins, this suggests that there is functional constraints at
protein level for these overlappings (Morrisson, 2010) . When, as
in viruses, a DNA region codes for multiple protein products in separate
reading frames (called overprinted genes), in this area, the mutation rate
appears to be very weak, the frame is said “close
off”, indeed, the probability that a
point mutation would not be disadvantageous in at least one of the frames is
low. The situation is similar, although this is in the same frame, when there
is a partial overlap between a protein encoding gene and a ss-trn gene. This type of strategy therefore makes it possible to
preserve the identity of the extremities of both protein and tRNA sequences
because when a mutation occurs the risk of counter-selection is higher compared
to the sequences that do not overlap. At the ss-tRNA level, this lock almost
only concern the top half, so this could be a strategy to greatly limit the
changes of the tRNA sequence in the region which interact with several
processing enzymes.

The ss-trn genes could also play a role in regulation of translation but
far upstream, e.g., bicistronic mRNA/ss-tRNA transcripts could be more stable,
likewise, ss-trn genes could also be
play a role in replication and transcription.

Methylation of trn genes and tRNAs
and there possible roles in transcription and translation

The methylation rate of the mt-genome is much
lower than that of nuclear (van
der Wijst et al., 2017) ; however, the location of
these modifications at the level of trn
genes (particularly around TAR10 and ATR49) and their possible consequences
should be studied. This all the more, as differential
mtDNA methylations have been linked to aging and diseases, including diabetes
and cancers (van der Wijst et al., 2017) . Moreover, methylation of nts of TAR10 and ATR49 are known
as those of A9 and G10 which can be
important for correct folding of tRNAs (Lorenz et al., 2017) . It is not known to us if
post-transcriptional modifications can occur on a bicistronic mt-transcript
containing a tRNA or a part of this latter but this would be worth looking for
as well as the possible consequence at the level of on maturation and

3.8 Reassignments of codons and

Several reassignments
from the standard code
are known and most of them imply mitochondria. Among the most frequent changes,
onto 11 different mt-codes, note that, unlike the
universal code, UGA codes for tryptophan instead of termination and AUA codes
for methionine instead of isoleucine in 8 and 5
of them respectively (NCBI,
2016) . Both
types of reassignments allow to avoid potential errors linked to the use of
traditional rules of wobble. Moreover, the evolution from UGA-stop to UGA-Trp
has been explained with the “capture”
hypothesis, in which, all UGA codons mutate first to its synonymous codon UAA
in genomes which high AT%. Then, when through mutation UGA re-appears, it is
free to be “captured”
by an amino acid, in this case Trp (Osawa and Jukes, 1989) . In
addition, AUA is frequently used as alternative initiation codons and the reassignment of this previous Ile
codon to internal sense Met
codon could also have evolved in AT-rich genomes. Moreover, in standard code, four
codons are assigned to arginine whereas two would be sufficient in relation to
the relatively low frequency of this amino acid in current proteins (Wallis, 1974)
In 8 out of 11 mt-codes, the number of Arg codons was reduced to two but with
different strategies, reassignments
of AGA and AGG
to other amino acids for 6 codes, lacking of two Arg codons (CGA
and CGC) in yeast mt-code and AGA and AGG were considered
terminators instead of coding for arginine in vertebrate.
However, at least in humans, it has now been shown that AGA and AGG sequences
are not recognized as termination codons, only UAA and UAG are use as stop
codons (Temperley et al.,
2010) suggesting that the AGR codons
would rather without assignment. The
reassignments of the AGR codons were thought to have become mt-stop
codons early in vertebrate evolution (Osawa et al., 1989) . All of these data lead us to think that the mt-code of
vertebrates could be the most optimized of all those known to date (the yeast
mt-code was not retained because 4 codons Leu were reassigned in Thr codons).
Besides, characteristics of the nt triplets at the position 8-10 and ending at
position 49 should be analyzed for each type of mt-code.

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

models have been raised to explain the origin of the tRNA molecule (see reviews
Di Giulio, 2009, 2012;
Fujishima and Kanai, 2014) and it is a too
large topic to discuss them here. Many studies have suggested
that the modern cloverleaf structure of tRNA may
have arisen through direct duplication of a primordial RNA hairpin (e.g., Di Giulio, 2009) . However, there is
remarkable and lends strong support to the ‘two halves” hypothesis (Maizels and Weiner, 1994) in which tRNA
consists of two coaxially stacked helices that appear to be independent
structural and functional domains: those referred to as the “top half” contains the
acceptor-stem and the T-arm whereas the “bottom half” contains the D- and anticodon-arms
(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-arm that interact with the catalytic
domain of specific aaRSs; moreover, it is also recognized by RNases P and
Z, and CCA-adding enzyme which are mainly RNA
end processing reactions (Maizels and Weiner, 1999;
Fujishima and Kanai, 2014) . Moreover, during translation, this domain can also
interact with elongation factor Tu and
one rRNA (Maizels and Weiner,
1999) . The importance of this domain in most of the
macromolecular interactions involving tRNA and in vitro even when it is detached from the bottom half suggests
that the specificities of this half was established before the bottom half of
tRNA which was incorporated later into the molecule (Sun and Caetano-Anollés, 2008) . Moreover, the growing evidence of tRNA elements involved in both
RNA and DNA replication in which its 3′-end plays a determinant role has led to
the idea that top half initially evolved for replication in the RNA world
before the advent of protein synthesis (Weiner and Maizels, 1999) . The
supposed evolutionarily recent “bottom half” of tRNA holds the standard code and its essential role is to interact specifically by complementary
interactions with a codon bearing by the mRNA and the late implementation of the standard genetic code with the late
appearance of interactions between the bottom half and the ribosome is
supported by recent studies (Caetano-Anollés
et al., 2013) . 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 is a question that is not resolved
today (Maizels
and Weiner, 1999) . However, several authors lean rather of independent
evolutionary origins (Maizels and Weiner, 1999; Sun and Caetano-Anollés, 2008) .

The study of ss-tRNAs makes it possible to propose a
model to partially explain the origin of canonical tRNAs (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.

On the other hand, the integration of the bottom half/cherry
bob structure could also be done at the RNA level, either in the RNA world by intermolecular RNA-RNA recombination or template switches or later with retrotranscription
events. Fujishima and
Kanai (2014) had
also proposed an equivalent model in which a long hairpin corresponding to
about the top half region merged with a viral RNA element corresponding to the
bottom half (cherry bob) to give the TLS found in modern viral genomes who
however possessed a pseudoknotted
aminoacyl-acceptor stem. A pseudoknot is
a RNA secondary structure containing at least two stem-loop structures in which
half of one stem is intercalated between the two halves of another stem (Colussi et al., 2014) .
Besides, rare pre-tRNA molecules found in the three domains of life exhibit an
intron. The origin of these introns is still being debated, in the ‘introns-early’
scenario almost all of these introns were lost during evolution, whereas the
opposite scenario named ‘introns-late’
theorizes that introns would have inserted
into some tRNA genes after their emergence (Yoshihisa, 2014) . 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

In tRNAs, the two first nts of both UAR10 and AUR49
belongs to a connector, 1 and 2 respectively, they are thus at the junction
between the top and bottom halfes 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 will not be discussed here
especially since this theoretical model is applicable whatever the ends of the
bottom half. However, as the V-R is
important for the aminoacylation (Larkin et al., 2002) , the 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 tRNAs, the G10 can establish
potential tertiary interactions with a nt of the V-R upstream the putative
start codon (Helm et al.,
2000) . At least in cytosolic tRNAs,
frequently U8 and sometimes U48 are involved in non Watson-Crick pairing.
Moreover, generally, base pair 15-48
is more conserved in mt-tRNAs than 8-14, this is probably due to the
fundamental role played by the first in maintenance of the tRNA L-shape (Lang et al, 2012) . TAR10 and ATR49 had to play
firstly only a role in the L-shaped tertiary
structure of tRNAs, their implication as codons, if it exists, would be only a
derivative character. The first triplet probably already play a structural role
in proto-tRNAs whereas the latter only during the evolution of organelle tRNAs.
The emergence of ATR49 triplets would be much later, it may be related to L-shaped tertiary structure of organelle tRNAs and to
consequences of severe genome reduction and extreme base compositions. The
opposite hypothesis would imply that the ATR49 triplet would have been a plesiomorphic character
that would have been counter-selected in large genomes but kept in certain
bacterial genomes up to the ancestors of mitochondria.

3.10 tRNA molecules at the origin of all the nucleic members of the RNA/protein

Some authors have hypothesized that tRNAs may be the
precursors of mRNAs, rRNAs (and therefore proto-ribosomes) and also of the
first genomes, and this is briefly
summarized here. A similar origin between tRNA and rRNA
has been suggested several times (Farias et al.,
2017) Analyses of sequences and secondary structures
of the ribosome suggested that the ribosomal peptidyl transferase
center (PTC), which forms peptide bonds between adjacent amino acids, was
originated by the fusion of proto-tRNAs (Farias et al., 2014) ; strikingly, the ribosome
is a ribozyme, since only RNA catalyses peptide bond formation (Noller, 2012) . Otherwise current
eubacterial rRNAs themselves could encode several tRNAs Root-Bernstein and Root-Bernstein, 2015 . Although eubacterial 5S rRNAs contained a TLS similar to the
alanine and arginine tRNA Root-Bernstein and Root-Bernstein, 2015 and so exhibit a tRNA-like 2D structure (Mathews et al., 2004). Authors had come to the conclusion that
rRNAs may have originated from the fusion of tRNA molecules (Farias et al., 2014) .

Among the great dogmas of molecular
biology there is that “tRNA
genes are of course entirely noncoding” (Bean et al., 2016) . However, the idea that in the RNA world to the RNA/protein world
transition, ancestral tRNAs could have worked like mRNAs was
proposed for the first time by Eigen and Winkler-Oswatitsch (1981) . Assuming that the first mRNAs had been recruited from
proto-tRNAs, it follows that TLSs have been found inside viral but also
cellular mRNAs (Ariza-Mateos
and Gómez, 2017) . The self-recognition between tRNA-like mRNAs
and canonical cloverleaf tRNAs could increase the stability of these molecules
and allow to obtain proto-proteins (Farias et al. 2016a) . Farias
et al. (2016a) suggested
that the first proteins 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. Otherwise eubacterial rRNAs could also encode
several active sites of key protein involved in forming the translation
machinery Root-Bernstein and
Root-Bernstein, 2015 . Then, analyses of sequences and secondary structures
of the ribosome suggested that it was derived from a set of tRNAs which could
have functioned as a primitive genome Root-Bernstein and Root-Bernstein, 2015 . Moreover, Farias et al.
(2016b) proposed a very parsimonious syncretic model (tRNA core hypothesis) in which some proto-tRNAs played the classical
role assigned to these molecules while others playing those of rRNAs and mRNAs,
a self-recognition between these molecules allowed to obtain proto-proteins.

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 would be too
speculative, but RNA structures having characteristics of ss-tRNA could have
accumulated many advantages in the RNA/protein world. Indeed, a structure in which both start and stop codons are
partially in a stem-loop which constitute basic signals for translation could
be one of the missing links of the RNA world hypothesis. Furthermore, in these proto-tRNAs, 3D structures could act as
initiation and termination signals before the emergence of the standard codons.
Moreover, mRNAs in the form of ss-tRNA or a combination of these several these
molecules they 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”) has been firstly proposed by Ohnishi (1990) . Furthermore,
mt-tRNAs of Kinetoplastida protist, which are nuclear encoded, are imported into the mitochondrion
and circularized mature tRNA molecules are produced probably by the mt-endogenous RNA
ligase activity either in vivo or
during mt-isolation (Aphasizhev
and Karmarkar, 1998) . 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 generate the typical cloverleaf structure with functional
termini (Soma, 2014)
. 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 (ATR49) which potentially codes for at least a peptide of 12 amino acids
(stop codons can be downstream the TAR10). However, the circularization could
be done elsewhere than at levels of nts 72 and 1 and thus TAR10 could no 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 more than 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 (Naoko
and Hiroshi, 2017) . So, 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.

the fusion of tRNA-like mRNA and classical tRNAs 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 proto-tRNAs may also be
selected (Wolf and Koonin, 2007) , it has even
been proposed that the
activity of the
juxtaposed 2?/3?-OHs of the tRNA A76 ribose qualifies tRNA as a ribozyme (Safro and Klipcan, 2013)
and some RNAs (the early tRNA adaptor) must have had the
ability to undergo 3′-aminoacylation. It
has been previously shown that many hairpin structured RNAs bear ribozyme
activity, which catalyzes self-cleavage and ligation reactions (Gwiazda et al. 2012)
. In addition, it remains possible that circular
ss-tRNA with amino acid-anchored structure could be at the origins of tmRNA.
Indeed, two-piece bacterial tmRNAs (e.g., in ?-Proteobacteria) are encoded by a
circularly permuted gene sequence implying that pre-tmRNA is processed and that
the two pieces are held together by non-covalent interactions. Moreover, in
line with an ?-proteobacterial origin of mitochondria, it has been discovered
probable mt-encoded circular permuted tmRNA
genes in the oomycete (water mold) Phytophthora sojae and in the jakobid
Reclinomonas americana (Hafez et al., 2013) . However, it has been hypothesized that a proto-trnA gene could possibly be at the origin of modern tmRNAs (Macé and Gillet, 2016) , at the level of metazoan mt-trnA genes, they are those which 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
was found in Eubacteria and none in Archaea.