Identification of BHB splicing motifs in intron-containing tRNAs from 18 archaea: evolutionary implications

Abstract

Most introns of archaeal tRNA genes (tDNAs) are located in the anticodon loop, between nucleotides 37 and 38, the unique location of their eukaryotic counterparts. However, in several Archaea, mostly in Crenarchaeota, introns have been found at many other positions of the tDNAs. In the present work, we revisit and extend all previous findings concerning the identification, exact location, size, and possible fit to the proposed bulge-helix-bulge structural motif (BHB, now renamed hBHBh') of the sequences spanning intron-exon junctions in intron-containing tRNAs of 18 archaea. A total of 103 introns were found located at the usual position 37/38 and 33 introns at 14 other different positions, that is, in the anticodon stem and loop, in the D-and T-loops, in the V-arm, or in the amino acid arm. For introns located at 37/38 and elsewhere in the pre-tRNA, canonical hBHBh' motifs were not always found. Instead, a relaxed hBH or HBh' motif including the constant central 4-bp helix H flanked by one helix (h or h') on either side generating only one bulge could be disclosed. Also, for introns located elsewhere than at position 37/38, the hBHBh' (or HBh') structure competes with the three-dimensional structure of the mature tRNA, attesting to important structural rearrangements during the complex multistep maturation-splicing processes. A homotetramer-type of splicing endonuclease (like in all Crenarchaeota) instead of a homodimeric-type of enzyme (as in most Euryarchaeota) appears to best fit the requirement for splicing introns at relaxed hBH or HBh' motifs, and may represent the most primitive form of this enzyme.

FIGURE 1.

Location, size, and BHB type of introns in the tRNA genes of 18 Archaea. The schematic cloverleaf sequence corresponds to the consensus sequence of all the tDNAs (257 altogether) from the six archaea that contain introns at positions other than 37/38 (33 altogether; see Table 1 ▶). The conventional IUB/IUPAC degenerate DNA alphabet (Cornish-Bowden 1985) is used in this and the following figures: 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. Key to base-pairing consensus is: (plus), Watson–Crick base pairing only; (asterisk), Watson–Crick or G-T/T-G pairings; (number sign), Watson–Crick pairing or mismatch; (minus), Watson–Crick pairing or G-T/T-G pairings or mismatches. Arrows show the positions where introns are located. Their exact locations are also indicated in the first line of each box (for example, 3/4 means that the intron is located between nt 3 and nt 4. Every intron is indicated with the abbreviated name of the species [with those of P. aerophilum (P. aero) highlighted in bold], the tDNA type (amino acid), the length of the intron, and its type of splicing motif (hBHBh′, hBH, or HBh′; see text). The 1/2 and 2/2 indications refer to the six cases of two introns within the same tDNA. In three of these six cases, one of the introns is located at 37/38 and is indicated accordingly. All tDNAs bearing introns in the 18 archaeons examined are essential and single-copy genes, except the case of tDNA-Glu (TTC) of M. kandleri (two copies; see also Table 1 ▶).

FIGURE 2.

Intron–exon sequences in selected archaeal tDNAs sorted out as a function of the type of their splicing motifs. The details of the sequences of all introns presented in Figure 1 ▶ are given here. Additionally, all introns (any type) of S. tokodaii are illustrated. (A) Only 30 sequences of intron–exon junctions (out of a total of 103 sequences in the 800 tDNAs analyzed; see Supplementary Material) corresponding to intron located at position 37/38 are listed. The sequences shown extend from base 27 to base 43 of the anticodon arm. Uppercase letters correspond to exonic sequence and the lowercase letters to the intronic sequence. The bases in the H helix (under headings H1 and H2; gray background) are in bold; complementary bases in h and h′ helices (strands h1, h′1, h′2 and h2) are in bold italics. Mismatches in H helix are shown with inverted case: lowercase in H1 (exon) and uppercase in H2 (intron). Those that exhibit an intronic h′ helix of two or more base pairs are designated as canonical hBHBh′, and those with an intronic h′ helix shorter than 2 bp as relaxed hBH (see examples of 2D structures in Fig. 3 ▶). Notice that, for the hBHBh′ type, all elements of the splicing motif can be defined (h1, H1, B1, h′1, loop, h′2, H2, B2, and h2) whereas for the hBH type, only h1, h2, H1, H2, and B2 can be defined; the intronic h′1, h′2, and B1 elements cannot. At the bottom of A, is the consensus sequence of the splicing motif corresponding to the 30 sequences shown here or in all the 103 sequences (see Supplementary Material). Mismatched bases were not taken into account for consensus computation. For the degenerate alphabet used, see legend to Figure 1 ▶. (B) The 33 sequences of intron–exon junctions corresponding to introns located at positions other than 37/38 in the 800 tDNAs analyzed are listed as a function of the decreasing size of their h helix (exonic). The sequences shown extend from five bases upstream of the 5′ end of strand H1 down to five bases downstream from the 3′ base of the intron. The same lettering conventions as above are used. Those that exhibit an h helix of two or more base pairs are designated as canonical hBHBh′, and those with an exonic h helix shorter than 2 bp as relaxed HBh′. In this latter case, only H1, B1, h′1, h′2, and H2 are present and, at variance with the above hBH motif, no exonic h1, h2, and B2 elements can be defined. Vertical linking lines at right show the six cases of two introns in the same pre-tRNA. Numbering at the extreme right of the figure corresponds to the figure numbers in which the 2D structures are shown. At the bottom of B is the consensus sequence of the splicing motif corresponding to the 33 sequences listed in this panel. (C) generalized consensus from sequences shown in A and B and from all 136 introns in tDNAs of the genomes of the 18 archaeons analyzed. Mismatched bases in H helix were not taken into account for consensus computation.

FIGURE 3.

Examples of splicing motifs present in archaeal pre-tRNAs bearing introns at positions 37/38 and others. (A,B,C) The cloverleaf sequences of three characteristic tDNAs harboring an intron between nt 37 and 38. (D,E) Introns located at unconventional positions. Small arrows show the intron splicing position, always located two bases downstream of the H1 and H2 strands of the central H helix. The central 4-bp H helix is highlighted with a gray background. (A) As examplified with pre-tRNA-Lys (UUU) from S. tokodaii, this panel shows the most often encountered splicing motif hBHBh′ (67 times over a total of 103 cases; see Supplementary Material). In this case, the exonic h helix (boxed) is formed by the antiparallel strands h1 and h2 of the anticodon stem. The central H helix (gray background) includes the first two bases of the anticodon (boxed). The third helix, h′ (boxed), is formed within the intron (here, 25 nt in length). The three h, H, and h′ helices allow for the formation of two bulges, B1 and B2. The intron is always spliced symmetrically two bases downstream of strands H1 and H2 of helix H. (B) In the pre-tRNA-Ser (UGA) of S. tokodaii, the intron (11 nt) located between nt 37 and 38 is too short to allow the helix h′ and the bulge B1 to be formed. This type of relaxed splicing motif is referred to as relaxed hBH. (C) In the pre-tRNA-Thr (UGU) from S. tokodaii, although this pre-tRNA harbors a 17-nt-long intron, the intron–exon junction can only fold into a relaxed hBH type of splicing motif. No complementary bases are present within the intron, preventing the formation of the intronic h′ helix. (D) Hypothetical folding of a 20-nt intron located at position 29/30 within the pre-tRNA-Val (GAC) of P. aerophilum. Bases 20–34 are replaced within the conventional cloverleaf scheme by asterisks and are drawn in an alternative 2D structure that fits the splicing motif requirement. In this pre-tRNA, formation of the canonical splicing motif hBHBh′ obviously competes with the formation of the regular D- and anticodon stems. (E) Hypothetical folding of a 36-nt intron located at position 45/46 in the pre-tRNA-Lys (CUU) of A. pernix. Here, bases 35–51 of the conventional cloverleaf are replaced by asterisks and are redrawn to fit a relaxed splicing motif HBh′, in which a helix h cannot be formed because of the absence of the needed complementary bases (dotted box). This putative folding obviously competes with the formation of the regular anticodon and T-stems.

FIGURE 4.

A novel intron is located at position 3/4 in three tDNAs of P. aerophilum. (A) The sequences of the three tDNAs of P. aerophilum coding for tRNA-Asp (GUC) (upper line), tRNA-Glu (CUC) (middle line), and tRNA-Glu (UUC) (lower line) are shown. They extend from the putative upstream TATA promoter element (black bold) down to position 76 of the mature tRNA. Bases in uppercase letters belong to the mature tRNA, those in lowercase to the intron and mature tRNA flanking sequences. The bases making up the putative intron located between nt 3 and nt 4 of the amino acid acceptor stem, including the intronic helix h, are shown in lowercase letters (within the box labeled “intron 3/4”). The sequence elements forming the canonical splicing motif hBHBh′ (h1, H1, B1, h′1, loop, h′2, H2, B2, and h2) are shown in bold (for a definition of these elements, see text and legend to Fig. 2 ▶). Bases forming the H1 and H2 strands are bold and highlighted with gray background. Bold italics are used to indicate the complementary bases forming the h1 and h2 antiparallel strands of the helix h, and the h′1 and h′2 antiparallel strands of the helix h′. Among these three tDNAs, only one, tDNA-Glu (TTC), harbors a second intron located between nt 58 and 59 (box labeled “intron 58/59”). (B) Hypothetical 2D structure of pre-tRNA-Glu (CUC) including the splicing motif within the amino acid acceptor stem. The sequence shown here, including the cloverleaf-like structure, extends from 31 bases upstream from tRNA base 1 down to base 73 at the 3′ end of the tRNA. Plus signs are used to indicate Watson–Crick base pairing and filled circles to indicate wobble GU/UG pairings. The intron (in lowercase) is shown folded under a canonical hBHBh′ splicing motif, using the same lettering conventions as in A. The small arrows indicate the splicing positions after nt 3 and before nt 4. The path of the nucleotidic chain is symbolized with thick gray lines. Remarkably, a peculiar gccccgg sequence is present in the 5′ region of the three tRNA genes, which is an exact replica of bases 1–7 of the mature tRNA. This sequence, designated h"1 is probably part of the pre-tRNA transcript, as it is located 20 bases 3′ of the TATA element. It is shown here facing the antiparallel strand of the acceptor stem (bases 66–72, designated h"2) because it is not known whether a helix between complementary strands h"1 and h"2 can form.

FIGURE 5.

Ordered sequential intron splicing has to occur in certain pre-tRNAs harboring two introns. Pre-tRNA-Pro (GGG) of M. thermoautotrophicum contains two introns in the anticodon loop, one between nt 32 and 33 (32 nt in length) and the second one between nt 37 and 38 (16 nt in length). In the conventional cloverleaf scheme, bases 21–39 are replaced by asterisks. They are redrawn in an alternative 2D structure that fits the splicing motif requirement. The same lettering conventions as in Figures 1 ▶–4 ▶ are used. Only the hypothetical folding of the noncanonically located 32-nt intron is shown. In this pre-tRNA, the formation of the canonical splicing motif hBHBh′ (allowing the intron at position 32/33 to be spliced) obviously competes with the formation of the splicing motif corresponding to the intron located at the usual position 37/38 (boxed). Only after removal of the intron at position 32/33, can the intron–exon junction corresponding to intron 37/38 be folded into another hBHBh′ splicing motif (folding not shown).

FIGURE 6.

Endonuclease-based phylogeny of pre-tRNA introns splicing motifs and related features. The homotetrameric α₄ endonuclease splicing at characteristic intron–exon junctions of archaeal pre-tRNAs (present in most Crenarchaeota and a few Euryarchaeota) is assumed to have evolved divergently towards a homodimeric β₂ enzyme in Euryarchaeota and into a heterotetrameric α,β,δ,γ enzyme in Eukarya (see text). The monomer of the homotetrameric ancestral splicing endonuclease is designated by “α”; that of the homodimeric enzyme is designated by “β” and the four different subunits of the eukaryotic heterotetrameric enzyme by “α, β, γ, and δ”. Note that the size of the β subunit is about twice that of the α subunit and probably results from gene duplication and subsequent fusion of an ancestral α-like subunit gene (Lykke-Andersen and Garrett 1997; Bujnicki and Rychlewski 2000; Li and Abelson 2000).