Figure 1.
RNA guanylyltransferase mechanistic pathway and associated structures.
The phosphoryltransfer catalysis requires conformational changes between the open (blue) and close (red) form of the RNA guanylyltransferase enzyme. The apo-enzyme (structure 1) first binds GTP (grey sphere, structure 2) which promotes the closure of the OB fold domain toward the NT domain (structure 3). In the catalytically active close conformation, the enzyme hydrolyzes the GTP to form the hallmark enzyme-GMP covalent intermediate complex (black sphere, structure 4). The lost of interactions between the bound guanylate and the OB fold domain, upon GTP hydrolysis, destabilizes the close conformation of the enzyme and leads to its reopening (structure 5) concomitant with the release of the pyrophosphate product. This exposes the RNA-binding site of the enzyme (exact location unknown), thereby allowing 5′-diphosphate RNA binding (structure 6) and subsequent GMP moiety transfer onto the acceptor RNA (structure 7). The capped RNA is then released and the apo-enzyme (structure 1) is regenerated allowing reinitiation of the pathway. (PDB: 1CKN).
Figure 2.
(A) Structure of mizoribine 5-monophosphate (MZP) and guanosine-5′-triphosphate (GTP). (B) An aliquot of the purified HCE protein (P) was analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS and visualized by staining with Coomassie blue dye. The position and size (in kDa) of the molecular weight marker (M) are indicated on the left. (C) RNA capping assay by the sequential RTase and GTase activity of HCE. A 5′-triphosphate RNA and [α-32P]GTP were incubated in presence of HCE in a buffer containing 5 mM MgCl2. As a control, EDTA was added to a final concentration of 50 mM to prevent RNA capping. Radiolabeled RNA products were analyzed by SDS-PAGE, an autoradiogram of the gel is shown. (D and E) Capping assays were performed in the presence of increasing concentration of MZP or GMP and the capped RNA products were analyzed by SDS-PAGE and quantified by autoradiography.
Figure 3.
(A) MZP does not inhibits the RTase activity. An RNA substrate radiolabeled at its 5′-terminal γ-phosphate was incubated with HCE and increasing concentration of MZP. 32Pi product was separated from RNA substrate by polyethyleneimine-cellulose TLC plate and quantified by autoradiography. (B) The GTase activity is inhibited by MZP. A 5′-diphosphate RNA was incubated with [α-32P]GTP, HCE and increasing concentration of MZP. Radiolabeled RNA products were analyzed on denaturing polyacrylamide gel and quantified by autoradiography. (C) The formation of HCE-GMP complex, the first step of GTase activity, is inhibited to a lesser extent then the complete GTase reaction by MZP. HCE was incubated with [α-32P]GTP and increasing concentration of MZP. Radiolabeled EpG complex were analyzed by SDS-PAGE and quantified by autoradiography. (D) MZP does not significantly inhibit the transfer of a GMP on an acceptor RNA; the second step of GTase activity. HCE was preincubated with [α-32P]GTP to allow EpG formation, a 5′-diphosphate RNA and increasing concentration of MZP were next added to the reaction. Formation of radiolabeled RNA products was analyzed on denaturing polyacrylamide gel and quantified by autoradiography. (E) Lineweaver-Burk representation of the enzyme-GMP covalent complex formation (GTase first step) as a function of the substrate concentration in presence of various MZP concentration. The maximal velocity of EpG formation is not affected by various MZP concentration (curves crossing on the Y axis). (F) Lineweaver-Burk representation of the complete GTase reaction velocity as a function of the substrate concentration in presence of various MZP concentration. HCE Km for GTP is independent from the MZP concentration (curves crossing on the X axis) for the complete reaction. MZP concentration : ✯ 0 µM, □ 50 µM, ▵ 200 µM, ▿ 500 µM, ✵ 1 mM, ★ 2 mM, ▪ 5 mM, ▴ 10 mM, ▾ 20 mM.
Figure 4.
Specificity of MZP inhibition toward RNA guanylyltransferase.
(A) Complete GTase reaction. 5′-diphosphate RNA was capped with HCE, HCE-G229–597, D1R, A103R or Ceg1 GTases in presence of increasing concentration of MZP. (B) Formation of EpG complex. The GTases from human, vaccinia virus, Chlorella virus or S. cerevisiae were allowed to form enzyme-GMP covalent complex in presence of increasing concentration of MZP. (C) Amino acids alignment of the human (HCE), S. cerevisiae (Ceg1), C. albicans and Chlorella virus (A103R) GTases highlighting the sequence conservation. Representation of the six conserved GTase signature motifs (I, III, IIIa, IV, V, VI) and their associated secondary structures (above). Numbering is based on the HCE amino acids. Amino acids predicted to coordinate MZP low affinity binding site are highlighted by an asterisk. (D) GTases harbor a similar three-dimensional architecture. Ribbon diagram of HCE GTase domain in close conformation (PDB: 3S24), in open conformation (homology model base on PDB: 1P16), Chlorella virus GTase (PDB: 1CKN), S. cerevisiae GTase (PDB: 3KYH), C. albicans GTase (PDB: 1P16). Active site color code is representative of the GTase signature motif as displayed in panel C.
Figure 5.
In cellulo mizoribine capping inhibition is rescued by HCE over-expression.
(A) Timeline of the experiments. (B) Cells over-expressing HCE-WT-HA, HCE-K294A-HA, GFP, or control cells were pre-treated with mizoribine for 42 h and transfected with the luciferase encoding vector PGL3. The RNA pol II transcribe reporter gene was allowed to express for 30 h prior to luciferase assay. Luciferase intensity, as a mesure of efficient capping of the reporter protein mRNA, was normalized for untreated cells and plotted against mizoribine concentration. For both mizoribine concentrations tested, the luciferase activity was significantly higher in cells over-expressing HCE-WT-HA as compared to control cells (cells over-expressing the GTase defective HCE-K294A-HA mutant, over-expressing GFP, or transfected with the control Vector). Shown is the mean of 18 experiments for each condition. (C) Western blots using anti-HA, anti-GFP and anti-actin antibody were performed on protein extracts from each cell line. (**P<0.01, ***P<0.001).