Comprehensive Analysis of Archaeal tRNA Genes Reveals Rapid Increase oftRNA Introns in the Order Thermoproteales
Junichi Sugahara,* 1 Kaoru Kikuta,*à1 Kosuke Fujishima,* Nozomu Yachie,*
Masaru Tomita,* à and Akio Kanai* à*Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan; Systems Biology Program, Graduate School ofMedia and Governance, Keio University, Fujisawa, Japan; and àFaculty of Environment and Information Studies, Keio University,
Fujisawa, JapanThe analysis of archaeal tRNA genes is becoming more important to evaluate the origin and evolution of tRNA
molecule. Even with the recent accumulation of complete genomes of numerous archaeal species, several tRNA genes
are still required for a full complement of the codon table. We conducted comprehensive screening of tRNA genes from47 archaeal genomes by using a combination of different types of tRNA prediction programs and extracted a total of
2,143 reliable tRNA gene candidates including 437 intron-containing tRNA genes, which covered more than 99.9% of
the codon tables in Archaea. Previously, the content of intron-containing tRNA genes in Archaea was estimated to beapproximately 15% of the whole tRNA genes, and most of the introns were known to be located at canonical positions(nucleotide position between 37 and 38) of precursor tRNA (pre-tRNA). Surprisingly, we observed marked enrichment
of tRNA introns in five species of the archaeal order Thermoproteales; about 70% of tRNA gene candidates were foundto be intron-containing tRNA genes, half of which contained multiple introns, and the introns were located at various
noncanonical positions. Sequence similarity analysis revealed that approximately half of the tRNA introns found at
Thermoproteales-specific intron locations were highly conserved among several tRNA genes. Intriguingly, identical
tRNA intron sequences were found within different types of tRNA genes that completely lacked exon sequencesimilarity, suggesting that the tRNA introns in Thermoproteales could have been gained via intron insertion events at
a later stage of tRNA evolution. Moreover, although the CCA sequence at the 3# terminal of pre-tRNA is added by
a CCA-adding enzyme after gene transcription in Archaea, most of the tRNA genes containing highly conserved introns
already encode the CCA sequence at their 3# terminal. Based on these results, we propose possible models explaining the
rapid increase of tRNA introns as a result of intron insertion events via retrotransposition of pre-tRNAs. The sequences
and secondary structures of the tRNA genes and their bulge-helix-bulge motifs were registered in SPLITSdb (http://
splits.iab.keio.ac.jp/splitsdb/), a novel and comprehensive database for archaeal tRNA genes.

Introduction
As tRNA is a key molecule in translation, its origin
and evolution are important topics widely discussed in
the field of molecular evolution (Maizels and Weiner
1994; Di Giulio 1999, 2006; Fujishima et al. 2008; Randau
and Söll 2008). Around 15% of all archaeal tRNA genes,
which are estimated to be the most ancestral (Sun andCaetano-Anolles 2008), have been reported to contain endogenous introns (intron-containing tRNA genes). According to Marck and Grosjean (2003), in a total of 800 archaealtRNA genes analyzed, 103 out of 136 introns of tRNA
genes are located at the nucleotide position between 37
and 38 (canonical position, 37/38); the remaining 33 introns
are located at other sites (noncanonical positions); 6 of thesetRNA genes reportedly harbor 2 introns. Recently, we havereported that computational analysis using SPLITS, a program for detecting various types of archaeal tRNA genes(Sugahara et al. 2006), has predicted three tRNA genes that
contain triple introns (Sugahara et al. 2007). The tRNA introns are known to form a structural motif consisting ofa bulge-helix-bulge (BHB) consensus sequence aroundthe intron–exon junction (Kaine et al. 1983; Diener andMoore 1998; Marck and Grosjean 2003). The canonical
BHB motif is a single hairpin structure that consists of
two bulges (B), which are separated by a central helix
1

Equal contribution to this work.
Key words: tRNA, Archaea, intron, bulge-helix-bulge (BHB) motif,
Thermoproteales, Bioinformatics.
E-mail: akio@sfc.keio.ac.jp.
Mol. Biol. Evol. 25(12):2709–2716. 2008
doi:10.1093/molbev/msn216
Advance Access publication October 1, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: journals.permissions@oxfordjournals.org(H) within consensus motif sequences (hBHBh#), and isrecognized and cleaved by heterotetrameric (a2b2), homodimeric (a2), or homotetrameric (a4) splicing endonucleases during precursor tRNA (pre-tRNA) processing(Lykke-Andersen and Garrett 1997; Li et al. 1998; TocchiniValentini et al. 2005a; Yoshinari et al. 2005). Relaxed
BHL and HBh# motifs including the constant central helix
H flanked by one helix (h or h#) on either sides are also
known motifs of pre-tRNA (Marck and Grosjean 2003)
and coevolution between the types of the BHB motifs andtheir corresponding splicing endonucleases have beenpreviously studied (Tocchini-Valentini et al. 2005b).
Unique types of introns carrying tRNA genes have recently been reported in Archaea and primitive Eukaryote.
It has been reported that the hyperthermophilic archaealparasite, Nanoarchaeum equitans, encodes split tRNA
genes with 5# and 3# halves on separate genes (Randau,
Calvin, et al. 2005; Randau, Munch, et al. 2005). We havealso reported a novel type of tRNA gene, so-called permuted tRNA genes, with the 3# half of the tRNA lying
upstream of the 5# half in the nuclear genome of the primitive red alga, Cyanidioschyzon merolae (Soma et al. 2007).
Interestingly, both split and permuted tRNA genes form
BHB motifs around the intron–exon junctions (Randau,
Pearson, and Söll 2005; Soma et al. 2007); thus, having thiscommon intron-processing machinery suggests an evolutionary link between disrupted (intron-containing, split,
and permuted) tRNA genes in Archaea and primitive Eukaryote. Analysis of archaeal tRNA genes possessing
BHB motifs is thus becoming important to elucidate
the evolution of tRNA genes. Besides, it has been widely
discussed whether the tRNA introns represent a primitive
trait that is important in tRNA origin or whether they are2710 Sugahara et al.

acquired at a later stage of tRNA evolution (Di Giulio 2006;
Fujishima et al. 2008; Randau and Söll 2008; Di Giulio 2008).
However, current tRNA databases are still inadequate(Sprinzl and Vassilenko 2005; Andersen et al. 2006; Putz
et al. 2007; http://lowelab.ucsc.edu/GtRNAdb/) due to thedifficulty of detecting introns located at noncanonical positions of tRNA. Besides, several tRNA genes are still unknown
in certain archaeal species, suggesting the possibility of undocumented novel intron-containing or split tRNA genes.
In an attempt to identify all the tRNA genes in Archaea, we constructed an integrated tRNA gene prediction
system based on a combination of SPLITS, tRNAscan-SE(Lowe and Eddy 1997), ARAGORN (Laslett and Canback2004), Blast (Altschul et al. 1990), and tRNA-CM covariance model (Eddy and Durbin 1994). We then conducted
a comprehensive prediction of tRNA gene candidates fromthe complete genome sequences of 47 archaeal species,
which is presented here with the results of phylogenetic analysis of tRNA introns, as well as the number, location,
sequence conservation, and coevolution of splicing endonucleases. All sequences, secondary structures, and splicing
motifs of the tRNA genes have been registered in SPLITSdb(http://splits.iab.keio.ac.jp/splitsdb/), a novel and comprehensive web-based database for archaeal tRNA genes.
SPLITSdb is the only database that covers all types of archaeal tRNA genes including split and intron-containingtRNA genes and will contribute to further research on the origin and evolution of tRNA and tRNA intron processing.

Materials and Methods
Genome Sequences
A total of 47 complete archaeal genomes were obtained from National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The complete list of species and accession numbers are shown in supplementarytable S2 (Supplementary Material online).

Comprehensive Prediction of tRNA Genes
An overview of the comprehensive prediction oftRNA genes is shown in supplementary figure S1 (Supplementary Material online). The tRNA gene candidates are
predicted from each genome sequence based on four different programs. Split tRNA gene candidates were predicted
by SPLITS with ‘‘–t’’ search mode. Intron-containing tRNAand other tRNA gene candidates, including the selenocysteinyl tRNA gene, were predicted by SPLITS, tRNAscanSE, and ARAGORN. Pyrrolysyl tRNA gene candidates
were predicted based on the BlastN search using Methanosarcina barkeri DNA sequences encoding the pyrrolysyl
tRNA genes as a query. The results of ARAGORN and
BlastN were further evaluated using tRNA-CM covariancemodel (Eddy and Durbin 1994) for more detail about thecloverleaf structural features and consensus sequences of
the candidates. Next, all the output data were merged
and overlapping tRNA gene candidates were eliminated.
In addition, initial predictions of tRNA introns and the secondary structure of splicing motifs were made by SPLITS.

Then, the location of the introns and the type of splicing
motifs were manually annotated according to (Marck andGrosjean 2003); and all the predicted tRNA genes wereconfirmed manually to maintain a highly reliable tRNA
gene data set by satisfying the following conditions: 1)
tRNA gene candidates that fulfill the criteria of the cloverleaf structure of archaeal tRNAs (Marck and Grosjean
2002) and 2) tRNA gene candidates that are able to decode
all sense codons in each species.
Phylogenetic Analysis Based on rRNA Sequences
The 16S rRNA sequences from 47 archaeal species
were aligned (as rDNA sequence) using ClustalW 2.0(Larkin et al. 2007) with the following parameters: multiplealignment parameters, gap opening 5 22.5; gap extension
penalty 5 0.83, and delay divergent sequence 5 25%; andpairwise alignment parameter, gap opening 5 22.5. These
parameters gave the highest performance in RNA sequencealignment (Wilm et al. 2006). Aligned rRNA sequenceswere manually improved by trimming both ends to maintain the consensus sequence. Bayesian trees were constructed using MrBayes software version 3.1.2 with
C. merolae 18S rRNA as an out-group (Huelsenbeck
and Ronquist 2001). The model of sequence evolution
was determined by MrModeltest software version 2.2.
The phylogenetic tree files were visualized using Interactive
Tree Of Life (Letunic and Bork 2007).
Sequence Similarity Analysis of Archaeal tRNA Introns
In order to analyze the conserved tRNA introns thatseem to be created via intron insertion events, we calculated
the sequence similarity score (%) for every archaeal tRNA
intron. For this purpose, we aligned all possible pairs oftRNA introns having similar length (allowing ±1 nt length
differentiation), and the sequence having the highest sequence similarity was selected as the matched sequence.
If the length of the sequences was different, a single gap
was allowed to optimize the overall sequence similarity.
We excluded pairs of tRNA introns derived from tRNA
genes corresponding to the same amino acids (e.g.,tRNAGly in Pyrobaculum calidifontis and Pyrobaculum arsenaticum) to eliminate the introns conserved among theorthologous tRNA genes.

Results and Discussion
Construction of SPLITSdb System and Prediction ofArchaeal tRNA Genes
To extract complete tRNA gene sets in archaeal species, we conducted a comprehensive prediction of tRNA
genes from 47 archaeal genomes using the SPLITSdb system, which implemented multiple types of tRNA prediction
programs and manual confirmation (supplementary fig. S1,
Supplementary Material online). As a result, 2,143 tRNA
gene candidates including 437 intron-containing tRNA
genes were extracted (supplementary table S1, Supplementary Material online). The candidates covered an average ofIntron Increase during Archaeal tRNA Evolution 2711

99.9% of all 61 sense codons and the initiator codon in the
47 species using the SPLITSdb system, versus an averageof 91.8% and 91.2% using tRNAscan-SE and ARAGORN,
respectively (supplementary table S2, Supplementary Material online). The same numbers of tRNA gene candidates
were often predicted by tRNAscan-SE, ARAGORN, andSPLITSdb system in a single species; however, the codon
coverage of the SPLITSdb system exceeded that of otherprediction programs. This is because tRNAscan-SE andARAGORN mispredict the anticodon of tRNA genes
due to tRNA intron insertion at noncanonical positions
in certain cases (Sugahara et al. 2006) and also misidentifies
AUA decoding tRNAIle genes possessing the posttranscriptionally modified CAT anticodon. The SPLITSdb system
has accomplished reliable extraction of tRNA gene candidates with reasonable number and little redundancy due tothe combination of the prediction of tRNA genes containing
noncanonical introns, correct identification of tRNA anticodons, and elimination of irregular tRNA genes.
Only 26 archaeal tRNA genes containing multiple introns were previously reported (Marck and Grosjean 2003;
Sugahara et al. 2007). To determine novel tRNA gene candidates containing multiple introns, the predicted 437 intron-containing tRNA genes were classified into three
types of tRNA: 364 with a single intron, 63 with double
introns, and 10 with triple introns. As a result, we found
47 novel tRNA gene candidates containing multiple introns. Although tRNA gene candidates containing triple introns were only reported previously in tRNAPro genes ofThermofilum pendens (Sugahara et al. 2007), we further
documented 7 novel candidates containing triple intronsin other two species; tRNAGlu (UUC), tRNAGlu (CUC),tRNAGln (UUG), tRNAGln (CUG), tRNAThr (UGU), andtRNAPro (UGG) genes in P. calidifontis (fig. 1a and supplementary fig. S2, Supplementary Material online) andtRNAPro (UGG) genes in Pyrobaculum islandicum(fig. 1b). Although most of those tRNA genes contained
at least one intron at a canonical position (37/38), we found
two tRNA gene candidates with all three introns located at
noncanonical positions, tRNAGlu (CUC) and tRNAGlu(UUC) in P. calidifontis (fig. 1a).

Phylogenetic Analysis of tRNA Introns and Its Splicing
Endonucleases
The proportion of split and intron-containing tRNA
genes and the coevolution of tRNA introns and their splicing endonucleases were analyzed based on the phylogenyof small subunit rRNA among the 47 species (fig. 2). We
initially observed that the numbers of tRNA introns in archaeal species possessing heterotetrameric endonucleases(all Crenarchaeal and Nanoarchaeal species, and Methanopyrus kandleri in Euryarchaea) (Tocchini-Valentini et al.
2005a; Yoshinari et al. 2005) were significantly high.
Moreover, we also found marked enrichment of tRNA introns in the archaeal order Thermoproteales (T. pendens,
P. calidifontis, Pyrobaculum aerophilum, P. islandicum,and P. arsenaticum), a member of the Crenarchaeota encoding heterotetrameric endonucleases. A significant 68.7% of
all Thermoproteales tRNA genes were found to be intron

FIG. 1.—Examples of archaeal pre-tRNAs containing three introns.
(a) Pre-tRNAGlu (UUC) from P. calidifontis. (b) Pre-tRNAPro (UGG)
from Pyrobaculum islandicum. The locations and topological types of
BHB motifs are presented for each intron. Predicted intron sequences areshown in lower case. BHB motif abbreviations are according to Marckand Grosjean (2003).

containing, whereas 36.4%, 9.2%, and 5.8% of other archaeal species tRNA genes were found to be intron containing with either heterotetrameric, homodimeric, or
homotetrameric endonucleases, respectively (fig. 2). Furthermore, 85.7% (54 out of 63) of the tRNA genes containing double introns and 100% (10 out of 10) of the tRNA
genes containing triple introns found in all archaeal species
were concentrated in Thermoproteales. In particular, 87% of
both T. pendens and P. calidifontis tRNA genes were found2712 Sugahara et al.

FIG. 2.—Ratio of number of split and intron-containing tRNA genes in Archaea. The ratio of the number of split or intron-containing tRNA genes
per total number of tRNA genes in each archaeal genomes are shown for a total of 47 archaeal species (32 Euryarchaeota, 14 Crenarchaeota, and1 Nanoarchaeota). Endonuclease architectures of pre-tRNA in each species are also denoted (a2b2: heterotetramer; a4: homotetramer; and a2:
homodimer). Thermoproteales, which are found to possess the largest amount of intron-containing tRNA genes, are indicated in bold characters.
Posterior probability values are denoted at each branch.

to be intron-containing tRNA genes and half of these genes
contained more than two introns.
To analyze the relative effect of the number of tRNA
introns and their divergence of location in the tRNA genes,
we compared the distribution of tRNA introns based on the
nucleotide position of tRNA gene in Thermoproteales andother archaeal species (fig. 3a). In total, 32 nt positions oftRNA genes in the 47 archaeal species were found to contain introns; 20 out of the 32 positions were found to be
Thermoproteales-specific intron location, 2 were found tobe specific to other archaeal species, and the remaining
10 positions were found to be common between Thermoproteales and other archaeal species. Although 83.4% oftRNA introns in archaeal species except Thermoproteales
were concentrated at a canonical position (nucleotide position at 37/38) of tRNA genes, only 24.6% of tRNA intronsin Thermoproteales were found at a canonical position, indicating wide diversity of tRNA intron locations in thisspecific order. Moreover, with the exception of Thermoproteales, all archaeal species tended to containintrons in the 5# half of tRNA gene, whereas tRNA intronsin Thermoproteales tended to be equally located in both 5#
and 3# halves of tRNA gene. Especially, archaeal species
that encoded homotetrameric or homodimeric endonucleases completely lacked introns at the 3# half of the tRNA
gene (supplementary fig. S3, Supplementary Material
online).

Rapid Increase of tRNA Introns in the Order
Thermoproteales
In this paper, comprehensive prediction and analysisof the tRNA genes from 47 archaeal species have discovered an extensive number of tRNA introns in five species ofthe order Thermoproteales. Further, the locations of tRNA
introns in Thermoproteales were found to be widely spread
across the nucleotide positions of tRNA genes comparedwith those in other archaeal species, and surprisingly, 20
intron positions were specific to the order. It is important
to determine whether the tRNA introns located at Thermoproteales-specific positions represent an ancient form oftRNA genes or whether these introns have been gained during a later stage of archaeal evolution. Therefore, we further
conducted sequence similarity analysis of archaeal tRNA
introns and compared the conservation rate among thetRNA intron groups shown in figure 3a: tRNA introns inarchaeal species except Thermoproteales, tRNA intronsin Thermoproteales located at common positions with otherarchaeal species, and tRNA introns located at Thermoproteales-specific positions. As a result, the tRNA introns in
Thermoproteales were found to be highly conserved withindifferent tRNA species compared with those in other archaeal species (fig. 3b). Especially, tRNA introns locatedat Thermoproteales-specific nucleotide positions showed
the most significant sequence conservation. For example,

Intron Increase during Archaeal tRNA Evolution 2713

FIG. 3.—Summary of location and sequence conservation of tRNA introns in Archaea. (a) Distribution of tRNA introns in archaeal tRNA genes.
Numbering of the nucleotide position of tRNA genes is according to Sprinzl and Vassilenko (2005). tRNA introns were classified into three groups:tRNA introns in all archaeal species except Thermoproteales (gray), tRNA introns in Thermoproteales located at common positions with other archaealtRNA introns (black), and tRNA introns in Thermoproteales located at Thermoproteales-specific positions (red). (b) Conservation rate of tRNA introns
for each group. Conservation rate is defined as the number of tRNA introns above the sequence similarity score threshold per total number of tRNA
introns in each group. Colors are the same as those in (a). See Materials and Methods for details of sequence similarity score calculation.

at sequence similarity thresholds above 80%, 42.7% (44 out
of 103) of tRNA introns located at Thermoprotealesspecific positions were conserved in different tRNA species
versus 16.2% (21 out of 129) of tRNA introns of Thermoproteales located at common positions and 0% (0 out of288) of other archaeal tRNA introns.
Examples of identical introns conserved in differenttRNA genes in Thermoproteales are presented in figure 4.
The introns located at Thermoproteales-specific positions
22/23 and 43/44 of the tRNAAsn (GTT) and tRNAIle(GAT) genes in T. pendens have 100% sequence similarity,even though the exon sequences of the tRNA genes themselves lack overall similarity (83.1%), suggesting different
evolutionary backgrounds (fig. 4a). From the genetic pointof view, it is difficult to consider that these introns wereindividually created in two tRNA genes and maintained
their sequences through evolution because the mutation rateof the intron region could be higher than that of the exon

region and would be rapidly modified. Therefore, we consider that intron insertion events could explain why similar
introns are found in divergent tRNA species in Thermoproteales, and the high conservation rate of tRNA introns located at Thermoproteales-specific positions strongly
suggests that these introns have been newly gained at a laterstage of tRNA evolution.
Moreover, we also found a positive correlation between the ratio of the number of tRNA genes possessing
a 3#-terminal CCA sequence and the conservation of tRNA
introns (supplementary fig. S4, Supplementary Material online). Although the ratio of the number of CCA sequence–possessing tRNA genes remained 30.3% (651 out of 2,143)of all archaeal tRNA genes, approximately 70% (49 out of71) of the tRNA genes containing highly conserved introns(sequence similarity ranging from 80% to 100%) were
found to be encoding the CCA sequence at their 3# terminal.
In Archaea, the CCA sequence is generally added by

2714 Sugahara et al.

FIG. 4.—Sequence alignment of tRNA genes containing identical intron sequences. (a) Sequences of tRNAAsn (GTT) and tRNAIle (GAT) genes in
Thermofilum pendens. (b) Sequences of tRNAPro (TGG) and tRNAAla (TGC) genes in Pyrobaculum calidifontis. Asterisks denote matching nucleotides
in the aligned tRNA gene sequences. All intron sequences are shown in lower case. Identical intron sequences are shown as outline characters on
a colored background. The sequences were aligned using ClustalW 2.0 (Larkin et al. 2007) with default parameters; then sequences were manuallyconfirmed to conduct correct structural alignment of tRNA gene.

a CCA-adding enzyme after gene transcription (Yue et al.
1996); therefore, the high rate of CCA sequences found at
the 3# terminal of tRNA genes which contain highly conserved introns suggests that intron insertion could have occurred via a retrotransposition event (supplementary fig.
S5a, Supplementary Material online). Further, we speculatethat it is possible that the trans-splicing event found in
Nanoarchaea which generates two chimeric tRNAs from
two alternative 5#-tRNA halves corresponding to a single
3#-tRNA half (Randau, Calvin, et al. 2005) may also create
identical introns in different tRNA genes (supplementary
fig. S5b, Supplementary Material online). The sequencesof the 3# halves of tRNAPro (TGG) and tRNAAla (TGC)
genes possessing identical introns at 53/54 found in P. calidifontis (fig. 4b) had significantly higher similarity (87.7%)than those of the 5# halves (46.5%), suggesting the possibility that two different tRNA genes containing identical
introns are created by retrotransposition via trans-splicing
of intron-containing pre-tRNA fragments.
Why are tRNA introns of these species increased?
Considering the splicing endonuclease evolution, the heterotetrameric endonuclease is estimated to have emerged after a gene duplication event of homotetramericendonuclease that strictly recognizes only the hBHBh# motif at a specific region of tRNA genes (supplementary fig.
S3, Supplementary Material online) (Tocchini-Valentini
et al. 2005b). Because all species of the order Thermoproteales possess heterotetrameric endonuclease which is capable of splicing out tRNA introns forming a relaxed HBh#
motif located at various noncanonical positions, we speculate that the expansion of substrate specificity of splicing
endonucleases may have contributed to the increase oftRNA introns. As another explanation, it is suggested thatthe presence of an intron in archaeal tRNA genes is a protective mechanism against integration of mobile genetic elements such as conjugative plasmids and viruses because
nonintronic tRNA genes are ideal targets of site-specific recombination (Randau and Söll 2008). Further investigationof archaeal tRNA genes including their splicing endonucleases and mobile genetic elements are necessary to substantiate this view.
Implementation and Web Interface of SPLITSdb
We have successfully completed encoding of all tRNA
genes required for reading 61 sense codons and an initiatorcodon in 45 of the 47 species. All sequence and secondary
structure data of the tRNA genes and the introns are registered in SPLITSdb (PostgreSQL version 8.2.4 running on
a Mac OS X server), a user-friendly web interface databasedeveloped for easy viewing and retrieval of tRNA gene
information by CGI scripts as outlined in figure 5. The options, ‘‘tRNA search’’ or ‘‘BHB search,’’ enable the user tosearch and screen for the required tRNA genes or BHB motifs. This system allows users to search the tRNA genes or
BHB motifs by selecting from one or several search conditions including ‘‘Organism,’’ ‘‘Anticodon,’’ ‘‘tRNA
type,’’ ‘‘Intron motif,’’ or ‘‘Intron location’’ (fig. 5a and b).
The search result is initially displayed in a table format(fig. 5c), and more detailed information such as sequences
and secondary structures is displayed (fig. 5d). Zipped files
containing all the information of tRNA genes and their
BHB motifs are also downloadable (fig. 5c). The home
page gives an overview of SPLITSdb that allows for easy
navigation throughout the database interface (fig. 5e). Asaround 90 archaeal genomes are being sequenced and moreare continually being sequenced (Liolios et al. 2008),
SPLITSdb is regularly being update with the latest archaealtRNA sequences.
Comparative studies of disrupted tRNA genes possessing a common BHB splicing motif that is found in various
primitive species will be important to evaluate the origin
and evolution of tRNA genes during the early stages of life.
Our comprehensive data set containing novel tRNA
gene candidates having multiple introns, together withSPLITSdb, which is the only database that includes split

Intron Increase during Archaeal tRNA Evolution 2715

FIG. 5.—Illustration of SPLITSdb interface. Gray boxes with dotted arrows indicate the links to following pages. (a) tRNA and BHB search engine
page options. (b) Example of a tRNA and BHB search. (c) Result of a tRNA search. The information listed in the table is downloadable as a text file byclicking on the download icon. (d) Example of detailed information of the results. (e) Home page with overview of SPLITSdb.

and intron-containing tRNA genes and their BHB motifs, will
provide a new molecular basis for upcoming tRNA studies.
Supplementary Material
Supplementary tables S1 and S2 and figures S1–S5 areavailable at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/).

Yoshinari (University of Tokyo, Japan), and Mr Keisuke
Morita (Keio University, Japan). This research was supported
in part by the Japan Society for the Promotion of Science;a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (The 21st Century COE Program, entitled Understanding and Control of Life’s Function
via Systems Biology); and research funds from the Yamagata
Prefectural Government and Tsuruoka City, Japan.

Acknowledgments
Many of the ideas presented in this paper were inspiredby discussions with Dr Yasuhiko Sekine, Dr Akiko Soma
(Rikkyo University, Japan), Dr Yohichi Watanabe, Dr Shigeo

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Takashi Gojobori, Associate Editor
Accepted September 24, 2008