Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps

The methyl ester of m⁷G^5′ p was synthesized by a carbodiimide-catalyzed reaction of G^5′ p with methanol followed by dimethylsulfate alkylation. Comparative spectral analyses indicated that m⁷Gp · methyl ester retained the rigid conformation characteristic of the messenger RNA cap analog, m⁷G^5′ p but not its strong inhibitory activity against initiation of capped mRNA translation. Attachment of reovirus mRNA to wheat germ ribosomes, crosslinking of capbinding protein to the 5′-end of oxidized mRNA, and stimulation by this protein of capped mRNA translation in HeLa cell extract were all several-fold more sensitive to inhibition by m⁷G^5′ p than to m⁷Gp · methyl ester. Conversion of the esterified analog to m⁷G^5′ p by digestion with venom phosphodiesterase restored completely the ability to inhibit initiation complex formation. The results indicate that structural features of the 5′-terminal m⁷G cap of mRNA over and above preferred conformation are recognized during eukaryotic protein synthesis.     

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.     

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.     

Designing 1,5‐Naphthyridine‐2,6‐dione‐Based Conjugated Polymers for Higher Crystallinity and Enhanced Light Absorption to Achieve 9.63% Efficiency Polymer Solar Cells

Highly crystalline conjugated polymers represent a key material for producing high‐performance thick‐active‐layer polymer solar cells (PSCs). However, despite their potential, a limited number of crystalline polymers are used in PSCs because of the lack of highly coplanar acceptor building blocks and insufficient light absorptivity (α < 10⁵) of most donor (D)–acceptor (A)‐type polymers. This study reports a series of novel 3,7‐di(thiophen‐2‐yl)‐1,5‐naphthyridine‐2,6‐dione (NTDT) acceptor‐based conjugated polymers, PNTDT‐2T, PNTDT‐TT, and PNTDT‐2F2T, synthesized with 2,2′‐bithiophene (2T), thieno[3,2‐b]thiophene (TT), and 3,3′‐difluoro‐2,2′‐bithiophene (2F2T) donor units, respectively. PNTDT‐2F2T exhibits superior polymer crystallinity and a much higher absorption coefficient than those of PNTDT‐2T or PNTDT‐TT because of adequate matching between highly coplanar A (NTDT) and D (2F2T) building blocks. A bulk heterojunction solar cell based on PNTDT‐2F2T exhibits a power conversion efficiency of up to 9.63%, with a high short circuit current of 18.80 mA cm^?2 and fill factor of 0.70, when a thick active layer (>200 nm) is used, without postfabrication hot processing. The findings demonstrate that the polymer crystallinity and absorption coefficient can be effectively controlled by selecting appropriate D and A building blocks, and that NTDT is a novel and versatile A building block for highly efficient thick‐active‐layer PSCs.     

Capping of Viral RNA in Cultured Spodoptera frugiperda Cells Infected with Autographa californica Nuclear Polyhedrosis Virus

Viral RNA from fall armyworm (Spodoptera frugiperda) cells infected with Autographa californica nuclear polyhedrosis virus contains cap structures. Most of the cap labeled in vivo with [³H]methionine or ³²P_i cochromatographed on DEAE-cellulose with the −5 charge marker; a minor component appeared at −4 net charge. The former is probably a cap 1 structure (m⁷GpppX^m_pYp), and the latter is probably a cap 0 (m⁷GpppXp). On the basis of relative labeling of the two caps with [³H]adenosine and [³H]guanosine, we concluded that each cap contained at least one adenosine residue in addition to guanosine and, therefore, that cap 0 contained m⁷GpppAp. Cleavage of [³H]methionine-labeled viral RNA with tobacco acid pyrophosphatase released a labeled component that cochromatographed on polyethyleneimine-cellulose with m⁷Gp, confirming the m⁷GpppX linkage in the cap. These results were also seen with host polyadenylated RNA. The caps appeared in nearly equal abundance in viral polyadenylated and non-polyadenylated RNAs. The ratio of ³²P_i incorporated into the cap to that incorporated into mononucleotides suggested average lengths for the polyadenylated and non-polyadenylated RNAs of 1,800 and 1,200 nucleotides, respectively.     

Sequences within RNA coding for HIV‐1 Gag p17 are efficiently targeted to exosomes

HIV budding requires the interaction with cell factors involved in the biogenesis of exosomes. This implies the possibility that viral products undergo exosome incorporation. While this has been already described for both Gag and Nef HIV‐1 proteins, no conclusive results on HIV genome have been produced so far. Here, we report that unspliced, but not single or double spliced, HIV‐1 RNA species are incorporated in exosomes. Deletion mutant analysis indicated that the presence of a stretch of sequences within the 5′ end of the Gag p17 open reading frame is sufficient for HIV‐1 RNA exosome incorporation. These sequences were found associating with exosomes also out of the HIV‐1 context, thus indicating that the diversion towards the vesicular compartment can occur without need of additional HIV‐1 sequences. Finally, the incorporation of genomic HIV‐1 RNA in exosomes significantly increased when producer cells express HIV‐1 defective for viral genome packaging. Manipulating infected cells to favour the selective incorporation in exosomes of genomic HIV‐1 RNA might have therapeutic implications.     

Kinetics of formation of 5′ terminal caps in mRNA

A kinetic analysis of the labeling of the methylated components of messenger RNA and heterogeneous nuclear RNA in mouse L cells indicates that the 5′ terminal cap I structures (m⁷GpppX^mpYp) of mRNA are derived from 5′ terminal cap structures of hnRNA. Most of the hnRNA caps are conserved during processing, whereas only a portion of the internal m⁶A residues in hnRNA are conserved. The cap II structures (m⁷GpppX^mpY^mpZp), which constitute the 5′ termini of some mRNAs, arise by a “secondary” methylation that occurs after the mRNAs have entered the cytoplasm. This secondary methylation is apparently restricted to a particular subclass of mRNAs having a high frequency of pyrimidine nucleotides at position Y, a composition at position X which differs from that of the bulk of the cap I-terminated mRNAs, and a relatively slow rate of turnover.     

Biological and biochemical characterization of HIV‐1 Gag/dgp41 virus‐like particles expressed in Nicotiana benthamiana

The transmembrane HIV‐1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4+ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus‐like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant‐optimized HIV‐1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus‐based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV‐1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV‐1.     

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.     

Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase

The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2′-O positions of the viral RNA cap (GpppA-RNA → m⁷GpppA-RNA → m⁷GpppAm-RNA), using S-adenosyl-l-methionine (AdoMet) as a methyl donor. We report here that sinefungin (SIN), an AdoMet analog, inhibits several flaviviruses through suppression of viral MTase. The crystal structure of West Nile virus MTase in complex with SIN inhibitor at 2.0-Å resolution revealed a flavivirus-conserved hydrophobic pocket located next to the AdoMet-binding site. The pocket is functionally critical in the viral replication and cap methylations. In addition, the N7 methylation efficiency was found to correlate with the viral replication ability. Thus, SIN analogs with modifications that interact with the hydrophobic pocket are potential specific inhibitors of flavivirus MTase.     

Analysis of mRNA 5'-terminal cap structures and internal N6-methyladenosine by reversed-phase high-performance liquid chromatography

A simple procedure for determining the complete methylation profile of an mRNA molecule in a single chromatographic separation is described. The mRNA is selectively hydrolyzed to its component nucleosides leaving its cap 0 (m⁷GpppN′) or cap 1 (m⁷GpppN′m) structure intact. The hydrolysis products, which can include cap 0, cap 1, 2′-O-methylnucleosides (N″m) of cap 2 (m⁷GpppN′mpN″m) and internal N⁶-methyladenosine, are separated on an octadecyl reverse-phase column using a mobile phase containing acetonitrile and ammonium formate, a weak ion-pairing reagent. methyl-³H-labeled poly(A)-containing mRNA is used to demonstrate the efficacy of the procedure.