Tri-split tRNA is a transfer RNA made from 3 transcripts that provides insight into the evolution of fragmented tRNAs in archaea

Abstract

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.

Fig. 1.

Alternative splicing of 5 split tRNA^Gly transcripts in C. maquilingensis. (A) Schematic diagrams of the 3 novel tRNA^Gly assembly models. Three potential mature joined tRNAs formed by alternative assembly of 5 split tRNA^Gly transcripts (I–V) are shown: I + II, tRNA^Gly (CCC); III + IV + II, tRNA^Gly (TCC); and III + V + II, tRNA^Gly (GCC). Anticodons are boxed. The locations of bulge–helix–bulge (BHB) splicing motifs represent each leader–exon junction (arrowhead). (B) Nucleotide sequence alignment of the 3 pretRNA^Gly candidates with the inclusion of gaps (−). Identical nucleotide sequences are indicated by asterisks. Leader sequences are boxed. Exon sequences are colored by transcript (I, red; II, blue; III, light blue; IV, orange; and V, pink).

Fig. 2.

A combination of 10 novel split tRNAs and their gene expression. (A) Assembly models of 6 different mature joined tRNAs in C. maquilingensis. The combination of 5 transcripts forms 3 mature tRNA^Gly (CCC/TCC/GCC), 3 transcripts form 2 mature tRNA^Ala (CGC/TGC), and 2 transcripts form 1 mature tRNA^Glu (TTC). Exon sequences are colored by transcript (I, red; II, blue; III, light blue; IV, orange; V, pink; VI, gray; VII, light green; VIII, green; IX, purple; and X, brown). Expression of (B) each split tRNA gene and (C) mature joined tRNAs and intermediate tRNA^Gly's was confirmed by RT-PCR. Samples without reverse transcriptase were used as negative controls (RT−) to ensure that only RNA transcripts were amplified (RT+). EtBr-stained DNA bands were detected as negative images by Molecular Imager FX Pro (Bio-Rad Laboratories). Red marks indicate each position of target transcripts.

Fig. 3.

Consensus between split tRNAs and intron-containing tRNAs in Archaea. (A) Nucleotide sequence alignment of 26 archaeal tRNA intron and leader sequences intervening at location 29/30 of 6 species in the Thermoproteales order. The visualization of consensus sequences was created by WebLogo (31). Abbreviations of the species names are as follows: Cma, C. maquilingensis; Pae, Pyrobaculum aerophilum; Par, P. arsenaticum; Pca, P. calidifontis; Pis, P. islandicum; and Tne, Thermoproteus neutrophilus. (B) RNA secondary structure of the intron/leader–exon junction at position 29/30 of 3 intron-containing tRNAs and split tRNA^Ala (TGC). Sequence identity (percentage) is denoted for each intron/leader sequence compared with the intron sequence of intron-containing tRNA^Ala (TGC) from P. islandicum (100%).

Fig. 4.

Schematic of archaeal tRNA evolution. The relation of 5 unique types of tRNAs is represented with exon (red) and intron/leader (blue) sequences indicated. Possible evolutionary direction via tRNA splicing (black arrow), reverse direction of tRNA splicing (dotted black arrow), intron translocation (blue arrow), and sequence evidence indicating the relationship between split tRNA and intron-containing tRNA (see Fig. 3 for details) (pink arrows) are shown.