Hydrolysis of some mRNA 5′-Cap Analogs Catalyzed by the Human Fhit Protein - and Lupin ApppA Hydrolases

Abstract

Hydrolysis of the following four cap analogs: m⁷G(5′)ppp(5′)A, m⁷G(5′)ppp(5′)m⁶A, m⁷G(5′)ppp(5′)m^2′OG and m⁷G(5′)ppp(5′)2′dG catalyzed by homogeneous human Fhit protein and yellow lupin Ap₃A hydrolase has been investigated. The hydrolysis products were identified by HPLC analysis and the K_m and V_max values calculated based on the data obtained by the fluorimetric method.     

Interaction of wheat protein synthesis elongation factor 1 with GTP analogs

Wheat protein synthesis elongation factor 1 was tested for binding to GTP analogs, including structures resembling “caps” that are present at the 5′-termini of most eukaryotic mRNAs. The interaction was assayed by determining the capacity of the analogs to inhibit the binding of [³H]GTP to elongation factor 1. Significant interaction of elongation factor 1 with G(5′)ppp(5′)G, G(5′)pppp(5′)G, and G(5′)ppp(5′)A was observed. Methylation of a ribose 2′-hydroxyl had very little effect, but methylation of the 7 position of guanosine greatly diminished the affinity of elongation factor 1 for these compounds. m⁷G(5′)ppp(5′)Cm, m⁷G(5′)ppp(5′)Um, and m⁷G(5′)ppp(5′)Am gave no detectable binding with EF1.     

Characterization of Neurospora crassa polyadenylated messenger ribonucleic acid: structure of the 5'-terminus.

Polyadenylated (poly(A)⁺) mRNA from Neurospora crassa was isolated by affinity chromatography on poly(U) Sepharose and its structure was examined. Two 5′-terminal ·cap’ structures, m⁷G(5′)ppp(5′)Ap and m⁷G(5′)ppp(5′)Gp, occurring in a relative distribution of 75 and 25% were found. No evidence was obtained for 2′-O-methylation in a nucleotide adjacent to the 5′-terminal cap.     

Design and Facile Synthesis of New Dinucleotide Cap Analog Containing Both 2′ and 3′-OH Modification on M7Guanosine Moiety

The first example of the synthesis of new dinucleotide cap analog containing 2^′,3^′-diacetyl group on m⁷guanosine moiety is described. The desired modified cap analog, m^{7,2′,3′–diacetyl}G[5^′]ppp[5^′]G has been obtained by the coupling reaction of triethylamine salt of m^{7,2′,3′–diacetyl}GDP with ImGMP in presence of ZnCl₂ as a catalyst in 62% yield with high purity. The structure of new cap analog has been confirmed by ¹H and ³¹P NMR and mass data.     

"Cap" sequences in poly(A)-rich RNA from dry wheat embryos

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5'-terminal "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5')ppp(5')N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C),a nd uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5')ppp(5')A, with roughly equivalent amounts of m7G(5')ppp(5')G and m7G(5')ppp(5')C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; the differences have not been previously noted in the literature and are the subject of brief comment in this paper.     

SYNTHESIS AND PROPERTIES OF mRNA CAP ANALOGS CONTAINING PHOSPHOROTHIOATE MOIETY IN 5′,5′-TRIPHOSPHATE CHAIN

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the α, β, or γ position of 5′,5′-triphosphate chain [m⁷Gp(s)ppG, m⁷Gpp(s)pG, and m⁷Gppp(s)G]. These are the first examples of the biologically important 5′mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the α or γ position within the dinucleotide cap analog was achieved using PSCl₃ in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in β position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and ¹H and ³¹P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.     

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5′ cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn⁷GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N⁷-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5′-phosphorothioate (m⁷GMPS), which is a translation inhibitor with in vitro potency higher than bn⁷GMP.     

Ribavirin targets eIF4E dependent Akt survival signaling

The eukaryotic translation initiation factor eIF4E is dysregulated in many cancers. eIF4E, through its mRNA export and translation functions, combinatorially modulates the expression of genes involved in Akt dependent survival signaling. For these activities, eIF4E must bind the 7-methyl guanosine (m⁷G) cap moiety on the 5′-end of mRNAs. We demonstrate that a physical mimic of the m⁷G cap, ribavirin, inhibits eIF4E dependent Akt survival signaling. Specifically, ribavirin impairs eIF4E mediated Akt activation via inhibiting the production of an upstream activator of Akt, NBS1. Consequently, ribavirin impairs eIF4E dependent apoptotic rescue. A ribavirin analog with distinct physico-chemical properties, tiazofurin, does not impair eIF4E activity indicating that only analogs that mimic the m⁷G cap will inhibit eIF4E function. Ribavirin represents a first-in-class strategy to inhibit eIF4E dependent cancers, through competition for m⁷G cap binding. Thus, ribavirin coordinately impairs eIF4E dependent pathways and thereby, potently inhibits its biological effects.     

Flavivirus RNA cap methyltransferase: structure,function, and inhibition

Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5′ terminal cap 1 structure (m⁷GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m⁷GpppA → m⁷GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2′-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2′-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.     

A Metazoan/Plant-like Capping Enzyme and Cap Modified Nucleotides in the Unicellular Eukaryote Trichomonas vaginalis

The cap structure of eukaryotic messenger RNAs is initially elaborated through three enzymatic reactions: hydrolysis of the 5′-triphosphate, transfer of guanosine through a 5′-5′ triphosphate linkage and N7-methylation of the guanine cap. Three distinctive enzymes catalyze each reaction in various microbial eukaryotes, whereas the first two enzymes are fused into a single polypeptide in metazoans and plants. In addition to the guanosine cap, adjacent nucleotides are 2′-O-ribose methylated in metazoa and plants, but not in yeast. Analyses of various cap structures have suggested a linear phylogenetic trend of complexity. These findings have led to a model in which plants and metazoa evolved a two-component capping apparatus and modification of adjacent nucleotides while many microbial eukaryotes maintained the three-component system and did not develop modification of adjacent nucleotides. Here, we have characterized a bifunctional capping enzyme in the divergent microbial eukaryote Trichomonas vaginalis using biochemical and phylogenetic analyses. This unicellular parasite was found to harbor a metazoan/plant-like capping apparatus that is represented by a two-domain polypeptide containing a C-terminus guanylyltransferase and a cysteinyl phosphatase triphosphatase, distinct from its counterpart in other microbial eukaryotes. In addition, T. vaginalis mRNAs contain a cap 1 structure represented by m⁷GpppAmpUp or m⁷GpppCmpUp; a feature typical of metazoan and plant mRNAs but absent in yeast mRNAs. Phylogenetic and biochemical analyses of the origin of the T. vaginalis capping enzyme suggests a complex evolutionary model where differential gene loss and/or acquisition occurred in the development of the RNA capping apparatus and cap modified nucleotides during eukaryote diversification.