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Putative anticodons in mitochondrial tRNA sidearm loops: PocketknifetRNAs?

Hervé Seligmann a,b,n,1Q1

a National Natural History Museum Collections, The Hebrew University of Jerusalem, 91904 Jerusalem, Israelb Department of Life Sciences, Ben Gurion University, 84105 Beer Sheva, IsraelQ3

H I G H L I G H T S

� tRNA sidearm loops might functionas anticodon loops.

� Putative sidearm anticodons coe-volve with codon usages.

� Enlarged sidearm anticodons coe-volve with predicted tetracodonnumbers.

G R A P H I C A L A B S T R A C T

tRNA pocketknife hypothesis

a r t i c l e i n f o

Article history:Received 29 May 2013Received in revised form15 August 2013Accepted 26 August 2013

Keywords:tRNA synthetaseMisacylationLoop–loop interactiontRNA L-shapeCloverleaf secondary structure

a b s t r a c t

The hypothesis that tRNA sidearm loops bear anticodons assumes crossovers between anticodon andsidearms, or translation by expressed aminoacylated tRNA halves forming single stem-loops. Only thelatter might require ribosomal adaptations. Drosophila mitochondrial codon usages coevolve withsidearm numbers bearing matching putative anticodons (comparing different codon families in onegenome, macroevolution) and when comparing different genomes for single codon families (micro-evolution). Coevolution between Drosophila and yeast mitochondrial antisense tRNAs and codon usagespartly confounds microevolutionary patterns for putative sidearm anticodons. Some tRNA sidearm loopshave more than seven nucleotides, putative expanded anticodons potentially matching quadrupletcodons (tetracodons, codons expanded by a fourth silent position, forming tetragenes (predicted byalignment analyses of Drosophila mitochondrial genomes)). Tetracodon numbers coevolve withexpanded tRNA sidearm loops. Sidearm coevolution with amino acid usages and tetragenes occurs forputative anticodons in 5′ and 3′ sidearms loops (D and TΨC loops, respectively), are stronger for theD-loop. Results slightly favour isolated stem-loops upon crossover hypotheses. An alternative hypothesis,that patterns observed for sidearm ‘anticodons’ do not imply translational activity, but recognitionsignals for tRNA synthetases that aminoacylate tRNAs, is incompatible with tetracodon/tetra-anticodoncoevolution. Hence analyses strengthen translational hypotheses for tRNA sidearm anticodons, tetra-genes, and antisense tRNAs.

& 2013 Published by Elsevier Ltd.

1. Introduction

The concept of modularity has been applied to developmentalprocesses (Raff, 1996), evolutionary patterns (Wagner, 1996;Wagner and Altenberg, 1996), and their ‘evo-devo’ integration(Bolker, 2000). This concept can also be applied at the level of

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Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/yjtbi

Journal of Theoretical Biology

0022-5193/$ - see front matter & 2013 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.jtbi.2013.08.030

n Correspondence address: National Natural History Museum Collections, TheHebrew University of Jerusalem, 91904 Jerusalem, Israel.

E-mail address: varanuseremius@gmail.com1 Current address: Unité de Recherche sur les Maladies Infectieuses et

Tropicales Émergentes, Faculté de Médecine, URMITE CNRS-IRD 198, Universitéde la Méditerranée, Marseille, UMER 6236, France.

Please cite this article as: Seligmann, H., Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs? J. Theor. Biol.(2013), http://dx.doi.org/10.1016/j.jtbi.2013.08.030i

Journal of Theoretical Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

(sub)molecular structures, such as the tRNA’s secondary structure,which consists of four arms, among which three are stem-loopstructures that might be considered each as modules, or as twomodular tRNA halves. Some evidence supports the latter, tRNA halfmodule hypothesis: genes templating for tRNAs frequently includeconserved introns, sometimes template for split tRNA halves,which are occasionally permuted (Randau and Söll, 2008). Thisindicates that fusion of tRNA parts created modern tRNAs(Fujishima et al., 2008, 2009). Split tRNAs, as well as tRNAs withintrons might be a derived situation (Fujishima et al., 2010).However, it seems most parsimonious to consider these asancestral states (Di Giulio 2008a, 2008b, 2009, 2012), suggestingpolyphyletic origins of tRNAs. Polyphyly of tRNA halves fits mostparsimoniously available data (Di Giulio, 1992, 1995, 1999, 2004,2006, 2013), and the fact that pseudocodonic usages of the 3′ halfmatches that of protein coding genes, while that of the 5′ half,does not (Michel, 2012, 2013). According to this evolutionaryscenario, an ancestral, stem-loop hairpin (the module) duplicated,fused, forming tRNAs as we know them today. The tRNA mole-cule’s modular structure is not an isolated case as a similarduplication and fusion scenario fits the evolution of 5S rRNA(Di Giulio, 2010).

According to this approach, the general structure of theancestral stem-loop hairpin might have resembled modern nema-tode armless tRNAs. Armless tRNAs are derived from ‘normal’tRNAs but probably secondarily re-evolved translationally activesmall (t)RNAs (Jühling et al., 2012). These resemble aminoacylatedRNA hairpins templated by the light strand replication origin(Yu et al., 2008). D- and TΨC-arms of modern tRNAs are putativelyhomologous to the presumed ancestral tRNA halves.

It is difficult to corroborate ancestral origin hypotheses ofmodern molecules. However, if one assumes that similar mole-cules function in modern organisms, one can at least indicate thatthe hypothetical ancestral state was functional. Hence evidence forsuch translational activity by tRNA halves could corroborate thepolyphyly of tRNA origins by fused 5′ and 3′ halves. Explicitly, thishypothesis predicts that D- and TΨC-sidearm loops function asanticodons. This hypothesis, if corroborated by bioinformaticsanalyses, implies potential experimental confirmation becausethese stem-loop hairpins are putatively still active in proteintranslation of modern mitochondria. Mitochondria have beenchosen for these analyses because preliminary examinations showthat for most nucleus-encoded tRNAs (from Archeans, Bacteria andEukaryota), putative anticodons in tRNA sidearm loops are almostexclusively matching codons for leucine and serine. This observa-tion is not yet understood but deserves further examination at anulterior point. Mitochondria, bearing the greatest variation insidearm loop anticodon identities, seem most adequate for a firstexploration of the tRNA sidearm loop anticodon hypothesis, alsotermed pocketknife tRNA hypothesis because pocketknives havemultiple exchangeable ‘heads’ enabling polyvalent functions.

This hypothesis assumes that the confirmed mitochondrialtRNA pool is incomplete. This is in line with observations ofvarious tRNA import mechanisms (Schneider and Maréchal-Drouart, 2000) from the cytosol into mitochondria (Tarassovet al., 1995; Tarassov and Martin, 1996; Rubio and Hopper, 2011),and which coevolve with mitochondrial codon usages (Salinaset al., 2012).

Here, the potential anticodons in the lateral arms of the 22tRNAs from 15 complete mitochondrial genomes of various Dro-sophila species are compared. A less extensive analysis of thetRNAs in mitochondrial genomes of two fungi is also presented.Analyses suggest that the sidearm loops function as anticodonloops because numbers of anticodons matching one codonfamily coevolve with codon usages, and because predicted tetra-gene lengths (genes putatively formed by tetracodons, codons

expanded by one silent nucleotide) coevolve with numbers ofexpanded tRNA sidearm loops (loops with more than sevennucleotides).

These results in Drosophila mitochondria converge with similarcomparative analyses of mammalian mitochondrial genomes andtRNAs that suggest anticodon loop activity by mitochondrial tRNAsidearm loops for a sample of widely diverging mammal species(chosen across various mammal groups, from primates, rodentsand carnivore, among others, Seligmann, 2013a). Hence analyses ofDrosophila mitochondria, beyond independent confirmation ofresults from mammals, enable to examine the phenomenon at ashorter evolutionary scale, that of variation among closely relatedDrosophila species. Results are compatible with both the ancestraltRNA half hypothesis and that of exchanges between anticodonarms and sidearms events resembling chromosomal crossingovers,though the former might be most parsimonious and slightly bettersupported by results. They also strengthen the hypothesis thattetragenes are functional protein coding genes.

2. Results

2.1. Sidearm anticodons and genome-wide amino acid usages:Comparisons between cognates

A preliminary analysis uses the mitochondrial tRNA databasemitotRNAdb (http://mttrna.bioinf.uni-leipzig.de/, Jühling et al.,2009, accessed 20 of May 2013) to detect all mitochondrial tRNAswith sidearm loops with exactly seven nucleotides among the30,525 available, which yields 5100 (16.7%) and 19,094 (62.6%) forthe D and TΨC loops, respectively. The numbers were brokenaccording to the amino acid matching the putative anticodonaccording to the genetic code. Fig. 1 plots the numbers of putativemitochondrial tRNA sidearm anticodons as a function of theknown amino acid usage frequencies for a large pool of proteins(Arqués and Michel, 1996), separately for D and TΨC loops.

Overall, sidearm anticodon numbers increase with amino acidusages, for D and TΨC loops separately as well as pooled according

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Fig. 1. Number of sidearm loops with putative anticodons matching specificcognate amino acids in mitochondrial tRNAs from tRNAdb database (accessedMay 2013) as a function of usages of codons matching these anticodons inGenbank’s proteins, as presented by Arqués and Michel (1996). Circles indicatenumbers of anticodons in D-sidearm loops (continuous regression line), trianglesfor TΨC-loops (discontinuous regression line). Filled symbols are for outliersexcluded from a secondary analysis designed to avoid outlier effects (see text).

H. Seligmann / Journal of Theoretical Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎2

Please cite this article as: Seligmann, H., Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs? J. Theor. Biol.(2013), http://dx.doi.org/10.1016/j.jtbi.2013.08.030i

to parametric (Pearson correlation coefficients r¼0.50, P¼0.012;r¼0.31, P¼0.09; r¼0.40, P¼0.043, one tailed tests) and the morerobust non-parametric tests (Spearman rank correlation coeffi-cient rs¼0.57, P¼0.0068; rs¼0.17, P¼0.23; rs¼0.373, P¼0.052,one tailed tests). This result could be due to some outliers withvery high numbers of anticodons. For D sidearms, anticodons forLeu are 34% of all D sidearm anticodons and are the only onesnumbering above 1000, the next highest value is for Thr (4 9 9).For TΨC sidearms, anticodons for Leu, Phe and Ser are outliers,numbering each above 3000 and representing 85.6% of all TΨCsidearms, anticodons. Hence correlation analyses are repeatedafter excluding these outliers, which yields qualitatively similarresults as when analysing the complete dataset (r¼0.63,P¼0.0019; r¼0.27, P¼0.15; r¼0.45, P¼0.035, one tailed tests;rs¼0.45, P¼0.0268; rs¼0.05, P¼0.423; rs¼0.418, P¼0.0518).Hence this preliminary test of the working hypothesis yieldstentatively positive results.

2.2. Sidearm anticodons and genome-wide amino acid usages:Comparisons between cognates for Drosophila

However, the amino acid usages are mainly derived fromcytosolic proteins, which differ in amino acid contents frommitochondrion-encoded proteins, as the later are to a muchgreater extent associated with membranes. Hence these analyseswere repeated, using all tRNAs from the complete mitochondrialgenomes of 15 Drosophila species available in GenBank (and usedin previous studies, Seligmann, 2012a, 2012b). These wereextracted using the mitochondrial model of tRNAscan SE (http://lowelab.ucsc.edu/tRNAscan-SE/, Lowe and Eddy, 1997; Schattneret al., 2005).

The secondary structure of each of these tRNAs, as predicted bytRNAScan SE, was visually examined and several types of informa-tion were recorded for each of the lateral arms (stem and loop):the size of the sidearm loop, the size of the sidearm stem, its GCcontents, and, if the loop counts seven nucleotides, nucleotides3–5 are recorded and considered as potential anticodon. Thesoftware extracted 388 predicted tRNA genes from these 15 mito-chondrial genomes. Fig. 2 presents the secondary structure of mttRNA Leu UAA of Drosophila pseudoobscura. The D loop has theanticodon UAG, which matches leucine’s codon CUA, the TΨC loophas the anticodon AGA, matching serine’s codon UCU. Note thatleucine and serine are the amino acids with the highest frequen-cies in proteins. The 5′ and 3′ sidearm loops have exactly sevennucleotides as in Fig. 2 (corresponding to the normal regularanticodon loop size) in 58 and 117 tRNA 5′ and 3′ sidearms,respectively. These putative anticodons match codons for leucinein 41 and 49% of the cases ( 5′- and 3′ sideloops, respectively),reminding observations for non-mitochondrial tRNAs (whereanticodons matching leucine and serine are more than 90% ofthe sidearm anticodons detected by this method (not shown)), andfor the pool of mitochondrial genomes across a wide range ofspecies analysed above for Fig. 1.

The mean mitochondrial genome-wide usage of codon familiesin Drosophila matching sidearm loop anticodons is positivelycorrelated with the mean number of sidearm loops with antic-odons matching these codon families, for each 5′- and 3′ loops (5′-loop: parametric Pearson correlation coefficient r¼62, P¼0.001;non-parametric Spearman rank correlation coefficient rs¼0.56,P¼0.005; 3′-loop: r¼0.80, P¼0.000, rs¼0.71, P¼0.0006, onetailed tests; Fig. 3a and b), strengthening results from Fig. 1. Thesecorrelations with mitochondrial amino acid usages are, asexpected, stronger than those with overall usages from Section2.1.1Q4 . This is because the amino acid usages in this case are specificto mitochondrial proteins, and sidearm anticodon counts are frommitochondrial tRNAs.

2.3. Sidearm anticodons and genome-wide amino acid usages:Comparisons between genomes

The previous section suggests that for mitochondrial tRNAsidearms with loop size matching that of regular anticodon loops(seven nucleotides), usage frequencies of amino acids and num-bers of matching sidearm loop anticodons per genomes arepositively correlated, and this for each 5′ and 3′ sidearm loops.This principle can also be explored at the level of specific codonfamilies, when comparing between different Drosophila mitochon-drial genomes (the previous section compares between codonfamilies, pooling genomes). This level of analysis correspondsto microevolution (short divergence times between Drosophilaspecies), analyses in the previous section correspond to divergencetimes between tRNA species (a macroevolutionary scale).

There was variation among Drosophila genomes in the numberof 5′ sidearm loops for 11 (among 22) codon families, for which acorrelation was calculated between D-loop anticodon numbersand genomic codon usages. This correlation is positive in 10among 11 codon families, which is a significant majority of cases(P¼0.00293, one tailed sign test). The correlation is significant atPo0.05 at the level of single codon families for two families(Ile: r¼0.45, P¼0.046; Thr: r¼0.57, P¼0.013). Both cases remainsignificant at Po0.05 when using the Benjamini and Hochberg(1995) adjustment for false positive discoveries, a more realisticadjustment for multiple testing than the overconservative Bonfer-roni adjustment (Perneger, 1998).

For 3′ TΨC-loops, similar correlations can be done only for9 codon families, and these yield positive correlations betweengenome-wide codon usage and TΨC anticodon numbers for sixcodon families, a non-significant majority of cases. None of thesecorrelations is significant. These analyses are repeated for the sum

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Drosophila pseudoobscura

D-loop:

Anticodon: UAG->Leucine

TΨC-loop:

Anticodon: AGA->Serine

Mt tRNA Leu UAA from

Fig. 2. Predicted secondary structure of mitochondrial tRNA Leu UAA of Drosophilapseudoobscura that matches leucine’s codon UUA. The putative anticodons in the 5′and 3′ sidearm loops (D- and TΨC loops) are UAG (matches leucine’s codon CUA)and AGA (matches serine’s codon UCU).

H. Seligmann / Journal of Theoretical Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

Please cite this article as: Seligmann, H., Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs? J. Theor. Biol.(2013), http://dx.doi.org/10.1016/j.jtbi.2013.08.030i

of 5′ and 3′ sidearm anticodons, enabling to calculate correlationsfor 13 codon families. The correlation between sidearm anticodonnumbers and codon usages is positive in 10 among 13 cases(P¼0.023, one tailed sign test). The only correlation significant atPo0.05 is the one already described above for Thr, as there are no3′ anticodons for Thr.

These analyses confirm the principle of coevolution betweencodon usages and numbers of anticodons described when compar-ing different codon families (Section 2.1), at the level of singlecodon families, comparing mitochondrial genomes of closelyrelated Drosophila species. Hence coevolution between sidearmanticodons and codon usages is observed for each 5′ and 3′sidearm loops, and at each micro- and macroevolutionary timescales. These results suggest that anticodons in tRNA sidearms arefunctional in translation.

2.4. Coevolution between expanded sidearm loops and tetragenes

Section 2.2 indicates that among 388 Drosophila mitochondrialtRNAs extracted by tRNAscan-SE from the 15 mitochondrial

genomes analysed, the majority of sidearm loops have lengthsthat differ from seven nucleotides, the size of regular anticodonloops. In this respect, mitochondrial tRNAs are known to differfrom non-mitochondrial tRNAs, for which most sidearm loops areseven nucleotides long. Among the Drosophila tRNA sidearm loops,98 have loops with more than seven nucleotides (79 for D- and 19for TΨC loops, see example in Fig. 4). Previous analyses suggestthat mitochondrial protein coding genes include regions usingtetracodons (codons expanded by a silent nucleotide) to code forproteins (or part of proteins), forming tetragenes. Predicted tetra-codon numbers coevolve (in each mammals and Drosophila) withtetragene GC contents, suggesting that tetracoding is an adapta-tion to conditions favouring strong codon–anticodon interactions(Seligmann, 2012a). For example, high temperatures could favourthe more stable interactions between four, rather than three pairsof nucleotides, as suggested for translation at the origin of life(Gonzalez et al., 2012). Experimental evidence for such phenom-ena exist at least at the single tetracodon level: nucleotideaddition in the anticodon of tRNAs suppresses frameshiftingnucleotide insertions (Riddle and Carbon, 1973; Sroga et al.,1992). Tetracoding coevolves with numbers of predicted antisensetRNAs with expanded anticodon loops, suggesting that expandedanticodons translate tetracodons (Seligmann, 2012a). Tetradecodingcan follow various other mechanisms (Walker and Frederick, 2008)such as tRNA hopping (O’Connor et al., 1989; O’Connor, 2003) andframeshifing (Barak et al., 1996).

These observations suggest the prediction, and empirical test ofthe tRNA sidearm anticodon hypothesis, that tetracodons aspreviously predicted (Seligmann, 2012a) should coevolve withnumbers of tRNAs with sidearms with expanded loops. Fig. 5aand b plots predicted tetracodon numbers from different Droso-phila species mitochondrial genomes as a function of numbers oftRNA sidearms with expanded (4seven nucleotides) sidearmloops in these Drosophila mitochondrial genomes. Positive correla-tions for each 5′ and 3′ sidearms (5′: r¼0.52, P¼0.023; rs¼0.603,

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Fig. 3. Mean number of putative sidearm loop anticodons in tRNAs per mitochon-drial genome calculated from 15 complete mitochondrial genomes of Drosophilaspecies, as a function of the mean usage of the codon family matching thatanticodon in these 15 Drosophila genomes, for anticodons in: (a) the D-loop(5′ tRNA half); and (b) the TΨC-loop (3′ tRNA half).

Mt tRNA Val UAC fromDrosophila erecta

D-loop: 8 nucleotides

Anticodon: UUAG ->Leucine

TΨC-loop: 7 nucleotides

Anticodon: CAA ->Leucine

Fig. 4. Predicted secondary structure of mitochondrial tRNA Leu UAA of Drosophilaerecta that matches valine’s codon GUA. The putative anticodons in the 5′ and 3′sidearm loops (D- and TΨC loops) are UUAG (matches leucine’s codon CUA) andCAA (matches leucine’s codon UUG). The 5′ sidearm loop is eight nucleoside long,hence the putative anticodon in the 5′ sidearm loop is expanded and has fournucleosides, putatively matching to decode tetracodons.

H. Seligmann / Journal of Theoretical Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎4

Please cite this article as: Seligmann, H., Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs? J. Theor. Biol.(2013), http://dx.doi.org/10.1016/j.jtbi.2013.08.030i

P¼0.012; 3′: r¼0.54, P¼0.018; rs¼0.387, P¼0.074; 5′þ3′:r¼0.58, P¼0.011; rs¼0.515, P¼0.027) independently confirm thecoevolution hypothesis between tetracoding and expanded side-arm anticodons. This non-trivial observation strengthens thehypothesis that anticodons in tRNA sidearm loops are active intranslation, and that of translation of tetracodons and tetragenes.Note that these results are in line with the prediction that thegenetic code based on quadruplet codons (tetracodons) originatedfrom mitochondria (Gonzalez et al., 2012), because tRNA sidearm

loops with more than seven nucleotides are frequent in mitochon-drial tRNAs, but very rare in cytosolic tRNAs.

3. Discussion

3.1. Sidearm length and translational activity

Results present evidence that putative anticodons in mitochon-drial tRNA sidearm loops function in translation. However, themechanism by which this occurs remains unclear. Three non-exclusive hypothetical mechanisms exist: (a) tRNA side- andanticodon arms are swapped by crossover-like events; (b) thetRNA’s three dimensional L-shape could be reached by unusualbending, where one sidearm takes the role usually filled by theanticodon, and the anticodon stem bends towards the remainingsidearm, with their loops interacting, forming the arch usuallyformed by interactions between the loops of the sidearms; and (c)isolated tRNA halves (stem-loop structures corresponding to thesidearms) function in translation.

Each of these mechanisms implies assumptions. Crossover andunusual arm bending (a and b) have the advantage over functionaltRNA halves (c) that they do not assume an unusual tRNA threedimensional shape. There is no evidence that crossovers betweentRNA arms occur. In relation to unusual arm bending, it seemsdoubtful that the tRNA molecule is flexible enough to enableforming a three dimensional L-shape where a sidearm takes theposition usually taken by the anticodon arm, and the anticodonarm the position taken usually by the sidearm, and this withoutcrossover. Though mechanism (b) cannot be ruled out, it seems atthis stage of knowledge less realistic than mechanism (a). Mechanism(c), that isolated stem-loops originating from tRNA halves functionin translation seems most probable, as isolated stem-loops corre-sponding to the light strand replication origin have been foundwith the adequate 5′ and 3′ processing and aminoacylated(Yu et al., 2008), and because stem-loop structures originatingfrom tRNA halves resemble the mitochondrial nematode armlesstRNAs predicted by alignments (Jühling et al., 2012). Analysesbelow only give partial hints in this respect.

One can assume that crossovers are more likely for longersidearms than for shorter ones. Hence if the mechanism subjacentto the phenomenon described here is based on crossovers, longersidearms should contribute more to the correlations described inSection 2 than shorter ones. The same accounts for unusual armbending, so that these to mechanisms cannot be differentiated atthis stage. Typically, anticodon arms have five paired bases in theirstem, hence sidearms bearing anticodons are weighted by thenumber of base pairs in their stem divided by five. Then the samecorrelation analyses as those done in the previous sections arerepeated. If sidearm length contributes to the sidearm loop’sfunction in translation, as expected according to the crossoverhypothesis, correlations with codon usages and tetracoding shouldbecome more positive than for previous, unweighted analyses.

For 5′ sidearms, only three correlations were altered byweighing, but correlation coefficients decreased in all three cases.A paired t-test for correlation coefficients (after z-transformation,z¼0.5xln((1þr)/(1�r)), Amzallag, 2001) yields a statistically sig-nificant difference between the correlation coefficients obtainedaccording to each weighted and unweighted analysis (two tailedpaired t-test P¼0.0092). For 3′ sidearms, results are qualitativelysimilar, but not statistically significant: seven correlations werealtered by the weighting procedure, four increased, but on average,z-transformed Pearson correlation coefficients decreased afterweighing according to stem length (two tailed paired t-testP¼0.629). Combining these independent tests using Fisher’smethod for combining P values, which sums �2xln(Pi) where i

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Fig. 5. Total number of predicted tetracodons in protein coding regions of 15Drosophila mitochondrial genomes as a function of tRNA sidearms with loops withmore than seven nucleotides, weighed by GC contents of sidearm stem, for 5′ and3′ sidearms (Fig. 5(a) and (b), respectively). The x-axis is the (GC weighted) numberof expanded sidearm loops in mitochondrial tRNAs from a given Drosophila species,as counted from examining the secondary structures such as in Figs. 2 and 4 for allsense tRNAs in that mitochondrial genome. Note that the scales of the x-axes differbetween Fig. 5(a) and (b). The y axis is the number of predicted tetracodons in thatgenome, as determined by alignments between putative peptides translated fromthe Drosophila’s regular mitochondrial protein coding genes, but assuming tetra-coding, and GenBank’s non-redundant protein database. Alignment data fortetracodon numbers are the same as used for the x-axis of Fig. 3 and the y axesof Figs. 9 and 10 in Seligmann (2012a).

H. Seligmann / Journal of Theoretical Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5

Please cite this article as: Seligmann, H., Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs? J. Theor. Biol.(2013), http://dx.doi.org/10.1016/j.jtbi.2013.08.030i

ranges from 1 to k for k tests (in this case 2 tests) yields a chisquarestatistic with 2xk degrees of freedom¼10.32, two tailed P¼0.036.Results differ between 5′ and 3′ arms, but overall analyses suggestthat short arms have loops more likely to function as anticodonloops than longer arms. This is opposite to predictions by thehypothesized crossover mechanism. Unusual arm bending seemsalso less likely for shorter than longer arms. Hence mechanisms aand b which maintain the regular tRNA three dimensional L-shapeare not compatible with results from analyses that weigh sidearmanticodons by arm lengths.

Adding to this analysis data from correlations betweenexpanded putative sidearm anticodons and predicted tetragenessuggests, after weighing data according to stem length, a slightlyless positive correlation for 5′ sidearms (r¼0.521) but slightlymore positive correlation for 3′ sidearms (r¼0.571) than forunweighted analyses. Hence overall, longer stems do not contri-bute to anticodon function by sidearms, but the possibility existsthat shorter sidearms tend to function more as anticodon armsthan longer sidearms.

Hence overall these analyses do not support the hypothesesthat maintain the tRNA’s three dimensional L-shape and mighteven refute its prediction for 5′ sidearms.

3.2. Sidearm stability and translational activity

The third mechanism according to which sidearm loops mayfunction as anticodon loops in translation is that isolated stem-loop RNA structures corresponding to sidearms function in trans-lation. These small structures would probably be relativelyunstable, hence their functioning would depend on their struc-tural stability. Structural stability increases proportionally to stemlength, and to the fraction of GC base pairs in that stem. Theprediction with stem length is confounded with that used in theprevious section for crossovers. It is possible that more stablesidearms are more breakable, and hence lend themselves more tocrossovers than sidearms with lower GC contents. However, G andC are larger than A and U, which might decrease the flexibility ofthe arms and render unusual arm bending less likely. Hencecorrelation analyses that weigh data according to the proportionof GC base pairs in the sidearm’s stem may indicate whether datafit the unusual arm bending hypothesis. Correlations with codonusages become more positive upon weighing by GC stem contentsin about half the cases where correlations are altered by weighing.Hence analyses are inconclusive. Correlations between GC-weighed sidearm numbers with expanded anticodons and totallengths of predicted tetracodons per genome increase, as com-pared to unweighed analyses, for each 5′ and 3′ sidearms sidearms(5′: r¼0.74, P¼0.0008; rs¼0. 73, P¼0.0031; 3′: r¼0.56, P¼0.015;rs¼0.55, P¼0.0188; 5′þ3′: r¼0.73, P¼0.001; rs¼0.76, P¼0.0022,one tailed tests, see Fig. 5a and b). Overall, these analyses andthose in the previous section slightly favour the isolated tRNA halfhypothesis upon the crossover hypothesis if one assumes thatsidearm stability contributes more to translation by isolated tRNAhalves than to arm breakability. The data do not support theunusual arm bending hypothesis.

3.3. Antisense tRNAs confound correlations between sidearmanticodons and codon usages

Besides regular sense tRNAs and their putative sidearm sub-units, some evidence indicates that mitochondria also includetRNAs templated by the DNA sequence that is the inversecomplement of the tRNA’s gene, called the antisense tRNA(Seligmann, 2010a, 2010b, 2011a). Codon usages usually correlatepositively with properties of tRNAs with matching anticodonsand that promote tRNA translational activity, as previously

observed for cloverleaf secondary structure formation by Droso-phila mitochondrial sense tRNAs (Fig. 4 in Seligmann, 2012b).Hence correlations between antisense tRNA anticodon numbersand codon usages could confound correlations described inSection 2 between sidearm anticodons and codon usages. Thispredicts a strong decrease in correlations between sidearmanticodons and codon usages for codon families matched bynumerous antisense tRNAs, but no effect on codon families forwhich there are no antisense tRNAs to confound effects ofsidearm anticodons. Hence one expects a negative correlationbetween the strength of the association between sidearm antic-odon numbers and the mean number (per genome) of antisensetRNAs with anticodons matching a codon family. This is indeedthe case for 5′ sidearm anticodons: correlations between antic-odon numbers and codon usages are positive for codon familiesfor which there are no antisense tRNAs with matching antic-odons, and become weaker or negative the more matchingantisense tRNAs exist (r¼�0.593, P¼0.0272; rs¼�0.537, P¼0.044, one tailed tests, see Fig. 6). However, note that similaranalyses for the 3′ sidearm do not confirm this principle (r¼0.34,P¼0.38, rs¼0.35, P¼0.31, two tailed tests, data not shown).These analyses suggest that, at least for 5′ sidearms, positivecorrelations between numbers of sidearm anticodons and codonusages are stronger than observed if one was to account forconfounding effects from other types of tRNAs, such as theantisense tRNAs. In addition, these results are an additionalindirect confirmation of translational activity by antisense tRNAsin mitochondria (Seligmann, 2010a, 2010b, 2011a, 2011b, 2012a,2012b, 2012c, 2013a; Faure et al., 2011).

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Fig. 6. Association between 5′ sidearm anticodon numbers from sense tRNAs andgenome-wide codon usage in Drosophila mitochondria as a function of meannumbers of antisense tRNAs with anticodons matching that codon family. The yaxis is the Pearson correlation coefficient between numbers of 5′ sidearm antic-odons in sense tRNAs from 15 Drosophila mitochondria and codon usages for thecorresponding codon families in these mitochondrial genomes. The x axis is themean number of antisense tRNAs with regular anticodons (not in sidearms)matching these codons in the same genomes. The negative correlation indicatesthat codon usages are explained by numbers of sense tRNA 5′ sidearm anticodonsfor codon families for which there are no (or few) matching (regular) anticodons inantisense tRNAs. This indicates that positive correlations between codon usagesand sense tRNA 5′ sidearm anticodons are in part confounded by correlationsbetween regular anticodons from antisense tRNAs and codon usages. Hencepositive correlations between sidearm anticodons and codon usages are probablystronger than observed.

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Please cite this article as: Seligmann, H., Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs? J. Theor. Biol.(2013), http://dx.doi.org/10.1016/j.jtbi.2013.08.030i

3.4. Codon usages, tRNA sidearms and antisense tRNAs from yeast

Coevolution between anticodons and codon usages as pre-sented for Drosophila mitochondrial genomes is independentlytested by comparing the mitochondrial genomes of a pair of fungi,baker’s yeast Saccharomyces cerevisiae, and Yarrowia lipolytica(mitochondrial genomes in GenBank NC001224 and NC002659,respectively), for each sense, antisense, and putative sidearm(D- and TΨC) anticodons (Table 1). The approach used in this caseassumes that the tRNA pool coevolves with codon usages, so asto optimize translational efficiency (Ikemura, 1981; Gouy andGautier, 1982; Percudani et al., 1997). Tissue specific differencesin gene expression and associated codon biases covary with tRNAexpression (Plotkin et al., 2004; Dittmar et al., 2006), a phenom-enon also affecting growth rates (Dong et al., 1996). This does notmean that when there is no evidence for such associations, thetRNAs are not active in translation, but if a positive associationsuggesting codon-tRNA optimization is detected, the positiveresult would imply translational activity in the case of sidearmanticodons.

Hence when comparing the two fungi mitochondrial genomes,one expects that when there are more tRNAs with a specificcognate in yeast than in Yarrowia, the cognate amino acid is morefrequent in the composition of the mitochondrion-encoded pro-teins of yeast than those of Yarrowia, and vice versa. Thisqualitative effect should be observed systematically across allcases where tRNA numbers vary between the two species.

For sense tRNAs, the data in Table 1 indicate an increase inyeast tRNA numbers (as compared to Yarrowia) for those loaded bycognates Arg, Asn, Glu and Tyr, and a decrease for tRNAs loaded byLys and Thr. Amino acid usages follow qualitatively the samepattern as tRNAs in four among these five cases (80%), whichaccording to a one tailed sign test has P¼0.094). This means thatthere is a tendency for cognate usage and sense tRNA genenumbers to covary in fungi mitochondria, as expected by tRNA-codon usage optimization, though this effect is not statistically

significant at Po0.05, probably because there are only five caseswhere sense tRNA gene numbers vary between these species.

The same analysis for antisense tRNAs yields a qualitativepositive association between changes in antisense tRNA numbersand codon usages in 8 among 10 cases, which is again 80% of thecases (P¼0.027). Hence this analysis indicates that antisense tRNAnumbers coevolve with codon usages, a result whose mostparsimonious interpretation implies translational activity by anti-sense tRNAs. A similar analysis for anticodons in sense tRNAsideloops is not conclusive, because positive covariation isobserved in 50% of the four cases where variation exists inD-sidearm anticodon numbers between the two species, as wellas 50% of the four cases for TΨC sidearm loops. However, whenconsidering antisense tRNAs and sidearm loops together, thepositive covariation occurs in 79% of the 14 cases, which hasP¼0.002. Hence taking into account antisense tRNAs, resultscorroborate in principle for fungi the positive association betweencodon usages as was described in Section 2.2. for Drosophilamitochondrial genome. The results from fungi are not incompa-tible with those from Drosophila, which overall confirm those fromsimilar analyses of mammal tRNAs (Seligmann, 2013a). Hence theprinciple that putative anticodons in tRNA sidearm loops functionin translation seems corroborated by all currently available results.

The analysis of expanded sidearm anticodons is not repeated infungi because this analysis is less adapted to these taxa: No stricthomology exists between mitochondrial protein coding genes ofthese two species, which makes the comparative analysis ofnumbers of predicted tetragenes embedded within the regularprotein coding genes a less adequate natural experiment than it isfor Drosophila.

4. General discussion

It is probable that the extreme genome compression thatoccurs in mitochondria is at the origins of the phenomenon ofanticodon activity by tRNA sidearms. This is by no means the onlycase where mitochondrial tRNAs have additional functions:besides templating for sense, and also antisense tRNAs (as notedabove), secondary structures formed by DNA sequences templat-ing for the heavy strand DNA of mitochondrial tRNA genesfunction as additional light strand replication origins (Seligmannet al., 2006a, 2010b; Seligmann, 2008, 2010c, 2011c), especiallythose located around the regular light strand replication origin(Seligmann and Krishnan, 2006). This polyvalence is not restrictedto mitochondrial tRNAs: mitochondrial protein coding genes alsoseem to have alternative coding properties, in addition to thealready noted tetragenes: proteins seem also encoded in the 3′-to-5′direction (Seligmann, 2012d, 2013b) and additional protein codinggenes seem revealed by permuting polymerization, polymerizationthat systematically exchanges between nucleotides (Seligmann,2013c, 2010b). This stresses that numerous unexpected functionsby mitochondrial sequences probably exist, in addition to the antic-odon function by mitochondrial tRNA sidearm loops described here.It is also probable that similar phenomena of multiple gene functionsoccur for other genomes, notably nuclear ones, but the extremereduction of mitochondrial genomes probably increased the densityof multiple functions.

There exists an alternative hypothesis to that of anticodons insidearm loops functioning in translation. These putative antic-odons might function as determinants (and antideterminants) intRNA recognition by tRNA synthetases (Pütz et al., 1991; Giegé,2006). The fact that the structural model of tRNA interaction withthe tRNA synthetase does not suggest direct contacts betweensidearm loops and the tRNA synthetase (Tsunoda et al., 2007, Fig. 1therein) does not preclude such a possibility. Hence the putative

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Table 1Codon usages and anticodon numbers in the mitochondrial genomes of Sacchar-omyces cerevisiae (S) and Yarrowia lipolytica (Y). Columns are: 1. Identity ofpredicted cognate amino acid matching anticodon; 2–3. Mitochondrial codonusages; 4–5. Sense tRNA anticodon numbers; 6–7. Antisense tRNA anticodonnumbers; 8–9. D-loop sidearm anticodon numbers; 10–11. TΨC-loop sidearm tRNAanticodon numbers.

Usage Sense Anti 5′ 3′

Aa S Y S Y S Y S Y S YAla 43 56.2 1 1 1 2 0 0 0 0Arg 29.9 23.5 2 1 2 1 0 0 0 0Asp 32.1 28.8 1 1 1 0 0 0 0 0Asn 95.4 75.2 2 1 1 1 1 0 0 0Cys 9.3 8.4 1 1 2 2 0 1 0 0Gln 21.6 18.1 2 1 0 0 0 0 0 0Glu 28.4 26.1 1 1 2 0 0 0 0 0Gly 54.6 58.8 1 1 0 0 0 0 0 0His 19.6 19.3 1 1 1 1 0 0 0 0Ile 90.8 56.1 1 1 1 0 0 0 0 0Leu UUR 108.4 127.7 1 1 0 1 0 0 12 0Leu CUN 16.1 11.5 0 0 0 0 1 0 1 0Lys 69.8 48.2 1 2 0 0 0 0 0 0Met 54.6 91.8 2 2 1 3 0 0 0 0Phe 56.5 68.8 1 1 2 0 0 0 1 0Pro 34.8 27.8 1 1 0 0 5 9 5 0Ser AGY 14.1 22.5 1 1 0 0 0 0 0 0Ser UCN 46.8 50.4 1 1 3 0 0 0 12 19Thr 42 54.3 1 2 1 1 0 0 0 0Trp 15 15 1 1 1 1 0 0 0 0Tyr 61.3 57.2 5 1 1 0 0 0 1 0Val 50.4 51.4 1 1 4 4 0 0 0 0

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sidearm anticodons would actually be determinants in tRNArecognition, and the coevolution between these and codon usagesmight be the indirect result of effects of codon usages ondeterminants. This hypothesis does not seem very plausible andis furthermore ruled out by the coevolution between expandedsidearm anticodons and tetragenes (Section 2.3), which is notpredicted by tRNA recognition.

Hence the major point at this stage is to indicate which, amongthe three hypotheses that assume anticodon activity in translationby tRNA sidearm loops, crossover, unusual armbending or tRNAhalves, is the most plausible. Results suggest some differencesbetween 5′ and 3′ sidearms: the crossover hypothesis is tenta-tively refuted for the former, but cannot be refuted for the latterfor anticodon–codon usage correlations, and tetracodon analysesfor stem stability (GC contents) support more the isolated stem-loop hypothesis for 5′ than 3′ sidearms. There is no support fromthe analyses for unusual arm bending. It is interesting to remind inthis context the differences existing in relation to the circular codebetween the tRNA halves (Michel, 2012, 2013): pseudocodoncontents of the 3′ half resembles that of protein coding genes,but not that of the 5′ half. Analyses presented here strengthenslightly the hypothesis of functional tRNA halves, as compared toexchanges between arms and unusual armbending.

Each of these hypotheses is most parsimonious as compared tothe other in relation to different aspects: the crossover hypothesisdoes not assume an unusual structure functioning as tRNA, theisolated tRNA half hypothesis does assume this. However, thispoint might be relatively weak due to the existence of aminoacy-lated short stem-loop RNAs corresponding to the light strandreplication origin (Yu et al., 2008) and armless minimal tRNAs inmitochondrial nematodes (Jühling et al., 2012). It is not clear howarmless tRNAs affect interactions between tRNAs and tRNAsynthetases (Tsunoda et al., 2007) during tRNA loading. The tRNAinteracts also with ribosomes (Peattie and Herr, 1981; Spahn et al.,2001) during translation, especially the ribosomal A (Ashraf et al.,1999), P (Rodnina et al., 1989) and E sites (Wower et al., 2001).These include crosslinking of the tRNA’s 3′end with specificnucleotides in these sites (Wower et al., 2001), but the implica-tions of armless tRNAs in this process remain unknown. Bothmitochondrial ribosomal 12s and 16s subunits evolved betweenthe nematodes Trichinella (whose mitochondrial tRNAs tend tohave sidearms) and Xiphinema (armless mitochondrial tRNAs,Jühling et al., 2012). Per se, this could but does not necessarilyimply ribosomal adaptation for armless tRNAs. The crossover andunusual arm bending hypotheses do not imply any specificadaptation in these respects, however they imply amino acidmisinsertions when the crossover between side- and anticodonarms, respectively unusual arm bending, occur after the tRNA wasloaded by its tRNA synthetase with its cognate amino acid, becausesidearm anticodons rarely match the tRNA’s regular cognate. A lastpoint associated with cost minimization and metabolic efficiencyprinciples and that favours the tRNA halves hypothesis is that suchhalves could be functional products of tRNA degradation by RNAendonucleases. Highly specific splicing sites exist for tRNAs fromthe cytosol (Calvin and Li, 2008). Intra-tRNA splicing producesRNAs derived from tRNAs with specific expression and functionand an abundant fraction of small RNAs that are not microRNAs(Lee et al., 2009). Such splicing, if it occurs at the base of sidearms,could produce structures resembling the armless tRNAs describedby Jühling et al. (2012). This stresses the modular aspect of thetRNA’s structure and the functional efficiency implied by modularstructures, in addition to the evolutionary advantages of modular-ity based on pure population genetic principles (Wagner andAltenberg, 1996). Overall, parsimony considerations seem toslightly favour the isolated tRNA half hypothesis, as compared tothe crossover hypothesis. The fact that no ESTs were detected in

Genbank and matching human mitochondrial tRNA sequencesafter exchanging between anticodon and sidearms (unpublishednegative results) is also indicative that crossovers between tRNAarms do not explain patterns described here. Overall, no clearmechanism on how putative anticodons in tRNA sidearms functionin translation can be indicated, but both parsimony considerationsand results suggest that the tRNA half hypothesis has a slightadvantage upon the crossover (and unusual arm bending) hypoth-eses. Nevertheless, results indicate that the unsuspected phenom-enon of sidearm loops functioning as anticodon loops occurs inmodern mitochondria.

Uncited references Q2

Schneider (2011), Seligmann et al. (2006b), Seligmann (2013d).

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H. Seligmann / Journal of Theoretical Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 9

Please cite this article as: Seligmann, H., Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs? J. Theor. Biol.(2013), http://dx.doi.org/10.1016/j.jtbi.2013.08.030i