The 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 from 47 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 be approximately 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 found to 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 sequence similarity, 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.

Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature tRNA Gly 's with synonymous codons are created from 1 constitutive 3 half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a ''tri-split tRNA.'' Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in intron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs.tRNA intron ͉ RNA processing ͉ molecular evolution ͉ trans-splicing ͉ Caldivirga maquilingensis T he origin and evolution of tRNA is one of the most important subjects being discussed in the field of molecular evolution, with varying hypotheses being proposed (1-6). Three types of tRNA genes have previously been identified in archaeal genomes: nonintronic tRNA, which is encoded on a single gene with no intron sequence; intron-containing tRNA, which is encoded on a single gene with a maximum of 3 introns punctuating the mature tRNA sequence at various locations (7-9); and trans-spliced tRNA-so-called split tRNA-which has 5Ј and 3Ј halves encoded on 2 separate genes found only in the hyperthermophilic archaeal parasite, Nanoarchaeum equitans (10). Interestingly, split tRNA and intron-containing tRNA share a common bulge-helix-bulge (BHB) consensus motif around the intron/leader-exon boundaries that can be cleaved by the same tRNA splicing endonucleases (11,12). BHB motifs are further classified by their structure into the canonical form (hBHBhЈ) and relaxed forms (HBhЈ and BHL). The discovery of these tRNA genes raised the question of whether ancestral tRNA was encoded on a single gene or on separate genes. Our previous study has shown clear phylogenetic relationships among these 3 types of archaeal tRNAs (13). Because N. equitans is a parasite with indications of a massive genome reduction (14), whether its tRNAs represent the ancient form of tRNA or a later product of genome reduction is still unclear. Therefore, we have been conducting comprehensive prediction and analysis of tRNA sequences in various species on the basis of our original software, SPLITS (8,9,13,15,16). We have especially focused on the hyperther...

Transfer RNA (tRNA) is widely known for its key role in decoding mRNA into protein. Despite their necessity and relatively short nucleotide sequences, a large diversity of gene structures and RNA secondary structures of pre-tRNAs and mature tRNAs have recently been discovered in the three domains of life. Growing evidences of disrupted tRNA genes in the genomes of Archaea reveals unique gene structures such as, intron-containing tRNA, split tRNA, and permuted tRNA. Coding sequence for these tRNAs are either separated with introns, fragmented, or permuted at the genome level. Although evolutionary scenario behind the tRNA gene disruption is still unclear, diversity of tRNA structure seems to be co-evolved with their processing enzyme, so-called RNA splicing endonuclease. Metazoan mitochondrial tRNAs (mtRNAs) are known for their unique lack of either one or two arms from the typical tRNA cloverleaf structure, while still maintaining functionality. Recently identified nematode-specific V-arm containing tRNAs (nev-tRNAs) possess long variable arms that are specific to eukaryotic class II tRNASer and tRNALeu but also decode class I tRNA codons. Moreover, many tRNA-like sequences have been found in the genomes of different organisms and viruses. Thus, this review is aimed to cover the latest knowledge on tRNA gene diversity and further recapitulate the evolutionary and biological aspects that caused such uniqueness.