Sequence evidence in the archaeal genomes that tRNAs emerged through the combination of ancestral genes as 5' and 3' tRNA halves

Abstract

The discovery of separate 5' and 3' halves of transfer RNA (tRNA) molecules-so-called split tRNA-in the archaeal parasite Nanoarchaeum equitans made us wonder whether ancestral tRNA was encoded on 1 or 2 genes. We performed a comprehensive phylogenetic analysis of tRNAs in 45 archaeal species to explore the relationship between the three types of tRNAs (nonintronic, intronic and split). We classified 1953 mature tRNA sequences into 22 clusters. All split tRNAs have shown phylogenetic relationships with other tRNAs possessing the same anticodon. We also mimicked split tRNA by artificially separating the tRNA sequences of 7 primitive archaeal species at the anticodon and analyzed the sequence similarity and diversity of the 5' and 3' tRNA halves. Network analysis revealed specific characteristics of and topological differences between the 5' and 3' tRNA halves: the 5' half sequences were categorized into 6 distinct groups with a sequence similarity of >80%, while the 3' half sequences were categorized into 9 groups with a higher sequence similarity of >88%, suggesting different evolutionary backgrounds of the 2 halves. Furthermore, the combinations of 5' and 3' halves corresponded with the variation of amino acids in the codon table. We found not only universally conserved combinations of 5'-3' tRNA halves in tRNA(iMet), tRNA(Thr), tRNA(Ile), tRNA(Gly), tRNA(Gln), tRNA(Glu), tRNA(Asp), tRNA(Lys), tRNA(Arg) and tRNA(Leu) but also phylum-specific combinations in tRNA(Pro), tRNA(Ala), and tRNA(Trp). Our results support the idea that tRNA emerged through the combination of separate genes and explain the sequence diversity that arose during archaeal tRNA evolution.

Figure 1. Phylogenetic tree of mature tRNA sequences in 45 archaeal species.

The phylogenetic neighbor-joining tree was constructed by using mature sequences of 1953 predicted tRNAs from the complete genomes of 45 archaeal species. Clusters are numbered from 1 to 22, and tRNAs within each cluster are denoted by amino acids corresponding to the anticodon. Clusters including tRNAs from all 3 archaeal phyla (Euryarchaeota, Crenarchaeota, and Nanoarchaeota) are shaded in blue. The amino acids corresponding to the split tRNAs are boxed.

Figure 2. Comparison of the 3 types of tRNA sequences.

(A) Full nucleotide sequences of pre-tRNAs (1 split tRNA^Lys [Neq] and 2 intronic tRNA^Args [Pae]) and 2 nonintronic tRNA^Lys (Pfu and Afu) were aligned. Black bar marks the intron of the intronic tRNAs and the leader sequences of the split tRNAs, which are inserted at tRNA nucleotide position 32/33. Red bar marks the anticodon. (B) Comparison of the secondary structures and nucleotide sequences around the exon–intron boundary of the 3 types of tRNAs. Nucleotides that are identical between leader sequence and intron are shown in red. Red bar marks the anticodon.

Figure 3. Network analysis based on the sequence similarities of 5′ and 3′ tRNA halves.

A total of 296 mature tRNA sequences from 7 archaeal species (Neq, Sso, Ape, Pae, Mka, Pfu, Mja) were artificially split into 5′ and 3′ halves at the anti-codon region. Each node (colored dot) represents a tRNA half, and its color indicates the charged amino acid's chemical properties (DE, mid-green; MNQ, light green; RKH, blue; FWY, purple; AGP, red; ILV, orange; CST, yellow; iMet, gray). Nodes are linked by a white line (edge) when the sequence similarity is above the threshold. (A) Network created by set of 5′ half sequences with thresholds of >70%, >75%, and >80%. The sequences are classified into 6 clusters (1–6) at a threshold of >80%. (B) Network created by set of 3′ half sequences with thresholds of >80%, >85%, and >88%. The sequences are classified into 9 clusters (A–I) at a threshold of >88%.

Figure 4. Distribution of networks based on the 5′ and 3′ tRNA sequences.

Relation between the number of tRNA sequences and the number of links is represented at 4 sequence similarity thresholds (A–D). Clustering coefficient c is denoted for each tRNA halves.

Figure 5. Representation of N. equitans codon table filled with 5′ and 3′ tRNA halves.

The table is filled with the group IDs (see Fig. 3) corresponding to each of the 5′ and 3′ tRNA halves in N. equitans (Neq). The anti-codon corresponding to the 6 split tRNAs is shown in red. An asterisk indicates that a sequence does not have a similar sequence above the threshold (5′ half, 80%; 3′ half, 88%).

Figure 6. Examples of tRNA sequences explained by specific combinations of 5′ and 3′ tRNA halves.

(A) Examples of tRNA sequences with different 5′ half sequences but a common 3′ half sequence. (B) Example of tRNA sequences with different 3′ half sequences but a common 5′ half sequence. Asterisk denotes matching nucleotides in the aligned tRNA sequences. Boxes delineate sequences belonging to the same group in the sequence similarity network (Fig. 3). The corresponding group ID is labeled in a black box.