Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

Abstract

Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing.

Figure 1

The tRNA splicing pathway of plants and fungi. Intron-containing pre-tRNAs are first cleaved by a tRNA endonuclease at the 5′ and 3′ boundaries of the intervening sequence producing paired tRNA halves with 2′,3′-cyclic phosphate, 5′-OH ends and a linear intron. Second, the halves are ligated by a complex reaction requiring GTP and ATP. Finally, the 2′-phosphate at the splice junction is removed by a NAD-dependent phosphotransferase (12,46).

Figure 2

Isolation of wheat germ tRNA ligase. (A) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. (B) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA^Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. (C) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc)₂, 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10⁴ c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.

Figure 3

Gel filtration on Superdex™ 200 and adenylyltransferase activity of wheat germ tRNA ligase. (A) Partially purified tRNA ligase from the Source S15 column was subjected to gel filtration on Superdex™ 200. The column (HiLoad™ 16/60) was run with a flow rate of 1 ml/min. Fractions of 2 ml were collected. Aliquots of the elution fraction were analysed on a 7.5% polyacrylamide/0.1% SDS gel. The proteins were visualized by silver staining. (B) Appropriate aliquots of the indicated fractions were incubated in the presence of [α-³²P]ATP for 15 min at 37°C. The ligase–[³²P]AMP adduct was detected by autoradiography of the dried gel. (C) The peak fractions from the tRNA Sepharose column were concentrated by ultrafiltration and 1/20 of this material was applied onto a 10% polyacrylamide/0.1% SDS gel and stained with Coomassie blue for analytical valuation. The arrows point to the position of the RNA ligase protein with an approximate molecular weight of 125 kDa. Protein size standards in kDa are indicated on the right.

Figure 4

Alignment of plant tRNA ligases. The amino acid sequences of the shortest ORFs of A.thaliana, cv. Columbia (accession no. AC026875) and O.sativa, cv. Japonica (accession no. AP005124) are aligned using the Blosum 62 matrix. Identical amino acids are indicated in black and similar amino acids are shaded in grey. Nucleotidyltransferase motifs (Lig I, Lig IIIa and Lig IV), a putative NTP-binding P-loop motif (PNK) and two conserved tetrapeptides (CPD) designating cyclic phosphodiesterase elements are boxed. The lysine residue of the AMP binding site (Lig I) is highlighted by a star. The KFEN sequence element upstream of the Lig I motif resembles an element found at the corresponding position in T4 Rnl1-like ligases (3) and in fungal tRNA ligases (15).

Figure 5

In vitro production of Arabidopsis tRNA ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [³⁵S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA agarose as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) (61).

Figure 6

RNA ligase activity of native and recombinant enzymes. (A) A chimeric pre-tRNA, containing most of the mature domain from the plant pre-tRNA^Tyr as shown in Figure 2B, and the intron and anticodon of pre-tRNA^Trp from M.jannaschii was used as a substrate for the archaeal splicing endonuclease to generate in vitro 3′ and 5′ tRNA halves and a linear intron. It was synthesized by T7 RNA polymerase in the presence of [α-³²P]UTP. In an ATP-dependent complex reaction, yeast and plant tRNA ligases convert tRNA halves and the linear intron into spliced tRNA and circular intron molecules, containing a 2′-phosphomonoester, 3′,5′-phosphodiester bond at the splice junction (–10,46). (B) Archeuka pre-tRNA-derived tRNA halves and linear intron molecules were incubated in 20 μl splicing buffer for 30 min at 37°C in the presence of RNA ligase of different origin. Lane a, undigested archeuka pre-tRNA. In lanes b–g, pre-tRNA after cleavage with splicing endonuclease was used as input RNA; lane b, incubation without protein; lane c, incubation with a protein fraction derived from the expression of a control vector (minus cDNA insert) in the RTS 100 wheat germ extract and subsequent Ni-NTA purification to test whether the observed ligase activities are in fact due to the recombinant tRNA ligases; lane d, incubation with a partially purified tRNA ligase (gel filtration fraction) from wheat germ; lane e, incubation with 50 ng recombinant Arabidopsis tRNA ligase; lane f, incubation with 50 ng recombinant yeast tRNA ligase; and lane g, incubation with a mixture of T4 RNA ligase and T4 polynucleotide kinase/3′-phosphatase. The reaction products were separated on a 12.5% polyacrylamide/8 M urea gel. Stars (*) indicate products that were examined in more detail. (C) Time course of inter- and intramolecular ligation reactions by plant and yeast recombinant tRNA ligases. Archeuka pre-tRNA (400 fmol) was cleaved with splicing endonuclease and the resulting tRNA halves and linear intron molecules were incubated either with 100 ng Arabidopsis (left panel) or with 100 ng yeast (right panel) recombinant tRNA ligase in 100 μl splicing buffer. At the times (min) indicated below, aliquots (10 μl) were removed and the RNA products were analysed on a 12.5% polyacrylamide/8 M urea gel.

Figure 7

Analysis of splicing products. (A) The [α-³²P]UTP-labelled spliced tRNAs (76 nt) synthesized in the presence of different RNA ligase preparations (see Figure 6), i.e. wheat tRNA ligase (lane a), recombinant Arabidopsis tRNA ligase (lane b), recombinant yeast tRNA ligase (lane c) and a mixture of T4 RNA ligase and T4 polynucleotide kinase/3′-phosphatase (lane d) were recovered from a preparative gel and digested with RNase T1. The labelled T1-oligonucleotides ranging in size from 1 to 15 nt were separated on a 40-cm-long 20% polyacrylamide/8 M urea gel. Oligonucleotides ≤6 nt migrated with the buffer front. (B and C) The 15mer T1-resistant oligonucleotides and the 8mer oligonucleotide were excised from the gel and digested with RNase T2 (B) or with RNase P1 (C). Analysis of labelled Nps or pNs was by thin-layer chromatography (t. l. c.) on cellulose plates in solvent A. (D) The UTP-labelled circular introns (21 nt) synthesized in the presence of different RNA ligase preparations [as described in (A)] were recovered from a preparative gel and digested with RNase T2. Analysis of Nps was by t. l. c. in solvent B. Positions of markers are indicated.