Chemical synthesis of 2″OMeNAD+ and its deployment as an RNA 2'-phosphotransferase (Tpt1) 'poison' that traps the enzyme on its abortive RNA-2'-PO4-(ADP-2″OMe-ribose) reaction intermediate

Abstract

RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1″ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 'poison' that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2'-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes.

Graphical Abstract

Figure 1.

Tpt1 mechanism. (A) The Tpt1 reaction pathway comprises the two chemical steps shown in which: (1) an RNA 2′-PO₄ oxygen attacks the C1″ atom of NAD⁺ to expel nicotinamide and form a 2′-phospho-ADP-ribosylated RNA intermediate and (2) transesterification of the ADP-ribose 2″-OH to the RNA 2′-PO₄ displaces the RNA 2′-OH and generates ADP-ribose-1″,2″-cyclic phosphate. (B) 2″OMeNAD⁺ is an NAD⁺ analogue in which the ribose 2″-OH is replaced by a 2″-OMe group that should not serve as a nucleophile for step 2 transesterification.

Figure 2.

Synthesis of 2′OMe-nicotinamide riboside (compound 5) using TMS-OTf mediated glycosylation. (A) (a) Iodomethane, Ag₂O, dimethylformamide, 80°C, 18 h, 84%. (b) tetrabutylammonium fluoride, tetrahydrofurane, r.t., 17 h; then acetic anhydride, pyridine, r.t., 19 h, 90% over two steps. (c) acetic acid, acetic anhydride, H₂SO₄, 10°C, 16 h, 71%. (d) nicotinamide, compound 4, acetonitrile, then TMS-triflate, r.t., 4 h. (e) nicotinamide, TMS-triflate, acetonitrile, r.t. 30 min, then compound 4, r.t., 4 h; then NH₃ in methanol, r.t., 18 h, 28% over two steps; (B) 1H-¹³C-HMBC NMR spectrum of compound 5-R, with highlighted cross signal between 1′-H and C-carbonyl. (C) 1H-¹³C-HMBC NMR spectrum of compound 5 showing the absence of the carbonyl-correlation and instead a cross signal between 1′-H and C-2 and 1H-1H NOESY spectrum of compound 5 with highlighted nuclear overhauser effects (NOEs) between 1′-H and 4′-H and 3′-H and aromatic protons.

Figure 3.

Synthesis of 2″OMeNAD⁺ (compound 7). (A) POCl₃, trimethylphosphate, 0°C, 4 h, 50 %; (B) AMP P-imidazolide, MgCl₂, formamide, r.t., 24 h, 48%.

Figure 4.

Utilization of 2″OMeNAD⁺ by Runella Tpt1 generates a dead-end 2′-phospho-ADP-ribosylated RNA. (A) Tpt1 titrations. Reaction mixtures (10 μl) containing 100 mM Tris–HCl (pH 7.5), 0.2 μM (2 pmol) 5′ ³²P-labeled 6-mer 2′-PO₄ substrate, either 50 μM NAD⁺ (left panel), no added NAD⁺ (middle panel), or 50 μM 2″OMeNAD⁺ (middle panel), and RslTpt1 as specified on the x-axes were incubated at 37°C for 30 min. The extents of formation of 2′-OH end-product and abortive 2′-P-ADPR intermediate are plotted as a function of input Tpt1. Each datum in the graphs is the average of three independent titration experiments ± SEM. (B) Kinetics of the reaction with 2″OMeNAD⁺. Reaction mixtures (100 μl) containing 100 mM Tris–HCl (pH 7.5), 0.2 μM 5′ ³²P-labeled 6-mer 2′-PO₄ substrate, 50 μM 2″OMeNAD⁺, and 0.5 μM RslTpt1 were incubated at 37°C. The reactions were initiated by adding RslTpt1 to a pre-warmed reaction mixture. Aliquots (10 μl, containing 2 pmol of 2′-PO₄ RNA substrate) were withdrawn at the times specified on the x-axis and quenched immediately with cold formamide/EDTA. The extents of formation of 2′-P-ADPR RNA and 2′-OH RNA are plotted as a function of reaction time. Each datum in the graphs is the average of three independent time course experiments ± SEM. The data were fit by nonlinear regression to a one phase association in Prism.

Figure 5.

2″OMeNAD⁺ traps Tpt1 on the RNA-2′-phospho-(ADP-2″OMe-ribose) dead-end product. (A) Reaction mixtures (10 μl) containing 100 mM Tris–HCl (pH 7.5), 0.2 μM (2 pmol) 5′ ³²P-labeled 6-mer 2′-PO₄ substrate, either no added NAD⁺, 50 μM NAD⁺, or 50 μM 2″OMeNAD⁺, and 5 pmol RslTpt1 (where indicated by + above the lanes) were incubated at 37°C for 15 min. The mixtures were adjusted to 5% (v/v) glycerol and then applied to a 15-cm native 8% polyacrylamide gel containing 0.25x TBE that had been pre-run at 110 V for 30 min. Separation of RslTpt1-bound and free RNA was achieved by electrophoresis for 2.5 h at 110 V at room temperature. The radiolabeled species were visualized by scanning the gel with a Typhoon FLA-7000 imaging device. The positions of the sample well and xylene cyanol (XC) and bromophenol blue (BPB) dye makers run in a parallel lane are indicated on the right. (B) Reaction mixtures (10 μl) containing 100 mM Tris–HCl (pH 7.5), 0.2 μM (2 pmol) 5′ ³²P-labeled 6-mer 2′-PO₄ substrate, 50 μM 2″OMeNAD⁺ and RslTpt1 as specified above the lanes were incubated at 37°C for 15 min, adjusted to 5% glycerol, and then analyzed by native PAGE.

Figure 6.

2″OMeNAD⁺ is not an effective substrate for Tpt1 from three fungal pathogens. Reaction mixtures (10 μl) containing 100 mM Tris–HCl (pH 7.5), 0.2 μM (2 pmol) 5′ ³²P-labeled 6-mer 2′-PO₄ substrate, either 50 μM NAD⁺ or 50 μM 2″OMeNAD⁺ (where indicated by +), and 0.5 μM (5 pmol) Tpt1 from the species specified on the x-axis were incubated at 37°C for 30 min. The extents of formation of 2′-OH RNAs in reactions containing NAD⁺ (blue bars) and formation of RNA-2′-phospho-(ADP-2″OMe-ribose) in reactions containing 2″OMeNAD⁺ (red bars) are shown. Data are the average of three independent experiments ± SEM.