Discovery of permuted and recently split transfer RNAs in Archaea

Abstract

Background: As in eukaryotes, precursor transfer RNAs in Archaea often contain introns that are removed in tRNA maturation. Two unrelated archaeal species display unique pre-tRNA processing complexity in the form of split tRNA genes, in which two to three segments of tRNAs are transcribed from different loci, then trans-spliced to form a mature tRNA. Another rare type of pre-tRNA, found only in eukaryotic algae, is permuted, where the 3' half is encoded upstream of the 5' half, and must be processed to be functional.

Results: Using an improved version of the gene-finding program tRNAscan-SE, comparative analyses and experimental verifications, we have now identified four novel trans-spliced tRNA genes, each in a different species of the Desulfurococcales branch of the Archaea: tRNA(Asp(GUC)) in Aeropyrum pernix and Thermosphaera aggregans, and tRNA(Lys(CUU)) in Staphylothermus hellenicus and Staphylothermus marinus. Each of these includes features surprisingly similar to previously studied split tRNAs, yet comparative genomic context analysis and phylogenetic distribution suggest several independent, relatively recent splitting events. Additionally, we identified the first examples of permuted tRNA genes in Archaea: tRNA(iMet(CAU)) and tRNA(Tyr(GUA)) in Thermofilum pendens, which appear to be permuted in the same arrangement seen previously in red alga.

Conclusions: Our findings illustrate that split tRNAs are sporadically spread across a major branch of the Archaea, and that permuted tRNAs are a new shared characteristic between archaeal and eukaryotic species. The split tRNA discoveries also provide new clues to their evolutionary history, supporting hypotheses for recent acquisition via viral or other mobile elements.

Figure 1

Predicted secondary structures of trans-spliced and permuted precursor tRNAs. (a) Mature tRNA^Asp(GUC) in A. pernix and T. aggregans are formed by joining the 5' half and the 3' half at position 37/38 after splicing at the bulge-helix-bulge (BHB) motif. (b) The 5' half and the 3' half of trans-spliced tRNA^Lys(CUU) in Staphylothermus hellenicus and S. marinus marinus join at position 30/31, same as the previously identified split tRNA^Lys(CUU) in N. equitans [5]. (c) Circularized permuted tRNA^iMet(CAU) and tRNA^Tyr(GUA) in Thermofilum pendens have the 3' half located upstream of the 5' half separated by intervening sequences represented in green. The two fragments join at position 59/60, same as the T-Ψ-C loop permuted tRNAs in the red alga C. merolae [9]. Pre-tRNA^Ala(UGC) in C. merolae is shown for comparison. The 5' half of tRNA transcripts are represented in blue, the 3' halves in orange. Black arrows indicate positions of splicing. Anticodons are boxed in light blue.

Figure 2

RT-PCR and northern analysis of tRNA^Asp(GUC) in A. pernix. (a) Expression analysis of mature tRNA^Asp(GUC) (Mat), 5' half of pre-tRNA^Asp(GUC) (5' h), and 3' half of pre-tRNA^Asp(GUC) (3' h) using RT-PCR. M represents the 10-bp DNA ladder. The band sizes correspond to the sizes of the PCR products based on selected primers, but not the transcript sizes of the mature tRNA and the halves. Negative controls without reverse transcriptase (RT (-)) displayed no PCR products in comparison. (b) Northern analysis of tRNA^Asp(GUC) using radiolabeled DNA probe that spans the mature tRNA^Asp(GUC) and the region between the two fragments. The mature tRNA, 5' half transcript and the 3' half transcript are as marked. No expression was found corresponding to the 199-nucleotide transcript originally predicted as pre-tRNA^Asp(GUC) with a 121-nucleotide intron. Bands at approximately 90 nucleotides and 125 nucleotides are expected due to cross-hybridization of highly similar tRNA sequences in other tRNA transcripts. M1 and M2 represent the 10-bp and 100-bp RNA ladders, respectively.

Figure 3

Proposed evolutionary relationship between tRNA^Asp(GUC) in D. kamchatkensis, A. pernix, and T. aggregans. D. kamchatkensis has a typical linear tRNA^Asp(GUC) which represents the likely ancestor of the split tRNA^Asp(GUC) genes in A. pernix and T. aggregans. The 5' and 3' halves of pre-tRNA^Asp(GUC) are located adjacent to each other in A. pernix and T. aggregans. The two halves in A. pernix are transcribed on the forward strand while those in T. aggregans are transcribed on opposite strands. Breaks in synteny were observed between the tRNA halves and upstream of the 5' half in A. pernix.

Figure 4

Phylogenetic distribution of trans-spliced and permuted tRNAs in Archaea. Trans-spliced tRNAs were identified in A. pernix, T. aggregans, S. marinus, and S. marinus in this study, in N. equitans by Randau and colleagues [4,5], and in C. maquilingensis by Fujishima and colleagues [8] (highlighted in gray). Permuted tRNAs were found in T. pendens in this study (yellow), and are most similar to the permuted tRNAs found in Cyanidioschyzon merolae, a eukaryotic algal species that is not included in the tree but would be a distant outgroup to the archaea shown here. The phylogenetic tree was generated based on the concatenation of 23S and 16S ribosomal RNAs. Sequences were aligned using ClustalW [48]. Alignments were manually adjusted using Jalview [49] to remove introns. The maximum likelihood tree was computed using PhyML [50] with general time-reversible model of sequence evolution. Numbers at nodes represent non-parametric bootstrap values computed by PhyML [50] with 1,000 replications of the original dataset.