Deciphering the molecular basis for nucleotide selection by the West Nile virus RNA helicase

The West Nile virus RNA helicase uses the energy derived from the hydrolysis of nucleotides to separate complementary strands of RNA. Although this enzyme has a preference for ATP, the bias towards this purine nucleotide cannot be explained on the basis of specific protein–ATP interactions. Moreover, the enzyme does not harbor the characteristic Q-motif found in other helicases that regulates binding to ATP. In the present study, we used structural homology modeling to generate a model of the West Nile virus RNA helicase active site that provides instructive findings on the interaction between specific amino acids and the ATP substrate. In addition, we evaluated both the phosphohydrolysis and the inhibitory potential of a collection of 30 synthetic purine analogs. A structure-guided alanine scan of 16 different amino acids was also performed to clarify the contacts that are made between the enzyme and ATP. Our study provides a molecular rationale for the bias of the enzyme for ATP by highlighting the specific functional groups on ATP that are important for binding. Moreover, we identified three new essential amino acids (Arg-185, Arg-202 and Asn-417) that are critical for phosphohydrolysis. Finally, we provide evidence that a region located upstream of motif I, which we termed the nucleotide specificity region, plays a functional role in nucleotide selection which is reminiscent to the role exerted by the Q-motif found in other helicases

Virtual High-Throughput Screening Identifies Mycophenolic Acid as a Novel RNA Capping Inhibitor

The RNA guanylyltransferase (GTase) is involved in the synthesis of the m7 Gppp-RNA cap structure found at the 59 end of eukaryotic mRNAs. GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed

The immunosuppressive agent mizoribine monophosphate is an inhibitor of the human RNA capping enzyme.

Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5'-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5'-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5' end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 5'-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells, which could account for the global mechanism of action of this therapeutic agent. Together, our study clearly demonstrates that mizoribine monophosphate inhibits the human RNA guanylyltransferase in vitro and impair mRNA capping in cellulo

Enzymatic Synthesis of RNAs Capped with Nucleotide Analogues Reveals the Molecular Basis for Substrate Selectivity of RNA Capping Enzyme: Impacts on RNA Metabolism

<div><p>RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated <i>in cellulo</i>. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model <i>Paramecium</i><i>bursaria</i><i> Chlorella virus-1</i> RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties <i>in cellulo</i>. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.</p> </div

<i>In</i><i>cellulo</i> and <i>in</i><i>vitro</i> properties of the novel cap analogues

<p>(A) Schematic representation of the experimental procedure for the determination of the translation efficiency of differentially capped lucA₆₀ RNA in HEK293 cells. (B) The relative translation efficiency was experimentally determined by quantifying <i>firefly</i> luciferase activity relative to the amount of total protein 6 hr post-transfection. Experimental data was adjusted relative to the capping efficiency (as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075310#pone-0075310-t001" target="_blank">Table 1</a>) of each analogue, and rationalized onto the ^m7G cap. The error associated with each data set is less than ± 0.1. (*) indicates more than 1.5 fold difference relative to the translation efficiency of a naturally capped RNA. (C) The relative RNA level was evaluated by quantifying the amount of lucA60 RNA relative to the GAPDH RNA by qRT-PCR 0 hr and 6 hr post-transfection. (D) Binding to eIF4E was determined by fluorescence spectroscopy with a 30 nt long differentially capped RNA molecule. (*) indicates more than 1.5 fold difference relative to the binding observed for the natural ^m7G capped RNA.</p

<i>In</i><i>vitro</i> characterization of 3’ O-methyl GTP (A₂₂) as an inhibitor of translation and as a binding partner to eIF4E

<p>(A) To the rabbit reticulocyte lysate (Promega) <i>in </i><i>vitro</i> translation system, ^m7G capped lucA₆₀ RNA (1µg) and increasing amounts of NTP was added. Luciferase activity was measured after 10 minutes and plotted relative to the activity in the absence of any nucleotides. (B) Increasing amounts of NTP were added to a 2 µM solution of the purified protein in a binding buffer (50 mM Tris/HCl, pH 8.0, and 50 mM KOAc) and following excitation of tryptophan residues at 290 nm the emission spectrum was scanned from 310 to 440 nm. A saturation isotherm was generated from these data by plotting the change in fluorescence intensity at 333 nm as a function of added NTP. The data was fit to a binding curve. (■) indicates ^m7GTP; (▲) indicates 3’ O methyl GTP (A₂₂); and (●) represents GTP.</p

The RNA capping mechanism and the nucleotide analogues tested

<p>(A) The two-step RNA guanylyltransferase reaction. (B) The GTP binding site of the PBCV-1 GTase (PDB 1CKN). Residues shown are those interacting with the base and the sugar moiety. (C) Nucleotide analogues used in this study.</p

Inhibition of capping by MZP.

<p>(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.</p