Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme

Abstract

Programmed RNA breakage is an emerging theme underlying cellular responses to stress, virus infection and defense against foreign species. In many cases, site-specific cleavage of the target RNA generates 2',3' cyclic phosphate and 5'-OH ends. For the damage to be repaired, both broken ends must be healed before they can be sealed by a ligase. Healing entails hydrolysis of the 2',3' cyclic phosphate to form a 3'-OH and phosphorylation of the 5'-OH to form a 5'-PO4. Here, we demonstrate that a polynucleotide kinase-phosphatase enzyme from Clostridium thermocellum (CthPnkp) can catalyze both of the end-healing steps of tRNA splicing in vitro. The route of tRNA repair by CthPnkp can be reprogrammed by a mutation in the 3' end-healing domain (H189D) that yields a 2'-PO4 product instead of a 2'-OH. Whereas tRNA ends healed by wild-type CthPnkp are readily sealed by T4 RNA ligase 1, the H189D enzyme generates ends that are spliced by yeast tRNA ligase. Our findings suggest that RNA repair enzymes can evolve their specificities to suit a particular pathway.

Figure 1.

Pathway choice in tRNA splicing is dictated by the outcome of 3′ end healing. (A) Distinctive phage and yeast pathways of tRNA repair. In phage tRNA restriction-repair, the 2′,3′ cyclic phosphate and 5′-OH ends of the broken tRNA are healed by T4 Pnkp, which completely removes the phosphate at the 3′ end and phosphorylates the 5′ terminus. T4 Rnl1 then seals the 3′-OH and 5′-PO₄ termini to form a standard 3′–5′ phosphodiester linkage. In yeast tRNA splicing, the ends are healed by Trl1 kinase-CPD domain (389–827), which generate a 3′-OH, 2′-PO₄ on the proximal tRNA half and a 5′-PO₄ on the distal half, and then sealed by a Trl1 ligase domain (1–388). The yeast pathway leaves a 2′-PO₄ at the splice junction. (B) Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM DTT, 25 µM ATP, 25 µM GTP, 140 fmol ³²P-labeled cleaved tRNA and 1 pmol of T4 Rnl1, T4 Pnkp, Trl1(1–388) or Trl1(389–827), where indicated by + over the lanes, were incubated for 30 min at 37°C. The reactions were quenched by adding 10 µl of 95% formamide, 50 mM EDTA. The samples were heated at 95°C for 1 min and then analyzed by electrophoresis through a 15-cm 15% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA. The products were visualized by autoradiography.

Figure 2.

Purification of recombinant RNA repair enzymes. Aliquots (5 µg) of the indicated recombinant proteins were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left.

Figure 3.

Wild-type CthPnkp elects the phage tRNA repair pathway. Reaction mixtures (20 µl) containing 50 mM Tris-acetate (pH 6.5), 5 mM MgCl₂, 0.5 mM MnCl₂, 50 µM ATP, 200 fmol radiolabeled cleaved tRNA substrate, either 1 pmol Trl1(1–388) or 1 pmol T4 Rnl1, and CthPnkp as specified were incubated for 30 min at 37°C. The products were resolved by PAGE and visualized by autoradiography.

Figure 4.

CthPnkp-H189D opens a 2′,3′ cyclic phosphodiester to form a 2′-phosphomonoester. (A) Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM 2′,3′ cAMP (Sigma) and either 4 µg wild-type CthPnkp, 1.4 µg CthPnkp-H189D or 1 unit CIP (New England Biolabs) where indicated by + were incubated for 30 min at 45°C. The reactions were quenched by adding 1 µl of 0.5 M EDTA. Phosphate release was measured colorimetrically using the Malachite green reagent (BIOMOL Research Laboratories). (B) Reaction mixtures containing (per 10 µl) 50 mM Tris-HCl (pH 8.0), 0.5 mM MnCl₂, 10 mM 2′,3′ cAMP and either 1.4 µg of CthPnkp-H189D and 1 U of CIP (closed circle), 1.4 µg of CthPnkp-H189D alone (closed triangle) or 1 U of CIP alone (open circle) were incubated at 45°C. Aliquots (10 µl) were withdrawn at the times specified and quenched immediately with EDTA. Phosphate release is plotted as a function of time. (C) A reaction mixture containing (per 10 µl) 50 mM Tris-HCl (pH 8.0), 0.5 mM MnCl₂, 10 mM 2′,3′ cAMP and 1.4 µg of CthPnkp-H189D was incubated at 45°C. Samples (10 µl) were withdrawn at the times specified and quenched immediately with EDTA. Aliquots (1 µl) of each sample were applied to a cellulose-F TLC plate (EMD Chemicals). Markers 2′AMP, 3′AMP and 2′,3′ cAMP (5 nmol each) were spotted in lane M. The TLC plate was developed with buffer containing saturated ammonium sulfate:3 M sodium acetate:isopropanol (80:6:2), in which the order of migration away from the origin (R_f) is 2′,3′ cyclic phosphate <3′-phosphate <2′-phosphate (28,29). The nucleotides were visualized by photography under UV light. All species on the TLC plate that were detected by UV are shown in the photograph.

Figure 5.

The H189D change redirects CthPnkp down the yeast tRNA-splicing pathway. Reaction mixtures (20 µl) containing 50 mM Tris-acetate (pH 6.5), 0.5 mM MnCl₂, 50 µM ATP, 5 mM MgCl₂, 200 fmol of cleaved tRNA substrate, 1 pmol Trl1(1–388) and wild-type CthPnkp, or mutated versions as specified were incubated for 30 min at 37°C. The products were resolved by PAGE and visualized by autoradiography.

Figure 6.

The K21A mutation of CthPnkp abolishes the 5′ kinase activity without affecting the 2′,3′ phosphodiesterase. (A) Phosphodiesterase reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM 2′,3′ cAMP and either 0.8 µg CthPnkp(1–472)-H189D, 0.8 µg CthPnkp(1–472)-K21A-H189D or 1 unit CIP (where indicated by +) were incubated for 30 min at 45°C. Phosphate release was measured by using the Malachite green reagent. (B) Polynucleotide kinase reaction mixtures (10 µl) containing 50 mM Tris-acetate (pH 7.0), 5 mM DTT, 100 µM [γ³²P]ATP, 10 µM 5′-OH oligodeoxynucleotide 12-mer 5′-CACTATCGGAAT and CthPnkp(1–472)-H189D or CthPnkp(1–472)-K21A-H189D as specified were incubated for 30 min at 45°C. The reactions were quenched with 10 µl of 95% formamide, 50 mM EDTA. The products were analyzed by urea-PAGE and visualized by autoradiography.