The mechanisms of codon reassignments in mitochondrial genetic codes

“The rRNA sequences were aligned initially using ClustalX ( Thompson et al. 1997 ).”

“Dictyostelium discoideum and Acanthamoeba castellanii are thought to be more distant relatives of this group that diverge before the split of fungi and metazoa ( Baldauf et al. 2000; Van der Peer et al. 2000; Lang et al. 2002).”

“The identity elements and antideterminants of tRNAs are key aspects of the molecules that play a crucial role in the proper aminoacylation and prevention of misacylation of a tRNA (Giegé et al. 1998).”

“The branching order of the species within Ascomycota agrees with Bullerwell et al. (2003a) and Kurtzman and Robnett (2003), and that within Chytridiomycota agrees with Bullerwell et al. (2003b).”

“The grouping of fungi and metazoa, proposed by Baldauf & Palmer (1993), is supported by all the studies cited in the previous paragraph.”

“Winchell (2002) and Halanych (2004) for the relationship between the animal phyla.”

“(v) Ascomycota (group containing Yarrowia and relatives) Knight et al. (2001a) list a single change in the ancestor of Metazoa, Acanthamoeba and Fungi.”

“xa, the relationships between groups in the very earliest parts of the eukaryotic tree have proved difficult to resolve (Lang et al. 1999; Baldauf et al. 2000; Van der Peer et al. 2000; Baldauf 2003; Philippe et al. 2004). Our aim here was not to resolve these early branches, but to obtain trees that would allow for a…”

“Dictyostelium discoideum and Acanthamoeba castellanii are thought to be more distant relatives of this group that diverge before the split of fungi and metazoa (Baldauf et al. 2000; Van der Peer et al. 2000; Lang et al. 2002).”

“A reviewer of this paper pointed out that Osawa and Jukes (1989) , the original proponents of the CD model, insisted that, according to their theory, every single occurrence of the codon must disappear from the genome before the reassignment occurred.”

“This mechanism is expected in the theory due to the analogy of the problem with compensatory mutation theory (Kimura, 1985; Higgs, 1998, 2000).”

“This mechanism is expected in the theory due to the analogy of the problem with compensatory mutation theory (Kimura 1985; Higgs 1998 2000).”

“We have followed Mallatt & Winchell (2002) and Halanych (2004) for the relationship between the animal phyla.”

“Telford et al. (2000) have also discussed these two alternative scenarios in a parsimony treatment, however they did not consider the position of the tRNA-Ile(CAU) gene loss, or the fact that there are several independent modifications to the tRNA-Met, both of which are essential points in our…”

“Amoebidium parasiticum and Monosiga brevicollis are thought to be the closest unicellular relatives of the metazoa, and are therefore also included in this group.”

“(vi). Rhodophyta (Chondrus, Porphyra): Fig. 2 and Burger et al. (1999) .”

“We attempted to answer this question with the kinetoplastids, Trypanosoma brucei and Leishmania tarentolae, which possess no mitochondrial tRNAs and where import is known to occur (Simpson et al. 1989; Hancock and Hajduk 1990; Schneider andMarechal-Drouard 2000).”

“4), which suggests that it was lost after the divergence of the phylum Porifera from other metazoan phyla.”

“It is known that many protein genes formerly present on the mitochondrial genome have been transferred to the nuclear genome (Blanchard and Lynch, 2000).”

“We believe that there is a good case that positive selection was important in the establishment of the canonical code (Freeland et al. 2003; Ardell & Sella, 2002) and that this accounts for the non-randomness and apparent optimization of the code.”

“This was proposed by Schultz and Yarus (1994, 1996).”

“2 and Turmel et al. (1999).”

“and Burger et al. (1995). (iii).”

“We attempted to answer this question with the kinetoplastids, Trypanosoma brucei and Leishmania tarentolae, which possess no mitochondrial tRNAs and where import is known to occur (Simpson et al. 1989; Hancock and Hajduk 1990; Schneider and Marechal-Drouard 2000 ).”

“For the arrangement of the species within the Stramenopiles, we used the results of a much larger scale study with the nuclear small subunit rRNA gene ( Van der Peer et al. 1996 ).”

“tRNA is imported to the mitochondrion and then undergoes a base modification so that it can translate both UGA and UGG (Alfonzo et al. 1999).”

“Now that many complete genomes of organisms and organelles are available, there is ample evidence that the genetic code is not as universal (Knight et al. 2001a; Yokobori et al. 2001; Santos et al. 2004) as previously believed (Crick 1968).”

“Kurzman & Robnett (2003) and Diezmann et al. (2004) have considered the phylogeny of these groups using nuclear genes, and find that Candida, Kluyveromyces, Saccharomyces and Pichia are all paraphyletic.”

“It has previously been shown that codon usage patterns differ between the intronic ORFs and the other genes (Bullerwell et al. 2003; Talla et al. 2005).”

“However, Massey et al. (2006) have now shown that there is selection against the ambiguous CUG codons that causes them to become very rare in their original Leu positions.”

“Nedelcu et al. (2000) identified a tRNA-Arg(UCU) that should interact with AGA, and a tRNA-Ile(UAU) that should interact with AUA.”

“However, it has been suggested that ambiguous translation may be favorable (Santos et al. 1999).”

“This was proposed by Schultz and Yarus (1994, 1996).”

“Reassignments of UAG stop codons Two cases of reassignment of UAG from Stop to Leu are known: one in the two chytrids, Rhyzophidium and Spizellomyces (Fig.”

“Seif et al. (2005) argue that this has occurred in Mortierella and Schizosaccharomyces, where there is evidence of a small number of UGA acting as Trp codons that are translated very inefficiently by the standard Trp tRNA with CCA anticodon.”

“(v) Ascomycota (group containing Yarrowia and relatives) Knight et al. (2001a) list a single change in the ancestor of Metazoa, Acanthamoeba and Fungi.”

“This point has been suggested previously (Saks et al. 1998), but as far as we are aware, there is no direct evidence that this occurs.”

“It has been suggested that deletion of a tRNA gene might be favored since it reduces the total length of the genome and hence allows more rapid replication ( Andersson and Kurland 1991, 1995 ).”

“Stramenopiles and alveolates are two groups that are thought to be monophyletic according to Wolters (1991) and Saunders et al . (1995) .”

“Although there is general agreement on the definitions of the major eukaryotic taxa, the relationships between groups in the very earliest parts of the eukaryotic tree have proved difficult to resolve (Lang et al. 1999; Baldauf et al. 2000; Van der Peer et al. 2000; Baldauf 2003; Philippe et al. 2004).”

“Multiple sequence alignments were carried out using T-COFFEE ( Notredame et al. 2000 ) and the alignment was edited to remove columns having more than 20% gaps.”

“These authors also show that the unmodified CAU anticodon can translate both codons if the base in the 37th position of the tRNA is t 6 A (N 6 - threonylcarbamoyladenosine) instead of A (see also Kuchino et al. 1987).”

“The codon usage of Chlamydomonas reinhardtii is markedly different from C. eugametos (Table 5 and Denovan-Wright et al. 1998).”

“It has previously been shown that codon usage patterns differ between the intronic ORFs and the other genes (Bullerwell et al. 2003; Talla et al. 2005).”

“Also in some echinoderms, the mutation of the base immediately adjacent to the 5’ end of the anticodon from a U to a C seems to help the GUU anticodon to recognize AAA (Castrasena et al. 1998).”

“floridae (Boore et al. 1999) and Epigonichthys maldivensis (Nohara et al.”

“In C. glabrata, Koszul et al. (2003) conclude that AUA is associated with Met based on comparison of C. glabrata sequences with S. cerevisiae sequences from which they find that 8 out of 16 AUA codons in C.glabrata appear at conserved Met sites encoded by AUA in S. cerevisiae.”

“The corresponding tRNA-Arg is also deleted in this species (Kerscher et al. 2001).”

“In the case of the tRNA-Asn(GUU), mispairing with AAA is prevented by post-transcriptional modification of the wobble position G to Queuosine (Q), which allows pairing with U and C but inhibits pairing with A (Morris et al. 1999).”

“) Another study suggesting that ambiguous translation is deleterious is that by Kim et al. (2000), who studied many examples of anticodon mutations in tRNA-Phe from E.”

“For this reason, wobble position U’s are often modified when they occur in tRNAs for two-codon families only (Yokobori et al. 2001).”

“There is evidence for this in Bacillus subtilis (Lovett et al. 1991, Matsugi et al. 1998), where UGA is ambiguous.”

“coli (Muramatsu et al. 1988) that the C at the wobble position undergoes a posttranscriptional base modification to K(2)C (lysidine), which allows this tRNA-Ile to pair with the AUA codon.”

“(xi) Bacillariophyta (Skeletonema, Thalassiosira) - Fig 3 and Ehara et al. (2000) (xii) Kinetoplastida (Trypanosoma, Leishmania) - Inagaki et al. (1998).”

“We interpret the decoding of AUA as Ile in Platyhelminths and Echinoderms/Hemichordates as indicating that an appropriate change in the tRNA-Met did not occur, and that AUA has always been Ile in these lineages.”

“We believe that there is a good case that positive selection was important in the establishment of the canonical code (Freeland et al. 2003; Ardell & Sella, 2002) and that this accounts for the non-randomness and apparent optimization of the code.”

“In the remaining Metazoa (i.e. Bilateria) the loss of the tRNA-Arg(UCU) gives rise to subsequent changes in the genetic code.”

“1999) and Epigonichthys maldivensis (Nohara et al. 2005), which also show no evidence of reassignment of AGA to Gly.”

“This mechanism is expected in the theory due to the analogy of the problem with compensatory mutation theory (Kimura, 1985; Higgs, 1998, 2000).”

“Since AGA was a moderately frequent codon for Ser prior to the reassignment, and since %A is high, we cannot argue that mutation pressure caused the disappearance of AGA and replacement by AGR prior to the change.”

“Many of the closest relatives to the species where UGA is reassigned have particularly low usage of UGA, e.g. Allomyces and Rhizopus, close relatives of the Basidiomycota, have 0; Cyanidioschyzon, close relative of Chondrus and Porphyra, has only 2; Scenedesmus and Chlamydomonas, close relatives of Pedinomonas, have 1…”

“Talla et al. (2005) argue that AUA is assigned toMet inK.”

“Before codon reassignment, tRNA-Met also has a CAU anticodon, which pairs with AUG only, but modification of the wobble-position C allows it to pair with both AUA and AUG ( Tomita et al. 1999b ).”

“A second similar case is the reassignment of UCA from Ser to Stop in Scenedesmus obliquus (Kück et al. 2000).”

“In the simulations of codon reassignment (Sengupta and Higgs 2005) it was possible to distinguish a few cases where this occurred.”

“The complete genome of Emiliana (Sanchez-Puerta et al. 2004) shows that it also possesses the reassignment.”

“Swire et al. (2005) did not distinguish between AI and UC.”

“Alternatively, in some Echinoderms (Matsuyama et al. 1998) and Molluscs (Tomita et al. 1998) a base modification from G to m 7 G also permits it to decode the entire AGN block as Ser.”

“(xi) Bacillariophyta (Skeletonema, Thalassiosira) - Fig 3 and Ehara et al. (2000) (xii) Kinetoplastida (Trypanosoma, Leishmania) - Inagaki et al. (1998).”

“It has previously been shown that codon usage patterns differ between the intronic ORFs and the other genes (Bullerwell et al. 2003; Talla et al. 2005).”

“This was originally proposed by Osawa and Jukes (1989, 1995) .”

“The fact that the tRNA-Thr(UAG) appears only in species where the tRNA-Leu(UAG) is absent suggests that the new gene evolved directly from the old tRNA-Leu(UAG) gene.”

“In mitochondrial genomes, the two strands of the genome are not equivalent, the four base frequencies are all different, and it is not true that πC = πG and πA = πU (see Urbina et al. 2006).”

“These species possess an unusual tRNA-Thr with anticodon UAG (Sibler et al. 1981; Osawa et al. 1990) and the usual tRNA-Leu(UAG) is not found in the genome.”

“There is evidence for this in Bacillus subtilis (Lovett et al. 1991, Matsugi et al. 1998), where UGA is ambiguous.”

“Another study suggesting ambiguous translation is deleterious is that of Kim et al. (2000), who studied many examples of anticodon mutations in tRNA-Phe from E. coli.”

“The three theories explaining codon reassignment are not exclusive considering that the ‘‘ambiguous intermediate’’ stage can be preceded by a significant decrease in the content of GCrich codons, so that codon reassignment might be driven by a combination of evolutionary mechanisms (56), often under the pressure for g…”

“changes, for reviews see [24–26]).”

“This tRNA-Trp mutation has also been observed in mitochondrial genomes that have the UGA StopRTrp recoding [24].”

“Splitting a two-codon family into separate single codons is possible in the case of Met and Trp codons, but this does not increase the number of amino acids encoded in either case; and, in many mitochondrial codes, Met and Trp both have two codons (Sengupta et al., 2007).”

“, 2009), as well as in mitochondria (Sengupta et al., 2007).”

“Remarkably, these mitochondrial protein-coding genes are translated using genetic codes different from the standard (nuclear) genetic code in many living eukaryotes 10 .”

“We have shown that the codon disappearance mechanism is a good explanation of some (but by no means all) the codon reassignments that occur in mitochondrial genomes [11].”

“This lack of phylogenetic signal may be caused by the strong A þ T bias of mitochondrial genomes or by multiple independent changes in the mitochondrial genetic code along the evolution of eukaryotes (such process is known to occur at a higher rate in the mitochondrial genome than in the nuclear genome; e.g., Knight e…”

“Still, any adaptive nature of the later changes in the genetic code has continued to be doubted, because there was apparently little to be gained from code alterations in modern organisms (9).”

“The changes corresponding to the gain and loss events are known in a large number of other cases too (Sengupta et al. 2007); thus, the biochemical details responsible for codon reassignments in the modern phase are fairly well understood.”

“These mechanisms fit the general ‘gain and loss’ framework, where ‘gain’ refers to acquisition of a new tRNA specificity, typically resulting from duplication of a tRNA gene, whereas ‘loss’ is elimination of a tRNA specificity, typically via deletion [11,23,24].”

“However, deviations in sense and nonsense codon use are found in bacteria, archaea, and both nuclear and organellar eukaryotic genomes (2, 3).”

“Loss of the Q modification and subsequent GA pairing is relevant with respect to the origin of several variant genetic codes in mitochondrial genomes (Yokobori et al. 2001; Sengupta et al. 2007).”

“While codon disappearance seems likely for the stop-to-sense codon changes of mitochondria, it is unlikely that the original codon would disappear entirely in sense-to-sense changes (Sengupta et al., 2007).”

“Simulation study on codon reassignment [54]suggested that the pathway of codon reassignment in favor of either “codon capture” or “ambiguous codon” depends on the initial conditions such as genome size and number of codons in interest.”

“This is why it is easier to detect codon reassignments that include stop codons, and might be the reason why most detected variations in genetic codes involve stop codons (Massey and Garey, 2007; Sengupta et al., 2007; McCutcheon et al., 2009; McCutcheon, 2010; Turmel et al., 2010).”

“All mitochondrial protein-coding genes commence with a classical methionine codon (ATG) and terminate with TAA, the preferred fungal mitochondrial termination codon [31].”

“…that the H. sapiens mitochondria with 22 tRNA species must gain some flexibility (each tRNA decodes two or four codons) in the decoding process (20–22) without loosing fidelity in contrast to E. coli with 45 tRNA species (or 41 anticodons) and H. sapiens cyto-plasms with 46 tRNA species (28,29).”

“These results offer empirical substantiation of previous models on stop codon reassignment (Osawa and Jukes 1989, 1995; Andersson and Kurland 1991; Sengupta and Higgs 2005; Sengupta et al. 2007).”

“Release factors 7,25 and tRNAs 26,27 with altered specificities suggest it is feasible to engineer attenuated UGA recognition, mitigating translation factor competition to free TGA alongside TAG for reassignment.”

“In Kluvyeromyces lactis, the mitochondrial genome lacks both CUN codons and any tRNA reading such codons (7).”

“Such avoidance of certain codon families would facilitate the evolutionary loss of the associated tRNA [33-35], although it is not always clear whether the avoidance is the cause or the consequence of the loss of the associated tRNA.”

“Unique features include RNA-poor mitoribosomes [25], partial stop codons in some transcripts that are completed by polyadenylation [21, 26], bicistronic transcripts [27], and unique genetic code [28].”

“Such avoidance of certain codon families would facilitate the evolutionary loss of the associated tRNA (Higgs et al. 2003; Sengupta and Higgs 2005; Sengupta et al. 2007), although it is not always clear whether the avoidance is the cause or the consequence of the loss of the associated tRNA.”

“Authors' response: There are a number of alternative genetic codes, for example in Candida albicans [38] and the ciliates Tetrahymena thermophila and Paramecium tetraurelia [39], not to mention in mitochondrial genomes [37].”

“Arg-tRNAUCU gene, which has been proposed as the initial step for these reassignments [68].”

“Full list o f a u th o r inform ation is available a t th e e n d o f th e article Two main models have been proposed to explain the evo­ lutionary changes in the genetic code [reviewed in [4-7]].”

“lactis; (Sengupta et al. 2007)].”

“According to previous studies that tried to explain the codon reassignments in mt genetic codes of eukaryotes, several mechanisms have been suggested to explain these alterations (Sengupta et al., 2005; Sengupta et al., 2007; Sengupta and Higgs, 2015).”

“Interestingly, codon reassignments are particularly frequent in metazoan mitochondria (Figure 1), where more than 10 variants have been described (Knight et al. 2001; Abascal et al. 2006a; Sengupta et al. 2007).”

“tionary scenario, which was based mainly on theoretical considerations and included a temporary ambiguous decoding system, was initially proposed by Schultz and Yarus (the ‘ambiguous intermediate’ theory; Schultz and Yarus, 1994; 1996) and further developed by others to explain the emergence of TilS in bacteria (Silva…”

“Stramenopiles universally lack mitochondrial-encoded threonine tRNA [41], contain a fused version of the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase [42, 43], and have relocalized the ATP-yielding part of glycolysis from the cytosol to the mitochondria…”

“For example, the UGA codon in mitochondria of many eukaryotes (other than land plants) codes for tryptophan rather than a stop (7); AGR codons (R=A or G) in inchordata mitochondria code for glycine rather than arginine (8); and CUN codons (N=A, U, G or C) in yeast mitochondria code for threonine instead of leucine (9).”

“AUA(Ile ! Met) has been described in the mitochondrial genome of yeast, metazoa, and Xanthophyceae (Ehara et al. 1997; Yokobori et al. 2001; Sengupta et al. 2007).”

“A connection between alterations of tRNA identity by RNA editing and variation of the genetic code was discussed in (12,22).”

“More recently, reassignment scenarios have been discussed within an extended gain– loss framework of codons and tRNAs, pointing out that it is necessary to consider the details of each case carefully (11).”

“This enrichment is effectuated by alterations in the mitochondrial genetic code that have occurred several times independently during the Cambrian radiation [26,27].”

“In addition, the structure of the genetic code implies a further mechanism against frameshifted translation.”

“For mitochondrial code variants this is recently described in [8].”

“Evolutionary emergence of these alterations is driven by genome reduction and mutational bias toward A?T residues, which are involved in the replacement of UAG(stop) by UAA and the emergence of UGA(Trp) from UGG(Trp) (Osawa and Jukes 1989; Sengupta et al. 2007).”

“…of how such codes could evolve, and the basic conclusion is that random events, like the disappearance of a codon, loss of a tRNA gene, or mutations in a tRNA gene that allow it to recognize other codons, drive this comparably modern evolution (Sengupta and Higgs 2005, Sengupta et al. 2007).”

“…fungi (3 times independently), chlorophytes, rhodophytes, stramenopiles (twice independently), kinetoplastids, diatoms, prymnesiophytes (surveyed in Sengupta et al. 2007), chlorarachniophytes (Inagaki et al. 1998) and prasinophytes (Turmel et al. 1999, 2010) Glutamine UAA and UAG Tetrahymena…”

“It should be noted that we do not attempt to explain the original emergence of the Standard Code itself or the codon reassignments during emergence of alternate codes (extensive studies can be found in Crick 1968; Ardell 1998; Freeland et al. 2000; Silva et al. 2004; Sengupta and Higgs 2005; Sengupta et al. 2007), or…”

“…lead to a reversion to the standard genetic code, as seems to be the case of AUA decoded normally as isoleucine in mitochondria of certain bilaterian invertebrates despite the fact that AUA encodes methionine in most bilaterians and is likely the ancestral state for the whole group 23,128 .”

“Almost all eukaryotes in which UGA is decoded by tRNA have the UCA anticodon [13].”

“Two main mechanisms have been proposed to explain how they may have evolved despite the large fitness cost that is expected to be associated with the modification of a codon [9,23,24]: through an ambiguous intermediate state and through the reassignment of a temporarily unused codon.”

“Further, several methanogenic archaea rewire the amber stop codon into a signal for incorporation of the 22nd amino acid, pyrrolysine (Pyl) (13,14), whereas the anomalous recognition of the AUA codon by tRNAMet alters the meaning of this particular triplet from isoleucine (Ile) to methionine (Met) in the mitochondria…”

“Simulation studies on codon reassignment (Sengupta et al. 2007) suggest that the pathway of codon reassignment in favour of either “codon capture” or “ambiguous codon” depends on initial conditions such as genome size and number of codons involved.”

“After a long debate on the origin of the modern genetic code variants [50,51], there is compelling evidence now to indicate that deviant genetic codes like those in animal mitochondria primarily evolve to match the amino acid requirements of the encoded proteins [18,51,52] rather than having their…”

“For instance, the mitochondrial code differs from the conventional code, shedding light on the coevolutionary dynamics between mitochondria and host cells [37].”

“Sengupta et al. (2007), for example, assume that sense-to-sense codon reassignment is deleterious in its initial stages and is followed by a lengthy period of adjustment in which an initially maladapted genome adapts to the new code.”

“The reappearance of UGA with the new meaning as a tryptophan codon in distantly related chlorophytes is then an exemplar manifestation of the codon disappearance model of codon reassignment [54].”

“The Codon Capture, also termed the Codon Disappearance theory, proposed by Osawa and Jukes in 1989, postulates that genetic code alterations are the result of neutral changes associated with the GC/AT content balance [35–38].”

“8, Moreover, during translation, mRNA triplets may, in some cases, have an additional meaning than they have according to the genetic code.”