Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5′- and 3′-halves

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Abstract
More evidence that gene discovery requires the skills of a detective. The hyperthermophile parasite Nanoarchaeum equitans has the smallest known genome. Too small, it seemed, when it was realized that it lacks genes for four transfer RNAs essential for protein biosynthesis. But the mystery of how N. equitans copes has been solved: it does things by halves. Computational analysis reveals the ‘missing’ tRNA genes as half-molecules in the genome. The structure of the tRNA pieces suggests a mechanism for joining the half-molecules, and the organism contains full-length active versions of these tRNAs. Split genes may be the mark of an ancient organism (N. equitans is a member of the Archaea) or may be a result of genome size reduction. Analysis of the genome sequence of the small hyperthermophilic archaeal parasite Nanoarchaeum equitans1,2 has not revealed genes encoding the glutamate, histidine, tryptophan and initiator methionine transfer RNA species. Here we develop a computational approach to genome analysis that searches for widely separated genes encoding tRNA halves that, on the basis of structural prediction, could form intact tRNA molecules. A search of the N. equitans genome reveals nine genes that encode tRNA halves; together they account for the missing tRNA genes. The tRNA sequences are split after the anticodon-adjacent position 37, the normal location of tRNA introns. The terminal sequences can be accommodated in an intervening sequence that includes a 12–14-nucleotide GC-rich RNA duplex between the end of the 5′ tRNA half and the beginning of the 3′ tRNA half. Reverse transcriptase polymerase chain reaction and aminoacylation experiments of N. equitans tRNA demonstrated maturation to full-size tRNA and acceptor activity of the tRNAHis and tRNAGlu species predicted in silico. As the joining mechanism possibly involves tRNA trans-splicing, the presence of an intron might have been required for early tRNA synthesis.