In silico screening of archaeal tRNA-encoding genes having multiple introns with bulge-helix-bulge splicing motifs

Abstract

In archaeal species, several transfer RNA genes have been reported to contain endogenous introns. Although most of the introns are located at anticodon loop regions between nucleotide positions 37 and 38, a number of introns at noncanonical sites and six cases of tRNA genes containing two introns have also been documented. However, these tRNA genes are often missed by tRNAscan-SE, the software most widely used for the annotation of tRNA genes. We previously developed SPLITS, a computational tool to identify tRNA genes containing one intron at a noncanonical position on the basis of its discriminative splicing motif, but the software was limited in the detection of tRNA genes with multiple introns at noncanonical sites. In this study, we initially updated the system as SPLITSX in order to correctly predict known tRNA genes as well as novel ones with multiple introns. By a comprehensive search for tRNA genes in 29 archaeal genomes using SPLITSX, we listed 43 novel candidates that contain introns at noncanonical sites. As a result, 15 contained two introns and three contained three introns within the respective putative tRNA genes. Moreover, the candidates completely complemented all the codons of two archaeal species of uncultured methanogenic archaeon, RC-I and Thermofilum pendens Hrk 5, with novel candidates that were not detectable by tRNAscan-SE alone.

FIGURE 1.

Codon tables of T. pendens showing the numbers of tRNAs of respective codons. The leftmost column, the top row, and the rightmost column indicate the first, second, and third bases of sense codons, and their coding amino acids are shown in respective fields. (A) Codon table including the 39 tDNA candidates predicted by tRNAscan-SE alone. (B) Codon table including the 46 tDNA candidates complemented and reassigned by SPLITSX, with 10 candidates identified only by tRNAscan-SE.

FIGURE 2.

Locations of introns of predicted tDNA genes in 11 Archaea. The schematic cloverleaf structure corresponding to the consensus sequence of archaebacterial tRNA sequences has been modified in accordance with the results of a previous work (Marck and Grosjean 2003). The conventional IUB/UPAC degenerate DNA alphabet is used in this figure: R (purine), A or G; Y (pyrimidine), C or T; S (strong), G or C; B (not A), C, G, or T; D (not C), A, G, or T; H (not G), A, C or T; V (not T), A, C, or G; N (any), A, C, G, or T. Base-pairing consensus is denoted by: (+) Watson–Crick base pairing only; (*) Watson–Crick or G-T/T-G pairings; (#) Watson–Crick pairing or mismatch; (−), Watson–Crick pairing or G-T/T-G pairings or mismatches. Intron positions of documented or novel candidates are shown by thin and bold arrows, and by clear and solid tabs over the text boxes, respectively. Each candidate is listed in the text boxes along with the isotype of the amino acid charge in parentheses. The intron length and type of bulge-helix-bulge (BHB) structure are indicated to the right of the colons. The number denotes the nucleotide length, and the capital letter denotes the type of BHB structure: (S) strict hBHBh′ motif, (R) relaxed HBh′ motif. Novel candidates are indicated by bold text.

FIGURE 3.

Schematic diagrams of novel tRNA-encoding genes including multiple introns predicted by SPLITSX, accompanied by COVE scores and free energies of BHB motif. Intron and exon sequences are represented by solid and clear circles, respectively. (Arrows) Location where introns are located. (A) TP21, a putative tRNA^Asp with two introns located between nucleotide positions 24 and 25 (24/25) and 45/46 encoded in the T. pendens genomic DNA. The respective position weight matrix (PWM) scores of the BHB motifs were 0.86 and 0.88. (B) TP09, a tRNA^Ile with two introns at 22/23 and 43/44 synthesized according to the BHB motif with PWM scores 0.85 and 0.63, in T. pendens. (C) MH01, tRNA^His with double introns at 34/35 and 37/38 synthesized according to the BHB motif with PWM scores of 0.67 and 0.90, in the Methanospirillum hungatei.

FIGURE 4.

Schematic representation of the synthesis procedure in the maturation of tRNA candidate TP29, with three introns located at 32/33, 37/38, and 45/46, along with the COVE score and the free energies of the BHB motifs. (Arrows) Locations where introns were inserted. The position weight matrix (PWM) scores of the respective BHB motifs were 0.74, 0.87, and 0.88. Intron and exon sequences are represented by solid and clear circles, respectively. Two introns that have the potential to form BHB motifs might initially be spliced out at positions 32/33 and 45/46 (A), then another BHB motif is predicted to occur and to be spliced out at position 37/38 (B). Finally, the putative tDNA region of TP29 may form tRNA^Pro (C).

FIGURE 5.

Comparison of the P. aerophilum tRNA^His (GUG) structure previously annotated by Marck and Grosjean (2003) with that reassigned by SPLITSX, along with their COVE scores and free energies of the BHB motif. Intron and exon sequences are represented by solid and clear circles, respectively. (A) The previous pre-tRNA^His (GUG) harboring the intron at 37/38 predicted by tRNAscan-SE, (C) the reassigned pre-tRNA^His (GUG) harboring the intron at 44/45 predicted by SPLITSX. Mature tRNA structures are displayed in B and D, respectively. (Arrows) Locations where introns were inserted, (x) a mismatch of base paring in the BHB motif.