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Elzbieta Bojarska

Bio: Elzbieta Bojarska is an academic researcher from University of Warsaw. The author has contributed to research in topics: DCPS & NAD+ kinase. The author has an hindex of 12, co-authored 35 publications receiving 499 citations.

Papers
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Journal ArticleDOI
01 Jun 2008-RNA
TL;DR: Synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain found that phosphorrothioate modifications generally stabilized the complex between eIF4E and the cap analog.
Abstract: Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain. Three of them were also modified with methyl groups at the 2'-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K(AS)) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the beta-substituted analog m(7)Gpp(S)pG) was characterized by a K(AS) that was more than fourfold higher than that of its unmodified counterpart (m(7)GpppG). All analogs modified in the gamma position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the alpha position (i.e., m(7)Gppp(S)G and m(2) (7,2'-O )Gppp(S)G) were established as S(P) and R(P) , respectively, using enzymatic digestion and correlation with the S(P) and R(P) diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.

121 citations

Journal ArticleDOI
TL;DR: The synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain make the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis.
Abstract: Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.

53 citations

Journal ArticleDOI
TL;DR: Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group, which should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes.

48 citations

Journal ArticleDOI
TL;DR: Comparison of experimental and computed bimolecular association rate constants indicate that the experimentally observed decrease of the rate constants with the increasing ionic strength is caused by two factors: the first is less effective steering of the ligand towards the binding site at higher ionic strengths, and the second is that for higher ionsic strengths theligand must be closer to the binding sites to induce the fluorescence quenching.
Abstract: The kinetics of binding 7-methyl-GpppG, an analogue of the 5′-mRNA cap, to the cap-binding protein eIF4E, at 20 °C, in 50 mM Hepes-KOH buffer, pH 7.2, and 50, 150 and 350 mM KCl, was measured using a stopped-flow spectrofluorometer, and was simulated by means of a Brownian dynamics method. For most of the stopped-flow measurements a single bimolecular step is an inadequate description of the binding mechanism and an additional step is required to accommodate the kinetic data. The rate constants derived from assumed one-step and two-step binding models were determined. The forward rate constants towards the complex formation decrease, and the reverse rate constants increase, with increasing ionic strength. The association rate constants derived from the stopped-flow measurements and the computed diffusional encounter rate constants agree, indicating that the first observed step can be viewed as a diffusionally controlled encounter of the protein and the ligand. Moreover, comparison of experimental and computed bimolecular association rate constants indicate that the experimentally observed decrease of the rate constants with the increasing ionic strength is caused by two factors. The first is less effective steering of the ligand towards the binding site at higher ionic strengths, and the second is that for higher ionic strengths the ligand must be closer to the binding site to induce the fluorescence quenching.

41 citations

Journal ArticleDOI
TL;DR: A synergistic characteristic of the DcpS structure and activity might be useful for better understanding of the HIT superfamily of pyrophosphatases catalytic mechanism, its regulatory role in gene expression, as well as for designing DCPS inhibitors of potential therapeutic application, e.g. in spinal muscular atrophy.

35 citations


Cited by
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TL;DR: This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.
Abstract: In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.

1,345 citations

Journal ArticleDOI
TL;DR: It is shown thatosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF3E, and a novel combination of two-dimensional isoelectric focusing and Western blotting with phosphospecific antibodies is established.
Abstract: In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5′ end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.

851 citations

Journal ArticleDOI
TL;DR: Emerging regulatory pathways that control mRNA capping and cap‐binding proteins in the cell are described that play a prominent role in the expression of eukaryotic, viral, and parasite mRNAs.
Abstract: The 5' mRNA cap structure is essential for efficient gene expression from yeast to human. It plays a critical role in all aspects of the life cycle of an mRNA molecule. Capping occurs co-transcriptionally on the nascent pre-mRNA as it emerges from the RNA exit channel of RNA polymerase II. The cap structure protects mRNAs from degradation by exonucleases and promotes transcription, polyadenylation, splicing, and nuclear export of mRNA and U-rich, capped snRNAs. In addition, the cap structure is required for the optimal translation of the vast majority of cellular mRNAs, and it also plays a prominent role in the expression of eukaryotic, viral, and parasite mRNAs. Cap-binding proteins specifically bind to the cap structure and mediate its functions in the cell. Two major cellular cap-binding proteins have been described to date: eukaryotic translation initiation factor 4E (eIF4E) in the cytoplasm and nuclear cap binding complex (nCBC), a nuclear complex consisting of a cap-binding subunit cap-binding protein 20 (CBP 20) and an auxiliary protein cap-binding protein 80 (CBP 80). nCBC plays an important role in various aspects of nuclear mRNA metabolism such as pre-mRNA splicing and nuclear export, whereas eIF4E acts primarily as a facilitator of mRNA translation. In this review, we highlight recent findings on the role of the cap structure and cap-binding proteins in the regulation of gene expression. We also describe emerging regulatory pathways that control mRNA capping and cap-binding proteins in the cell.

354 citations

Journal ArticleDOI
TL;DR: Recent improvements of mRNA gene delivery are summarized and its opportunities for the usage in gene therapy are discussed and vector-induced immunogenicity may be avoidable.

280 citations