Two reactions of Haloferax volcanii RNA splicing enzymes: joining of exons and circularization of introns

Abstract

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon-intron junctions, producing 2',3'-cyclic phosphate and 5'-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3' terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5' and 3' flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3',5'-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.

FIGURE 1.

Sequences of pre-tRNA^Trp (A), elongator pre-tRNA^Met (D), and their spliced products: ligated exons (B and E) and circularized introns (C and F). Arrowheads in A and D denote the splice sites within the bulge-helix-bulge (BHB) motifs. Anticodons are highlighted. Asterisks in B, C, E, and F denote the splice junctions. Positions corresponding to the oligonucleotides used for RT-PCR (Figs. 2B, 3A ▶ ▶) and Northern hybridizations (Fig. 4 ▶) are labeled and marked by the lines ending in half arrows. Bases within parentheses in C indicate the changes in various synthetic intron-like transcripts (Fig. 8 ▶).

FIGURE 2.

Analyses of the ligase reaction products. (A) [α-³²P]CTP labeled pre-tRNA^Trp transcripts (lanes 1 and 4) are used for endonuclease reactions (lanes 2 and 5) followed by ligase reactions (lanes 3 and 6). The products are resolved by denaturing PAGE. Lanes 1–3, 6% gel, and lanes 4–6, 8% gel. (P) Precursor; (LI) linear intron; (E) exons; (LE) ligated exons; (CI-6) circular intron in 6% gel; (CI-8) circular intron in 8% gel; (XC) xylene cyanol; (BB) bromophenol blue. (B) RT-PCR products using tRNA^Trp intron-specific primers H and I (Fig. 1C ▶), and gel-eluted circular introns (A, CI-6 and CI-8) as template are resolved by native 6% PAGE (lane 1). Lane 2 contains a 25-bp DNA ladder. (C) The 95-bp RT-PCR product (B, lane 1) is sequenced using primer I. Arrow denotes the position of the splice junction (asterisk in Fig. 1C ▶) in the sequence of the circularized intron.

FIGURE 3.

Determination of the in vivo presence of pre-tRNAs and circular introns. (A) RT-PCR products using H. volcanii total RNA and various primers are separated on a native 6% polyacrylamide gel. The primers (Fig. 1 ▶) used, expected sizes of the products, and the template regions corresponding to these products (thick lines in the schematic diagrams) are indicated below the lanes. Lane 4 contains a 25-bp DNA ladder. (B) The 69-bp RT-PCR product in A, lane 5, is sequenced using primer L. The arrow denotes the position of the splice junction (Fig. 1F ▶, asterisk) in the sequence of the circularized intron derived from elongator pre-tRNA^Met.

FIGURE 4.

Northern analysis to detect in vivo presence of circular introns. Northern blots of H. volcanii total RNA separated by denaturing 6% (lanes 1–3) and 8% (lanes 4–6) PAGE are hybridized to 5′ ³²P-labeled tRNA^Trp-specific oligonucleotides (marked in Figs. 1A,C). The oligonucleotides used are as follows: H (intron-specific) in lanes 1 and 6, N (circular intron-specific, overlapping splice junction region) in lanes 2 and 5, and B (exon-specific) in lanes 3 and 4. (P) Precursor; CI-6 and CI-8, circular introns in 6% and 8% gels, respectively; LI-6 and LI-8, linear introns in 6% and 8% gels, respectively; t, tRNA.

FIGURE 5.

Splicing of truncated elongator pre-tRNA^Met. Sequences of the precursor (A) and its circularized intron (B). The anticodon is highlighted in A. Arrows indicate the splice sites within the BHB motif in A and the junction in B. Phosphates derived from [α-³²P]ATP labeling of the precursor are denoted by asterisks in A and B. Products of endonuclease (C, lane 1) followed by ligase (C, lane 2) using [α-³²P]ATP-labeled precursor are separated on a 50-cm long, denaturing 15% gel. (P) Precursor; (LI) linear intron; (5′+3′) two exons together; (3′) only 3′ exon; (LE) ligated exons; (CI) circular intron; (XC) xylene cyanol. The gel-eluted precursor and its intron products (shown in C) were digested with nuclease P1 and resolved by TLC (D–F) using solvent a in the first dimension and solvent b in the second dimension. (D) Precursor (P). (E) Linear intron (LI). (F) Circular intron (CI). Outlines indicate the positions of nonradioactive markers in D–F.

FIGURE 6.

Analyses of splicing products of truncated elongator pre-tRNA^Met by different concentration gels. Reactions similar to those shown in Figure 5C ▶ using [α-³²P]CTP-labeled transcripts were resolved on 20-cm long, denaturing 15% (A) and 20% (B) gels. Lane 1, endonuclease products; lane 2, ligase products. (P) Precursor; (LE) ligated exons; (LI) linear intron; (CI) circular intron; (E) exons.

FIGURE 7.

Ligase reactions using purified substrates. Gel-eluted endonuclease products of [α-³²P]CTP labeled pre-tRNA^Trp are used in ligase reactions. The products are separated by denaturing 6% PAGE. Lane 1, Pre-tRNA; lane 2, standard endonuclease reaction; lane 3, standard ligase reaction; lanes 4 and 6, gel-eluted linear introns and exons, respectively; lanes 5 and 7, ligase reactions using gel-eluted linear introns and exons, respectively. (P) Precursor; (LI) linear intron; (E) exons; (LE) ligated exons; (CI) circularized intron; (XC) xylene cyanol; (BB) bromophenol blue.

FIGURE 8.

Ligation of synthetic intron-like transcripts. (A): [α-³²P]UTP-labeled synthetic tRNA^Trp intron transcripts are modified to create proper termini and then used as substrate for the ligase reactions. The products are analyzed on a denaturing 8% polyacrylamide gel. Lane 1, transcript; lane 2, dephosphorylated transcript; lane 3, dephosphorylated transcript after periodate oxidation and amine cleavage; lane 4, ligase reaction using substrate shown in lane 3; lane 5, substrate shown in lane 3 treated with RNA 3′ terminal phosphate cyclase; lane 6, ligase reaction using substrates shown in lane 5; lane 7, standard ligase reaction using endonuclease treated pre-tRNA^Trp as substrate. (LI) Linear intron and intron-like substrate; (CI) circular RNA; (P) precursor; (E) exons; (LE) ligated exons; (XC) xylene cyanol; (BB) bromophenol blue. Gel-eluted products from A are digested with nuclease P1 and separated by TLC in B (LI, lane 1) and C (CI, lane 6) using solvent a in the first dimension and solvent c in the second dimension. Outlines indicate the positions of nonradioactive markers in B and C. (The labeled spot near pU in B is derived from an unidentified contaminant present in [α-³²P]UTP; see Materials and Methods.) (D) Synthetic intron transcripts similar to those in A with a specific CC-to-AA change (Fig. 1C ▶) were used for ligation reactions like those shown in A. Lane 1, transcript; lane 2, dephosphorylated transcript; lane 3, dephosphorylated transcript after periodate oxidation and amine cleavage; lane 4, substrate shown in lane 3 treated with RNA 3′ terminal phosphate cyclase; lane 5, ligase reaction using substrates shown in lane 4. Ligated dimer of the substrate is indicated by an asterisk in D.

FIGURE 9.

A model for the reactions occurring during RNA splicing in Archaea. (A) Symmetric nature of the BHB-containing endonuclease substrate and two seven-base hairpin loops in ligase products. (B) Reactions involving specific phosphodiester linkages during RNA splicing. 1–9 and 1*–9* are residues involved in the formation of BHB in the substrates and hairpins in the products. Arrows in A indicate the splice sites in the substrate and products. The two phosphates (p1 and p2) and 2‘, 3′, and 5′ positions involved in the reactions are indicated. (E1 and E2) Two exons; (I) intron.