ABSTRACT
The recent COVID-19 pandemics have demonstrated the great therapeutic potential of in vitro transcribed (IVT) mRNAs, but improvements in their biochemical properties, such as cellular stability, reactogenicity and translational activity, are critical for further practical applications in gene replacement therapy and anticancer immunotherapy. One of the strategies to overcome these limitations is the chemical modification of a unique mRNA 5'-end structure, the 5'-cap, which is responsible for regulating translation at multiple levels. This could be achieved by priming the in vitro transcription reaction with synthetic cap analogs. In this study, we combined a highly efficient trinucleotide IVT capping technology with several modifications of the 5' cap triphosphate bridge to synthesize a series of 16 new cap analogs. We also combined these modifications with epigenetic marks (2'-O-methylation and m6Am) characteristic of mRNA 5'-ends in higher eukaryotes, which was not possible with dinucleotide caps. All analogs were compared for their effect on the interactions with eIF4E protein, IVT priming, susceptibility to decapping, and mRNA translation efficiency in model cell lines. The most promising α-phosphorothiolate modification was also evaluated in an in vivo mouse model. Unexpected differences between some of the analogs were analyzed using a protein cell extract pull-down assay.
ABSTRACT
Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA-based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5'-end, called 5'-cap. Depending on the 5'-terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5'-end variants. Here, a series of 8 new synthetic 5'-cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap-protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap-binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap-binding subunit in a multi-protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA-related processes.
ABSTRACT
Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Amâa common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.
Subject(s)
RNA Caps , Vaccines , Animals , Mice , RNA, Messenger/genetics , RNA Caps/chemistry , RNA Caps/genetics , RNA Caps/metabolism , Protein Biosynthesis , MethylationABSTRACT
Messenger ribonucleic acid (mRNA) is the universal cellular instruction for ribosomes to produce proteins. Proteins are responsible for most of the functions of living organisms, and their abnormal structure or activity is the cause of many diseases. mRNA, which is expressed in the cytoplasm and, unlike DNA, does not need to be delivered into the nucleus, appears to be an ideal vehicle for pursuing the idea of gene therapy in which genetic information about proteins is introduced into an organism to exert a therapeutic effect. mRNA molecules of any sequence can be synthesized using the same set of reagents in a cell-free system via a process called in vitro transcription (IVT), which is very convenient for therapeutic applications. However, this does not mean that the path from the idea to the first mRNA-based therapeutic was short and easy. It took 30 years of trial and error in the search for solutions that eventually led to the first mRNA vaccines created in record time during the SARS-CoV-2 pandemic. One of the fundamental problems in the development of RNA-based therapeutics is the legendary instability of mRNA, due to the transient nature of this macromolecule. From the chemical point of view, mRNA is a linear biopolymer composed of four types of ribonucleic subunits ranging in length from a few hundred to hundreds of thousands of nucleotides, with unique structures at its ends: a 5'-cap at the 5'-end and a poly(A) tail at the 3'-end. Both are extremely important for the regulation of translation and mRNA durability. These elements are also convenient sites for sequence-independent labeling of mRNA to create probes for enzymatic assays and tracking of the fate of mRNA in cells and living organisms. Synthetic 5'-cap analogs have played an important role in the studies of mRNA metabolism, and some of them have also been shown to significantly improve the translational properties of mRNA or affect mRNA stability and reactogenicity. The most effective of these is used in clinical trials of mRNA-based anticancer vaccines. Interestingly, thanks to the knowledge gained from the biophysical studies of cap-related processes, even relatively large modifications such as fluorescent tags can be attached to the cap structure without significant effects on the biological properties of the mRNA, if properly designed cap analogs are used. This has been exploited in the development of molecular tools (fluorescently labeled mRNAs) to track these macromolecules in complex biological systems, including organisms. These tools are extremely valuable for better understanding of the cellular mechanisms involved in mRNA metabolism but also for designing therapeutic mRNAs with superior properties. Much less is known about the usefulness/utility of poly(A) tail modifications in the therapeutic context, but it is clear that chemical modifications of poly(A) can also affect biochemical properties of mRNA. This Account is devoted to chemical modifications of both the 5'- and 3'-ends of mRNA aimed at improving the biological properties of mRNA, without interfering with its translational function, and is based on the authors' more than 20 years of experience in this field.
Subject(s)
COVID-19 , Protein Biosynthesis , Humans , RNA, Messenger/metabolism , SARS-CoV-2/genetics , Ribosomes/metabolismABSTRACT
Messenger RNA (mRNA)-based gene delivery is a powerful strategy for the development of vaccines and therapeutics. Consequently, approaches that enable efficient synthesis of mRNAs with high purity and biological activity are in demand. Chemically modified 7-methylguanosine (m7G) 5' caps can augment the translational properties of mRNA; however, efficient synthesis of structurally complex caps, especially on a large scale, is challenging. Previously, we proposed a new strategy to assemble dinucleotide mRNA caps by replacing the traditional pyrophosphate bond formation by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here, we used CuAAC to synthesize 12 novel triazole-containing tri- and tetranucleotide cap analogs with the aim of exploring the chemical space around the first transcribed nucleotide in mRNA and overcoming some of the limitations previously reported for the triazole-containing dinucleotide analogs. We evaluated the efficiency of incorporation into RNA for these analogs and their influence on the translational properties of in vitro transcribed (IVT) mRNAs in rabbit reticulocyte lysate and JAWS II cultured cells. The incorporation of the triazole moiety within the 5',5'-oligophosphate of trinucleotide cap produced compounds that were well incorporated into RNA by T7 polymerase while replacing the 5',3'-phosphodiester bond with triazole impaired incorporation and translation efficiency, despite a neutral effect on the interaction with the translation initiation factor eIF4E. One of the compounds (m7Gppp-tr-C2H4pAmpG), had translational activity and other biochemical properties comparable to natural cap 1 structure, thus being a promising mRNA capping reagent for potential in cellulo and in vivo applications in the field of mRNA-based therapeutics.
ABSTRACT
In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2'-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2'-O-methylated adenosine, it can be additionally modified to N6,2'-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between 'self' and 'non-self' in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2'-O-methylation of the second transcribed nucleotide within the mRNA 5' cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2'-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as 'self' and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.
Subject(s)
Nucleotides , RNA Caps , Animals , Methylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA Caps/genetics , RNA Caps/metabolism , Nucleotides/metabolism , Immune Evasion , Mammals/geneticsABSTRACT
Herein, we report a straightforward one-step procedure for modifying N-nucleophilic groups in the nucleobases of commercially available nucleoside phosphoramidites. This method involves the deprotonation of amide groups under phase-transfer conditions and subsequent reaction with electrophilic molecules such as alkyl halides or organic isocyanates. Using this approach, we obtained 10 different classes of modified nucleoside phosphoramidites suitable for the synthesis of oligonucleotides, including several noncanonical nucleotides found in natural RNA or DNA (e.g., m6A, i6A, m1A, g6A, m3C, m4C, m3U, m1G, and m2G). Such modification of nucleobases is a common mechanism for post-transcriptional regulation of RNA stability and translational activity in various organisms. To better understand this process, relevant cellular recognition partners (e.g., proteins) must be identified and characterized. However, this step has been impeded by limited access to molecular tools containing such modified nucleotides.
Subject(s)
Nucleosides , Oligoribonucleotides , Oligonucleotides , Organophosphorus CompoundsABSTRACT
Vaccinia virus (VACV) represents a family of poxviruses, which possess their own decapping machinery as a part of their strategy to eliminate host mRNAs and evade the innate immune response. D9 is one of the two encoded VACV decapping enzymes that is responsible for cap removal from the 5' end of both host mRNA transcripts and viral double-stranded RNAs. Little is known about the structural requirements for D9 inhibition by small molecules. Here, we identified a minimal D9 substrate and used it to develop a real-time fluorescence assay for inhibitor discovery and characterization. We screened a panel of nucleotide-derived substrate analogues and pharmacologically active candidates to identify several compounds with nano- and low micromolar IC50 values. m7GpppCH2p was the most potent nucleotide inhibitor (IC50 â¼ 0.08 µM), and seliciclib and CP-100356 were the most potent drug-like compounds (IC50 0.57 and 2.7 µM, respectively). The hits identified through screening inhibited D9-catalyzed decapping of 26 nt RNA substrates but were not active toward VACV D10 or human decapping enzyme, Dcp1/2. The inhibition mode for one of the compounds (CP-100356) was elucidated based on the X-ray cocrystal structure, opening the possibility for structure-based design of novel D9 inhibitors and binding probes.
Subject(s)
Vaccinia virus , Viral Proteins , Endoribonucleases/metabolism , Fluorescence , Humans , Nucleotides , RNA Caps/metabolism , RNA, Messenger/metabolism , Vaccinia virus/genetics , Viral Proteins/metabolismABSTRACT
Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral double-stranded RNA (dsRNA), a trigger for antiviral responses. Here we present a high-resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a domain organization different from other decapping enzymes in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap-binding proteins VP39, eIF4E, and CBP20, and distinct from eukaryotic decapping enzyme Dcp2.
Subject(s)
Poxviridae , Viral Proteins , Catalysis , Endoribonucleases/chemistry , Endoribonucleases/genetics , Endoribonucleases/metabolism , Poxviridae/genetics , Poxviridae/metabolism , RNA Caps/metabolism , RNA, Double-Stranded , RNA, Messenger/metabolism , Vaccinia virus/genetics , Viral Proteins/metabolismABSTRACT
The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.
Subject(s)
Lymphocyte Activation , Methyltransferases/physiology , Protein Biosynthesis , Ribosomes/metabolism , T-Lymphocytes/enzymology , Animals , Gene Knockout Techniques , Guanosine/metabolism , Lymphocyte Activation/genetics , Methyltransferases/biosynthesis , Methyltransferases/genetics , Mice , RNA Caps/chemistry , RNA Caps/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Small Untranslated/metabolism , RNA-Binding Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Up-RegulationABSTRACT
Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3'-end labeling of inâ vitro transcribed RNAs. We synthesized novel adenosine 3',5'-bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide-containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain-promoted alkyne-azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD-capped RNAs, and 5'-fluorescently labeled m7 Gp3 Am -capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence-based monitoring of decapping. This method will facilitate the use of various functionalized mRNA-based probes.
Subject(s)
Azides , RNA , Alkynes , Cycloaddition Reaction , RNA, Messenger/geneticsABSTRACT
Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the evaluation of compounds targeting mRNA 5' cap-binding proteins of therapeutic interest, including eukaryotic translation initiation factor 4E and scavenger decapping enzyme. For this purpose, a library of 19 carboxyfluorescein probes based on 7-methylguanine nucleotides was evaluated as FA probes for these proteins. Optimal probe:protein systems were further investigated in competitive binding experiments and adapted for high-throughput screening. Using a small in-house library of compounds, we verified and confirmed the accuracy of the developed FA assay to study cap-binding protein binders. The applications of the most promising probes were then extended to include evaluation of allosteric inhibitors as well as RNA ligands. From this analysis, we confirmed the utility of the method to study small molecule ligands and evaluate differently 5' capped RNAs.
Subject(s)
Fluoresceins/chemistry , Fluorescence Polarization/methods , Guanine/analogs & derivatives , Molecular Probes/chemistry , Guanine/chemistryABSTRACT
mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. Chemically modified 5' cap structures have provided access to mRNAs with superior translational properties that could benefit the currently flourishing mRNA field. Prime examples of compounds that enhance mRNA properties are antireverse cap analog diastereomers that contain an O-to-S substitution within the ß-phosphate (ß-S-ARCA D1 and D2), where D1 is used in clinically investigated mRNA vaccines. The compounds were previously found to have high affinity for eukaryotic translation initiation factor 4E (eIF4E) and augment translation in vitro and in vivo. However, the molecular basis for the beneficial "thio-effect" remains unclear. Here, we employed multiple biophysical techniques and captured 11 cap analog-eIF4E crystallographic structures to investigate the consequences of the ß-O-to-S or -Se substitution on the interaction with eIF4E. We determined the SP/RP configurations of ß-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the ß-S/Se atom occupies the same binding cavity between Lys162 and Arg157, indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. This finding explains why both ß-S-ARCA diastereomers have higher affinity for eIF4E than unmodified caps. Binding affinities determined for di-, tri-, and oligonucleotide cap analogs suggested that the "thio-effect" was preserved in longer RNAs. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Phosphorothioate Oligonucleotides/metabolism , RNA Caps/metabolism , Animals , Binding Sites , Cell Line , Crystallography, X-Ray , Eukaryotic Initiation Factor-4E/chemistry , Mice , Nucleic Acid Conformation , Organoselenium Compounds/chemistry , Organoselenium Compounds/metabolism , Phosphorothioate Oligonucleotides/chemistry , Protein Binding , RNA Caps/chemistry , Static Electricity , StereoisomerismABSTRACT
Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA body and poly(A) tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogs modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases-SP6, T7, and poly(A) polymerase. To verify the efficiency of incorporation of ATP analogs in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'-mononucleotides. The method also estimated the average poly(A) tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the poly(A) tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Phosphorothioate Oligonucleotides/chemistry , Poly A/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Adenosine Triphosphate/metabolism , Animals , DNA-Directed RNA Polymerases/genetics , Dendritic Cells/cytology , Dendritic Cells/metabolism , HeLa Cells , Humans , Mice , Poly A/chemistry , Poly A/genetics , Protein Processing, Post-Translational , RNA, Messenger/chemistry , RNA, Messenger/genetics , Transcription, GeneticABSTRACT
RNAs with 5' functional groups have been gaining interest as molecular probes and reporter molecules. Copper-catalyzed azide-alkyne cycloaddition is one of the most straightforward methods to access such molecules; however, RNA functionalization with azide group has been posing a synthetic challenge. This article describes a simple and efficient protocol for azide functionalization of oligoribonucleotides 5'-end in solid-phase. An azide moiety is attached directly to the C5'-end in two steps: (i) -OH to -I conversion using methyltriphenoxyphosphonium iodide, and (ii) -I to -N3 substitution using sodium azide. The reactivity of the resulting compounds is exemplified by fluorescent labeling using both copper(I)-catalyzed (CuAAC) and strain-promoted (SPAAC) azide-alkyne cycloaddition reactions, ligation of two RNA fragments, and cyclization of short bifunctionalized oligonucleotides. The protocol makes use of oligoribonucleotides synthesized by standard phosphoramidite approach on solid support, using commercially available 2'-O-PivOM-protected monomers. Such a protection strategy eliminates the interference between the iodination reagent and silyl protecting groups (TBDMS, TOM) commonly used in RNA synthesis by phosphoramidite approach. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of oligoribonucleotide 5'-azides Basic Protocol 2: CuAAC labeling of oligoribonucleotide 5'-azides in solution Alternate Protocol 1: CuAAC labeling of oligoribonucleotide 5'-azides on solid support Basic Protocol 3: SPAAC labeling of oligoribonucleotide 5'-azides Basic Protocol 4: CuAAC ligation of oligoribonucleotide 5'-azides Basic Protocol 5: CuAAC cyclization of oligoribonucleotide 5'-azides Support Protocol: HPLC Purification.
Subject(s)
Azides/chemical synthesis , Click Chemistry , Fluorescent Dyes/chemistry , RNA/chemistry , Solid-Phase Synthesis Techniques/methods , Azides/chemistry , Cyclization , SolutionsABSTRACT
The high sensitivity of 19F nucleus to changes in the chemical environment has promoted the use of fluorine-labeled molecular probes to study structure and interactions of nucleic acids by 19F NMR. So far, most efforts have focused on incorporating the fluorine atom into nucleobase and ribose moieties using either monomer building blocks for solid-phase synthesis, or nucleoside triphosphates for enzymatic synthesis. Here, we report a simple and efficient synthesis of 5'-fluoromonophosphorylated and 5'-fluorodiphosphorylated oligodeoxyribonucleotides, which combines solid-phase and in-solution synthesis methods and requires only commercially available nucleoside phosphoramidites, followed by their evaluation as 19F NMR probes. We confirmed that the fluorine atom at the oligonucleotide 5' end did not alter the secondary structure of DNA fragments. Moreover, at the same time, it enabled real-time 19F NMR monitoring of various DNA-related biophysical processes, such as oligonucleotide hybridization (including mismatch identification), G-quadruplex folding/unfolding and its interactions with thrombin, as well as formation of an i-motif structure and its interaction with small-molecule ligands.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Oligonucleotide Probes/chemistry , Fluorides , Fluorine Radioisotopes , Humans , Models, Molecular , Oligonucleotide Probes/chemical synthesisABSTRACT
7-Methylguanosine 5' cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5'-terminal nucleotides and additional methylations (2'-O-methylation and m6A). Currently available 5'-capping methods do not address this diversity. We report trinucleotide 5' cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (±2'-O-methylation). HPLC-purified mRNAs carrying these 5' caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5'-terminal A/Am and m6Am, whereas the lowest was observed for G and Gm. The mRNAs carrying 2'-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.
Subject(s)
Protein Biosynthesis/genetics , RNA Caps/genetics , RNA, Messenger/isolation & purification , Transcription, Genetic , Animals , Eukaryota/genetics , Eukaryotic Initiation Factor-4E/genetics , Gene Expression Regulation/genetics , HeLa Cells , Humans , Methylation , Nucleotides/genetics , Protein Processing, Post-Translational/genetics , RNA, Messenger/geneticsABSTRACT
In response to foreign RNA, cellular antiviral mechanisms stimulate high expression of interferon-induced proteins with tetratricopeptide repeats (IFITs). Two members of the IFIT protein family, IFIT1 and IFIT5, are capable of binding the very terminal 5' end of mRNA. In eukaryotes, these mRNA termini contain a cap structure (m7GpppN, cap 0) that is often subjected to further modifications. Here, we performed a thorough examination of IFIT1 and IFIT5 binding to a wide spectrum of differently capped as well as fully uncapped mRNAs. The kinetic analysis of IFIT1 and IFIT5 interactions with mRNA ligands indicates that the cap structure modifications considerably influence the stability of IFIT1/RNA complexes. The most stable complexes were formed between IFIT1 and GpppG/A- and m7GpppG/A-RNAs. Unexpectedly, we found that NAD+- and NADH-capped RNAs associate with IFIT5 with kinetic parameters comparable to pppG-RNA. Finally, we measured interactions of IFIT1 with mRNAs bearing modified synthetic cap analogs that start to become the important tools in biotechnological and medicinal research. We found that incorporation of modified cap analogs to the RNA protects the latter, to a certain degree, from the translational inhibition caused by IFIT1 protein.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Neoplasm Proteins/metabolism , RNA Caps/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Humans , Kinetics , NAD/genetics , Neoplasm Proteins/genetics , Protein Binding , RNA Cap Analogs , RNA-Binding Proteins/geneticsABSTRACT
We report the chemical synthesis of a set of nicotinamide adenine dinucleotide (NAD) cap analogues containing chemical modifications that reduce their susceptibility to NAD-RNA-degrading enzymes. These analogues can be incorporated into transcripts in a similar way as NAD. Biochemical characterization of RNAs carrying these caps with DXO, NudC, and Nudt12 enzymes led to the identification of compounds that can be instrumental in unraveling so far unaddressed biological aspects of NAD-RNAs.
Subject(s)
Adenine/pharmacology , NAD/antagonists & inhibitors , Niacinamide/pharmacology , RNA Caps/antagonists & inhibitors , Adenine/analogs & derivatives , Adenine/chemistry , Molecular Conformation , NAD/metabolism , Niacinamide/analogs & derivatives , Niacinamide/chemistry , RNA Caps/metabolismABSTRACT
The 5' cap consists of 7-methylguanosine (m7G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m7GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented ß-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and ß-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting ß-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.