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3.11 Why methionine
as the initiating amino acid?

Production of methionine
in the supposed prebiotic chemistry conditions, present no particular
challenges (Kim and
Benner, 2018) . Methionine could be initially incorporated into the primordial
RNA-dependent world as a cofactor because, among others, of its involvement in
methyltransferase activities although spontaneous emergence of S-Adenosylmethionine
in the RNA world is very conceivable (Laurino and Tawfik, 2017) .
In addition, thiol-rich
peptides can play various catalytic roles (Vallée et al., 2017) and compounds with sulfur and thioesters must have played a crucial role in
the origin of amino acid activation and peptide bond synthesis (Jakubowski, 2016) . Furthermore, methionine preferentially interacts
with its codon AUG (Mellersh,
1993) . Therefore,
several studies, not all mentioned here, suggest that methionine and its thiolated
precursor and derivative
molecules would have rapidly interacted with RNAs and play critical roles in
the RNA world to the RNA/protein world transition.

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On the other hand,
if one extrapolates data from current biological systems to proto-cell world,
various data suggest that it should be very advantageous to initiate the
synthesis of proteins with a methionine. For many proteins, the identity of
the first residue that remains after removal of the NH2-terminal
methionine residue has a profound influence on half-life. This is called the N-end rule. Even if it has
differences according to the type of proteins and the organisms, methionine is
one of the few amino acids that can significantly increase the half-life of
proteins (Varshavsky, 1995) . In current organisms, the rapid removal
of initial methionine allows an increased protein turnover that is an adaptive
mechanism, but in the distant past, it could be very advantageous for the
proteins (or only peptides) to remain functional for very long periods and thus
to retain the initiating amino acid. This could also be correlated to the relatively
high biologic half-life of methionine. Moreover, the metabolic cost of methionine synthesis is
highest among the amino acids (Kaleta et al., 2013) ; so, initiating synthesis by this amino acid almost ensuring
that the risk of deficiencies in other amino acids and energy intermediaries
was low.

Already in the RNA
world, the interactions with methionine could have to be numerous, moreover,
the advantage that would be obtained by the integration of this amino acid as
the first residue during the synthesis of the proteins is such that a codon
start coding for this amino acid (or a modified version as N-formylmethionine) might
have appeared very early in the course of evolution. In prebiotic conditions,
selection largely depended on the stability of the molecules. The cloverleaf
tRNAs and the rRNAs (constituting the proto-PTC and moreover being able to
derive from tRNA fusion) are very stable molecules (Megel et al.,
2015) . A mRNA in the form of ss-tRNA or a combination of several
of these last molecules would have been weakly fragile. In the supposedly conditions
of the prebiotic world, methionine could be protected from degradation by, in
part, interacting with the RNA, and under standard conditions of life, it
proved to be a relatively stable molecule and which moreover can stabilize the
proteins when it is the first incorporated residue and also the second. The
origin of codons of initiation attributed to methionine is thus very old and their presences at the
first stages of the RNA/protein world and in some proto-tRNAs can not be

funnel hypothesis and tRNAs

In the
living world, it exists mechanisms in order to avoid that in biological
systems, a molecule similar but not strictly identical (in term of sequence and
therefore of structure level) to another which is functional could interfere in
a given system, but making it inoperative. In the hypothesis called by us “double funnel”, the
first funnel, for which the larger hole is in the top symbolizes the fact that within
a multigene family, all members keep almost identical sequences over very long
periods of time. This suggests the presence active mechanisms allowing to the
sequences that begin to diverge to become again almost all identical. Gene
conversion is one of the mechanisms of this process called concerted evolution
(Duret and Galtier, 2009) .
Contrarily, to avoid that the equivalence principle which could apply between
molecules closely related at the sequence level, the opposite mechanism would
quickly accentuate differences and corresponds to the second funnel for which
the pipe is in the top. The natural mutation rate allows to induce variations
between sequences but it is probably too slow where the situation is critical
as, e.g., after an allopolyploidisation event. To our knowledge, to date no
active mechanisms allowed to quickly induce specifically mutations in
paralogous genes is known but, among others, allopolyploid organisms suggest
their existence, at least there would be a process preventing gene conversions,
indeed, paralog groups of nuclear ribosomal RNA and protein-encoding genes seem
to escape to concerted evolution mechanisms since may be hundreds of millions
of years (Barthélémy et al.,
2006, 2007ab) . Two examples suggest
also that the double funnel hypothesis would also apply to the tRNA world. When trn genes were duplicated
and a substitution occurred in the anticodon as in those specifying tRNA-Leu of
metazoa, this would effectively turn one type of tRNA into the other (Higgs et al., 2003) . As the mt-mutational
rate is frequently high, the two duplicons must
relatively quickly present changes at additional sites; moreover, the final functional
product is the tRNA which can be modified differently by methylation or other
types of process. An example, of accumulation of mutations after a duplication
event can be found in the insect Apis mellifera mtDNA (Cornuet et al., 1991) . Tandem
duplication of a segment encompassing the gene specifying tRNA-Leu gene had
generated an intergenic region whose the putative secondary structure exhibit 3
hairpins and a TLS. Without taking in account the three indels, the two
cloverleaf structures exhibit a level of nt identity of 73% and more than 50%
of the G or C of the trn sequence has
been substituted by A or T. Moreover, a point mutation occurred in both the
anticodon and the ATR49 triplet (in both sequences, there is no TAR10 triplet).
Therefore, the TLS can not interfere with the source tRNA; however, the TLS
might contain an origin of replication and/or transcription (Cornuet et al., 1991) . This is
an example in accordance with the double funnel hypothesis; the duplicated
cloverleaf structure can not give a functional tRNA but has potentially
acquired a new role.


Studies strongly suggest that the tRNA cloverleaf structure unfolded prior to the
appearance of a fully functional ribosomal core, making it one of the
most ancient RNAs of the RNA world (Harish and Caetano-Anollés, 2012;
Fujishima and Kanai, 2014)
or even the oldest (Megel et al., 2015) . Though the “RNA-world” hypothesis is well accepted,
the successive events leading to the emergence of different partners playing a
role in translation and the involvement of tRNAs in this evolution are highly
controversial but a coveted field of research (Bhattacharyya and Varshney, 2016) . However, some
hypotheses as those of the “tRNA
core” (Farias et
al., 2016b) strongly suggest that tRNAs would be at the origin of the
primitive genetic material, and gave rise to mRNA and rRNA, as well as, the
conformational structure of the first proto-ribozymes. The base module being a
pleiofunctional RNA that can adopt, among other things, the cloverleaf
structure which is still found today in various sequences that have no direct
link with translation. Inspired by a sporting saying, it would be possible to
conclude that “one
should not change a winning secondary structure”. In a precellular
context, a molecule with the characteristics of a ss-tRNA because of its small
ORF associated with the cloverleaf structure would have presented a serious
asset. A system based on molecules analogous to current ss-tRNAs, being the key
to start the organization of progenote and cumulating the function of both
mRNAs and tRNAs, they would have been the first molecules on earth to support
non-random protein synthesis.

antiquity of ss-tRNAs can be discussed, it is very likely that the TAR10 (and
especially TAG) triplets had very early to play a critical role in the tertiary
folding at least for certain types of tRNAs. The implication of these tRNAs in
the termination of the translation would be an exaptation in which the first
role was played by a structural signal. The age of the ATR49 triplets is more
problematic. This study traces the origin of these triplets to the first
endosymbioses and then suggests that it would be an apomorphy, a derived
character. Moreover, analyses by taxa and type of tRNA suggest a non-homogeneous evolution. At the beginning of the RNA/protein
world, it has quickly become essential to start peptide synthesis at particular
codons and and initiate protein synthesis with methionine has many advantages. However,
one can not exclude that ATR49 was an ancestral state which would have not been
retained as the size of the intergenic spaces increased. Analyses of known
tRNAs of ?-proteobacteria and cyanobacteria suggest that in the organelles,
ATR49 triplets would have been selected as and when the size of their genomes

It has been hypothesized that genomes
may be under increased pressure to minimize their size and that overlaps would
only be the consequence of this selection pressure (see, Johnson and Chisholm, 2004) . However, several features strongly suggest that overlapping genes are not a direct mechanism to
substantially reduce genome size. Gene overlaps allow mtDNA genome
compaction while avoiding the loss of tRNA genes (Doublet et al., 2015) . Nevertheless, overlaps may allow a more efficient control in
the regulation of gene expression, the regulatory pathways are simplified and
the number of proteins (and genes) required decreases (Johnson and Chisholm, 2004) . Among others, short
antiparallel overlaps may be involved in antisens regulatory mechanisms. Consequently
genomes with compact sizes enable a putatively less flexible but more efficient

The selection of tRNAs
had to be done mainly on two seemingly opposite criteria, stability and
plasticity, making it a kind of Swiss army knife of the RNA world. This explains that beyond their
central role in protein synthesis, tRNAs have many other crucial functions. To
date, it can be hypothesized that ss-tRNAs might play roles in the regulation
of gene expression, stress responses and metabolic processes. Indeed, in
silico analyses allowed to speculate that several overlapping sequences may
code simultaneously for mRNAs and tRNAs in most of the metazoan
mt-genomes. These overlaps can have a variable (sometimes large) number
of nts (see above); however, when annotating their genomes, several authors voluntary
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 the
obtaining of tRNAs and translation of mRNAs and/or the development of repair
system to keep the two genes functional due in some cases of a co-evolution of several hundred million years. 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, it must
also be added internal codons from the ATR49. To date, it is unclear what
biochemical mechanism would allow alternate transcription leading to the
complete tRNA rather than to the mRNA. However as shorter and expanded proteins
can be functional and that by various processes including editing, it can be
the same for incomplete tRNAs and despite any experimental evidence, a role,
certainly regulator, of TAR10 and ATR49 triplets is very likely. So as in silico analysis shows here its
limitations, experimental testing of this hypothesis is needed. This will
require analysis of the processed bicistronic transcripts (trn/protein-encoding or the contrary). Moreover, even if mt-trn genes are most often expressed at
very low levels (Doublet
et al., 2015) , only direct sequencing of tRNAs will allow the
validation of transcription and epitranscriptomic maturation and can
pinpointing the type of nt modifications including the positions that are
edited post-transcriptionally. Purified native, or even synthetic, tRNAs should
also be tested for their in vitro activity
to confirm the functionality of aberrant transcripts. Finally, similar
experiments must be made on the flanking mRNAs and of
their products. If as we think ss-tRNAs could play regulatory roles, initially
experiments should be done under stress and non-stress laboratory conditions.

In this study, the bias
for metazoan mtDNA does not allow for a complete picture of variation in the
entire eukaryotic world, e.g., it also must be take into careful consideration of protist mt-genomes. Special
attention should also be paid to the non-Watson-Crick pairings that could be established by nts
of TAR10 and ATR49 triplets and put this into perspective with structural
characteristics of tRNAs, including the size of V-R. Taking in account the
possible presence of TAR10 and ATR49 triplets in the algorithms use to predict
tRNAs could be a great help for annotations of mt-genomes, i.e., this would
reduce the number of false positives and negatives and more accurately
determine tRNA termini. However, the type of tRNA, taxa and genomic system
should be taken into account.

The mtDNA plays a central role in
apoptosis, ageing and cancer Ladoukakis and Zouros, 2017 ; moreover, mt-diseases are among
the most common group of inherited metabolic disorders and neurological
disorders (Sun and Wang,
2017) . In addition, as new functions and new mechanisms of action of
tRNAs are continuously being discovered (Barciszewska et al., 2016) and as ss-trn genes could affect the cellular
dynamic during normal and stress conditions which could lead to pathological
disorders, investigations should be undertaken in order to know all the
subtlety of the potential mechanisms of action and regulation of these genes and

of Interest

The authors declare that the
research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.

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