Intranet
Access
Contact
Publications
2024
Gomes Marina Vitoria, Landwerlin Pauline, Diebold-Durand Marie-Laure, Shaik Tajith B, Durand Alexandre, Troesch Edouard, Weber Chantal, Brillet Karl, Lemée Marianne Victoria, Decroos Christophe, Dulac Ludivine, Antony Pierre, Watrin Erwan, Ennifar Eric, Golzio Christelle, Romier Christophe
The cohesin ATPase cycle is mediated by specific conformational dynamics and interface plasticity of SMC1A and SMC3 ATPase domains Journal Article
In: Cell Rep, vol. 43, no. 9, pp. 114656, 2024, ISSN: 2211-1247.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{pmid39240714,
title = {The cohesin ATPase cycle is mediated by specific conformational dynamics and interface plasticity of SMC1A and SMC3 ATPase domains},
author = {Marina Vitoria Gomes and Pauline Landwerlin and Marie-Laure Diebold-Durand and Tajith B Shaik and Alexandre Durand and Edouard Troesch and Chantal Weber and Karl Brillet and Marianne Victoria Lemée and Christophe Decroos and Ludivine Dulac and Pierre Antony and Erwan Watrin and Eric Ennifar and Christelle Golzio and Christophe Romier},
doi = {10.1016/j.celrep.2024.114656},
issn = {2211-1247},
year = {2024},
date = {2024-09-01},
urldate = {2024-09-01},
journal = {Cell Rep},
volume = {43},
number = {9},
pages = {114656},
abstract = {Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP-dependent manner. The mechanisms underlying cohesin ATPase activity are poorly understood. Here, we characterize distinct steps of the human cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, whereas the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of the SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favors this flexibility, which is counteracted by NIPBL and DNA binding (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) stiffens the SMC3 proximal coiled coil, thus constricting together with that of SMC1A the ATPase module DNA-binding chamber. The plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables these structural rearrangements while keeping the ATP gate shut. VIDEO ABSTRACT.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP-dependent manner. The mechanisms underlying cohesin ATPase activity are poorly understood. Here, we characterize distinct steps of the human cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, whereas the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of the SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favors this flexibility, which is counteracted by NIPBL and DNA binding (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) stiffens the SMC3 proximal coiled coil, thus constricting together with that of SMC1A the ATPase module DNA-binding chamber. The plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables these structural rearrangements while keeping the ATP gate shut. VIDEO ABSTRACT.
Bussi Giovanni, Bonomi Massimiliano, Gkeka Paraskevi, Sattler Michael, Al-Hashimi Hashim M, Auffinger Pascal, Duca Maria, Foricher Yann, Incarnato Danny, Jones Alisha N, Kirmizialtin Serdal, Krepl Miroslav, Orozco Modesto, Palermo Giulia, Pasquali Samuela, Salmon Loïc, Schwalbe Harald, Westhof Eric, Zacharias Martin
RNA dynamics from experimental and computational approaches Journal Article
In: Structure, vol. 32, no. 9, pp. 1281–1287, 2024, ISSN: 1878-4186.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN, WESTHOF
@article{pmid39241758,
title = {RNA dynamics from experimental and computational approaches},
author = {Giovanni Bussi and Massimiliano Bonomi and Paraskevi Gkeka and Michael Sattler and Hashim M Al-Hashimi and Pascal Auffinger and Maria Duca and Yann Foricher and Danny Incarnato and Alisha N Jones and Serdal Kirmizialtin and Miroslav Krepl and Modesto Orozco and Giulia Palermo and Samuela Pasquali and Loïc Salmon and Harald Schwalbe and Eric Westhof and Martin Zacharias},
doi = {10.1016/j.str.2024.07.019},
issn = {1878-4186},
year = {2024},
date = {2024-09-01},
urldate = {2024-09-01},
journal = {Structure},
volume = {32},
number = {9},
pages = {1281--1287},
abstract = {Conformational dynamics is crucial for the biological function of RNA molecules and for their potential as therapeutic targets. This meeting report outlines key "take-home" messages that emerged from the presentations and discussions during the CECAM workshop "RNA dynamics from experimental and computational approaches" in Paris, June 26-28, 2023.},
keywords = {ENNIFAR, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Conformational dynamics is crucial for the biological function of RNA molecules and for their potential as therapeutic targets. This meeting report outlines key "take-home" messages that emerged from the presentations and discussions during the CECAM workshop "RNA dynamics from experimental and computational approaches" in Paris, June 26-28, 2023.
Imbernon Julia Revillo, Weibel Jean‐Marc, Ennifar Eric, Prévost Gilles, Kellenberger Esther
Structural analysis of neomycin B and kanamycin A binding Aminoglycosides Modifying Enzymes (AME) and bacterial ribosomal RNA Journal Article
In: Molecular Informatics, 2024, ISSN: 1868-1751.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{RevilloImbernon2024,
title = {Structural analysis of neomycin B and kanamycin A binding Aminoglycosides Modifying Enzymes (AME) and bacterial ribosomal RNA},
author = {Julia Revillo Imbernon and Jean‐Marc Weibel and Eric Ennifar and Gilles Prévost and Esther Kellenberger},
doi = {10.1002/minf.202300339},
issn = {1868-1751},
year = {2024},
date = {2024-06-10},
urldate = {2024-06-10},
journal = {Molecular Informatics},
publisher = {Wiley},
abstract = {<jats:title>Abstract</jats:title><jats:p>Aminoglycosides are crucial antibiotics facing challenges from bacterial resistance. This study addresses the importance of aminoglycoside modifying enzymes in the context of escalating resistance. Drawing upon over two decades of structural data in the Protein Data Bank, we focused on two key antibiotics, neomycin B and kanamycin A, to explore how the aminoglycoside structure is exploited by this family of enzymes. A systematic comparison across diverse enzymes and the RNA A‐site target identified common characteristics in the recognition mode, while assessing the adaptability of neomycin B and kanamycin A in various environments.</jats:p>},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
<jats:title>Abstract</jats:title><jats:p>Aminoglycosides are crucial antibiotics facing challenges from bacterial resistance. This study addresses the importance of aminoglycoside modifying enzymes in the context of escalating resistance. Drawing upon over two decades of structural data in the Protein Data Bank, we focused on two key antibiotics, neomycin B and kanamycin A, to explore how the aminoglycoside structure is exploited by this family of enzymes. A systematic comparison across diverse enzymes and the RNA A‐site target identified common characteristics in the recognition mode, while assessing the adaptability of neomycin B and kanamycin A in various environments.</jats:p>
D'Agostino Mattia, Simonetti Angelita, Motta Stefano, Wolff Philippe, Romagnoli Alice, Piccinini Astra, Spinozzi Francesco, Marino Daniele Di, Teana Anna La, Ennifar Eric
Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism Journal Article
In: Structure, 2024, ISSN: 1878-4186.
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Unité ARN
@article{pmid38582076,
title = {Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism},
author = {Mattia D'Agostino and Angelita Simonetti and Stefano Motta and Philippe Wolff and Alice Romagnoli and Astra Piccinini and Francesco Spinozzi and Daniele Di Marino and Anna La Teana and Eric Ennifar},
doi = {10.1016/j.str.2024.03.008},
issn = {1878-4186},
year = {2024},
date = {2024-03-01},
urldate = {2024-03-01},
journal = {Structure},
abstract = {The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.
Brillet Karl, Janczuk-Richter Marta, Poon Amanda, Laukart-Bradley Joanne, Ennifar Eric, Lebars Isabelle
Characterization of SLA RNA promoter from dengue virus and its interaction with the viral non-structural NS5 protein Journal Article
In: Biochimie, vol. 222, pp. 87–100, 2024, ISSN: 1638-6183.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{pmid38408720,
title = {Characterization of SLA RNA promoter from dengue virus and its interaction with the viral non-structural NS5 protein},
author = {Karl Brillet and Marta Janczuk-Richter and Amanda Poon and Joanne Laukart-Bradley and Eric Ennifar and Isabelle Lebars},
doi = {10.1016/j.biochi.2024.02.005},
issn = {1638-6183},
year = {2024},
date = {2024-02-01},
urldate = {2024-02-01},
journal = {Biochimie},
volume = {222},
pages = {87--100},
abstract = {The Dengue virus (DENV) is the most significant arthropod-borne viral pathogen in humans with 400 million infections annually. DENV comprises four distinct serotypes (DENV-1 to -4) which complicates vaccine development. Any of the four serotypes can cause clinical illness but with distinctive infection dynamics. Variations in sequences identified within the four genomes induce structural differences in crucial RNA motifs that were suggested to be correlated to the degree of pathogenicity among DENV-1 to -4. In particular, the RNA Stem-loop A (SLA) at the 5'-end of the genome, acts as a key regulator of the viral replication cycle by interacting with the viral NS5 polymerase to initiate the minus-strand viral RNA synthesis and later to methylate and cap the synthesized RNA. The molecular details of this interaction remain not fully described. Here, we report the solution secondary structures of SLA from DENV-1 to -4. Our results highlight that the four SLA exhibit structural and dynamic differences. Secondly, to determine whether SLA RNA contains serotype-specific determinants for the recognition by the viral NS5 protein, we investigated interactions between SLA from DENV -1 to -4 and DENV2 NS5 using combined biophysical approaches. Our results show that NS5 from DENV2 is able to bind SLA from other serotypes, but that other viral or host factors may be necessary to stabilize the complex and promote the catalytically active state of the NS5. By contrast, we show that a serotype-specific binding is driven by specific interactions involving conformational changes within the SLA RNA.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Dengue virus (DENV) is the most significant arthropod-borne viral pathogen in humans with 400 million infections annually. DENV comprises four distinct serotypes (DENV-1 to -4) which complicates vaccine development. Any of the four serotypes can cause clinical illness but with distinctive infection dynamics. Variations in sequences identified within the four genomes induce structural differences in crucial RNA motifs that were suggested to be correlated to the degree of pathogenicity among DENV-1 to -4. In particular, the RNA Stem-loop A (SLA) at the 5'-end of the genome, acts as a key regulator of the viral replication cycle by interacting with the viral NS5 polymerase to initiate the minus-strand viral RNA synthesis and later to methylate and cap the synthesized RNA. The molecular details of this interaction remain not fully described. Here, we report the solution secondary structures of SLA from DENV-1 to -4. Our results highlight that the four SLA exhibit structural and dynamic differences. Secondly, to determine whether SLA RNA contains serotype-specific determinants for the recognition by the viral NS5 protein, we investigated interactions between SLA from DENV -1 to -4 and DENV2 NS5 using combined biophysical approaches. Our results show that NS5 from DENV2 is able to bind SLA from other serotypes, but that other viral or host factors may be necessary to stabilize the complex and promote the catalytically active state of the NS5. By contrast, we show that a serotype-specific binding is driven by specific interactions involving conformational changes within the SLA RNA.
Compain Guillaume, Monsarrat Clément, Blagojevic Julie, Brillet Karl, Dumas Philippe, Hammann Philippe, Kuhn Lauriane, Martiel Isabelle, Engilberge Sylvain, Oliéric Vincent, Wolff Philippe, Burnouf Dominique Y, Wagner Jérôme, Guichard Gilles
Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp Journal Article
In: JACS Au, vol. 4, no. 2, pp. 432–440, 2024, ISSN: 2691-3704.
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, PPSE, Unité ARN
@article{pmid38425897,
title = {Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp},
author = {Guillaume Compain and Clément Monsarrat and Julie Blagojevic and Karl Brillet and Philippe Dumas and Philippe Hammann and Lauriane Kuhn and Isabelle Martiel and Sylvain Engilberge and Vincent Oliéric and Philippe Wolff and Dominique Y Burnouf and Jérôme Wagner and Gilles Guichard},
doi = {10.1021/jacsau.3c00572},
issn = {2691-3704},
year = {2024},
date = {2024-02-01},
urldate = {2024-02-01},
journal = {JACS Au},
volume = {4},
number = {2},
pages = {432--440},
abstract = {Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.},
keywords = {ARN-MS, ENNIFAR, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.
2023
Giuliodori Anna Maria, Belardinelli Riccardo, Duval Melodie, Garofalo Raffaella, Schenckbecher Emma, Hauryliuk Vasili, Ennifar Eric, Marzi Stefano
CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression Journal Article
In: Front Microbiol, vol. 14, pp. 1118329, 2023, ISSN: 1664-302X.
Abstract | Links | BibTeX | Tags: ENNIFAR, ROMBY, Unité ARN
@article{pmid36846801b,
title = { CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression},
author = {Anna Maria Giuliodori and Riccardo Belardinelli and Melodie Duval and Raffaella Garofalo and Emma Schenckbecher and Vasili Hauryliuk and Eric Ennifar and Stefano Marzi},
doi = {10.3389/fmicb.2023.1118329},
issn = {1664-302X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Front Microbiol},
volume = {14},
pages = {1118329},
abstract = { CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.},
keywords = {ENNIFAR, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.
2022
Mráziková Klaudia, Kruse Holger, Mlýnský Vojtěch, Auffinger Pascal, Šponer Jiří
Multiscale Modeling of Phosphate···π Contacts in RNA U-Turns Exposes Differences between Quantum-Chemical and AMBER Force Field Descriptions Journal Article
In: J Chem Inf Model, vol. 62, iss. 23, pp. 6182-6200, 2022, ISSN: 1549-960X.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{pmid36454943,
title = {Multiscale Modeling of Phosphate···π Contacts in RNA U-Turns Exposes Differences between Quantum-Chemical and AMBER Force Field Descriptions},
author = {Klaudia Mráziková and Holger Kruse and Vojtěch Mlýnský and Pascal Auffinger and Jiří Šponer},
doi = {10.1021/acs.jcim.2c01064},
issn = {1549-960X},
year = {2022},
date = {2022-12-01},
urldate = {2022-12-01},
journal = {J Chem Inf Model},
volume = {62},
issue = {23},
pages = {6182-6200},
abstract = {Phosphate···π, also called anion···π, contacts occur between nucleobases and anionic phosphate oxygens (OP2) in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). These contacts were investigated using state-of-the-art quantum-chemical methods (QM) to characterize their physicochemical properties and to serve as a reference to evaluate AMBER force field (AFF) performance. We found that phosphate···π interaction energies calculated with the AFF for dimethyl phosphate···nucleobase model systems are less stabilizing in comparison with double-hybrid DFT and that minimum contact distances are larger for all nucleobases. These distance stretches are also observed in large-scale AFF vs QM/MM computations and classical molecular dynamics (MD) simulations on several r(gcGNRAgc) tetraloop hairpins when compared to experimental data extracted from X-ray/cryo-EM structures (res. ≤ 2.5 Å) using the WebFR3D bioinformatic tool. MD simulations further revealed shifted OP2/nucleobase positions. We propose that discrepancies between the QM and AFF result from a combination of missing polarization in the AFF combined with too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms in addition to an exaggerated short-range repulsion of the LJ repulsive term. We compared these results with earlier data gathered on lone pair···π contacts in CpG Z-steps occurring in r(UNCG) tetraloops. In both instances, charge transfer calculations do not support any significant → π* donation effects. We also investigated thiophosphate···π contacts that showed reduced stabilizing interaction energies when compared to phosphate···π contacts. Thus, we challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be explained by larger London dispersion.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Phosphate···π, also called anion···π, contacts occur between nucleobases and anionic phosphate oxygens (OP2) in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). These contacts were investigated using state-of-the-art quantum-chemical methods (QM) to characterize their physicochemical properties and to serve as a reference to evaluate AMBER force field (AFF) performance. We found that phosphate···π interaction energies calculated with the AFF for dimethyl phosphate···nucleobase model systems are less stabilizing in comparison with double-hybrid DFT and that minimum contact distances are larger for all nucleobases. These distance stretches are also observed in large-scale AFF vs QM/MM computations and classical molecular dynamics (MD) simulations on several r(gcGNRAgc) tetraloop hairpins when compared to experimental data extracted from X-ray/cryo-EM structures (res. ≤ 2.5 Å) using the WebFR3D bioinformatic tool. MD simulations further revealed shifted OP2/nucleobase positions. We propose that discrepancies between the QM and AFF result from a combination of missing polarization in the AFF combined with too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms in addition to an exaggerated short-range repulsion of the LJ repulsive term. We compared these results with earlier data gathered on lone pair···π contacts in CpG Z-steps occurring in r(UNCG) tetraloops. In both instances, charge transfer calculations do not support any significant → π* donation effects. We also investigated thiophosphate···π contacts that showed reduced stabilizing interaction energies when compared to phosphate···π contacts. Thus, we challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be explained by larger London dispersion.
Mair Stefan, Erharter Kevin, Renard Eva, Brillet Karl, Brunner Melanie, Lusser Alexandra, Kreutz Christoph, Ennifar Eric, Micura Ronald
Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA Journal Article
In: Nucleic Acids Res, vol. 50, iss. 11, pp. 6038-6051, 2022, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{pmid35687141,
title = {Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA},
author = {Stefan Mair and Kevin Erharter and Eva Renard and Karl Brillet and Melanie Brunner and Alexandra Lusser and Christoph Kreutz and Eric Ennifar and Ronald Micura},
doi = {10.1093/nar/gkac477},
issn = {1362-4962},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Nucleic Acids Res},
volume = {50},
issue = {11},
pages = {6038-6051},
abstract = {Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.
Roovers Martine L, Labar Geoffray, Wolff Philippe, Feller Andre, Elder Dany Van, Soin Romuald, Gueydan Cyril, Kruys Veronique, Droogmans Louis
In: RNA, vol. 28, iss. 9, pp. 1185-1196, 2022, ISSN: 1469-9001.
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Unité ARN
@article{pmid35710145b,
title = {The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA},
author = {Martine L Roovers and Geoffray Labar and Philippe Wolff and Andre Feller and Dany Van Elder and Romuald Soin and Cyril Gueydan and Veronique Kruys and Louis Droogmans},
doi = {10.1261/rna.079131.122},
issn = {1469-9001},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {RNA},
volume = {28},
issue = {9},
pages = {1185-1196},
abstract = {A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) N-terminal domain.},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) N-terminal domain.
Bereiter R., Renard E., Breuker K., Kreutz C., Ennifar E., Micura R.
1-Deazaguanosine-Modified RNA: The Missing Piece for Functional RNA Atomic Mutagenesis Journal Article
In: J Am Chem Soc, vol. 144, iss. 23, pp. 10344-10352, 2022, ISBN: 35666572, (1520-5126 (Electronic) 0002-7863 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{nokey,
title = {1-Deazaguanosine-Modified RNA: The Missing Piece for Functional RNA Atomic Mutagenesis},
author = {R. Bereiter and E. Renard and K. Breuker and C. Kreutz and E. Ennifar and R. Micura},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35666572},
doi = {10.1021/jacs.2c01877},
isbn = {35666572},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {J Am Chem Soc},
volume = {144},
issue = {23},
pages = {10344-10352},
abstract = {Atomic mutagenesis is the key to advance our understanding of RNA recognition and RNA catalysis. To this end, deazanucleosides are utilized to evaluate the participation of specific atoms in these processes. One of the remaining challenges is access to RNA-containing 1-deazaguanosine (c(1)G). Here, we present the synthesis of this nucleoside and its phosphoramidite, allowing first time access to c(1)G-modified RNA. Thermodynamic analyses revealed the base pairing parameters for c(1)G-modified RNA. Furthermore, by NMR spectroscopy, a c(1)G-triggered switch of Watson-Crick into Hoogsteen pairing in HIV-2 TAR RNA was identified. Additionally, using X-ray structure analysis, a guanine-phosphate backbone interaction affecting RNA fold stability was characterized, and finally, the critical impact of an active-site guanine in twister ribozyme on the phosphodiester cleavage was revealed. Taken together, our study lays the synthetic basis for c(1)G-modified RNA and demonstrates the power of the completed deazanucleoside toolbox for RNA atomic mutagenesis needed to achieve in-depth understanding of RNA recognition and catalysis.},
note = {1520-5126 (Electronic)
0002-7863 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Atomic mutagenesis is the key to advance our understanding of RNA recognition and RNA catalysis. To this end, deazanucleosides are utilized to evaluate the participation of specific atoms in these processes. One of the remaining challenges is access to RNA-containing 1-deazaguanosine (c(1)G). Here, we present the synthesis of this nucleoside and its phosphoramidite, allowing first time access to c(1)G-modified RNA. Thermodynamic analyses revealed the base pairing parameters for c(1)G-modified RNA. Furthermore, by NMR spectroscopy, a c(1)G-triggered switch of Watson-Crick into Hoogsteen pairing in HIV-2 TAR RNA was identified. Additionally, using X-ray structure analysis, a guanine-phosphate backbone interaction affecting RNA fold stability was characterized, and finally, the critical impact of an active-site guanine in twister ribozyme on the phosphodiester cleavage was revealed. Taken together, our study lays the synthetic basis for c(1)G-modified RNA and demonstrates the power of the completed deazanucleoside toolbox for RNA atomic mutagenesis needed to achieve in-depth understanding of RNA recognition and catalysis.
2021
Bec G, Ennifar E
switchSENSE Technology for Analysis of DNA Polymerase Kinetics Book Chapter
In: Rederstorff, M (Ed.): vol. 2247, pp. 145-153, Springer Protocols, Humana Press, New York, NY, Small Non-Coding RNAs, 2021.
Abstract | BibTeX | Tags: Biosensor, DNA polymerase, ENNIFAR, HIV reverse transcriptase, Kinetics, switchSENSE technology, Unité ARN
@inbook{Bec2021,
title = {switchSENSE Technology for Analysis of DNA Polymerase Kinetics },
author = {G Bec and E Ennifar
},
editor = {M Rederstorff},
year = {2021},
date = {2021-01-01},
journal = {Methods in Molecular Biology },
volume = {2247},
pages = {145-153},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
edition = {Small Non-Coding RNAs},
series = {Methods in Molecular Biology},
abstract = {The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics, dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate of the enzyme. },
keywords = {Biosensor, DNA polymerase, ENNIFAR, HIV reverse transcriptase, Kinetics, switchSENSE technology, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics, dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate of the enzyme.
Chagot M E, Quinternet M, Manival X, Lebars I
Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs Journal Article
In: Methods Mol Biol, vol. 2300, pp. 251-266, 2021, ISBN: 33792884, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: 3D structure, dynamics, ENNIFAR, NMR, RNA, Unité ARN
@article{Chagot2021,
title = {Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs},
author = {M E Chagot and M Quinternet and X Manival and I Lebars},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33792884},
doi = {10.1007/978-1-0716-1386-3_19},
isbn = {33792884},
year = {2021},
date = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2300},
pages = {251-266},
abstract = {Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids.In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation.},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {3D structure, dynamics, ENNIFAR, NMR, RNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids.In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation.
Velours C, Aumont-Nicaise M, Uebel S, England P, Velazquez-Campoy A, Stroebel D, Bec G, Soule P, Quetard C, Ebel C, Roussel A, Charbonnier J B, Varela P F
Macromolecular interactions in vitro, comparing classical and novel approaches Journal Article
In: Eur Biophys J, vol. 50, no. 3-4, pp. 313-330, 2021, ISBN: 33792745, (1432-1017 (Electronic) 0175-7571 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Artificial binders, Double-stranded DNA breaks repair factors, ENNIFAR, Macromolecular interactions, Molecular scale biophysics, Unité ARN
@article{,
title = {Macromolecular interactions in vitro, comparing classical and novel approaches},
author = {C Velours and M Aumont-Nicaise and S Uebel and P England and A Velazquez-Campoy and D Stroebel and G Bec and P Soule and C Quetard and C Ebel and A Roussel and J B Charbonnier and P F Varela},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33792745},
doi = {10.1007/s00249-021-01517-5},
isbn = {33792745},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Eur Biophys J},
volume = {50},
number = {3-4},
pages = {313-330},
abstract = {Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France (https://mosbio.sciencesconf.org/). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.},
note = {1432-1017 (Electronic)
0175-7571 (Linking)
Journal Article},
keywords = {Artificial binders, Double-stranded DNA breaks repair factors, ENNIFAR, Macromolecular interactions, Molecular scale biophysics, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France (https://mosbio.sciencesconf.org/). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.
Bereiter R, Himmelstoss M, Renard E, Mairhofer E, Egger M, Breuker K, Kreutz C, Ennifar E, Micura R
Impact of 3-deazapurine nucleobases on RNA properties Journal Article
In: Nucleic Acids Res, vol. 49, no. 8, pp. 4281-4293, 2021, ISBN: 33856457, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Impact of 3-deazapurine nucleobases on RNA properties},
author = {R Bereiter and M Himmelstoss and E Renard and E Mairhofer and M Egger and K Breuker and C Kreutz and E Ennifar and R Micura},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33856457},
isbn = {33856457},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nucleic Acids Res},
volume = {49},
number = {8},
pages = {4281-4293},
abstract = {Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.
Schenckbecher E, Bec G, Sakamoto T, Meyer B, Ennifar E
Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE Journal Article
In: Methods Mol Biol, vol. 2263, pp. 341-350, 2021, ISBN: 33877606, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Biophysics, ENNIFAR, Kinetics, Ribosome, RNA, switchSENSE, Unité ARN
@article{,
title = {Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE},
author = {E Schenckbecher and G Bec and T Sakamoto and B Meyer and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33877606},
doi = {10.1007/978-1-0716-1197-5_15},
isbn = {33877606},
year = {2021},
date = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2263},
pages = {341-350},
abstract = {Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome, initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE((R)) technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome. This study provides a proof of concept for the application of this method on large complexes.},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {Biophysics, ENNIFAR, Kinetics, Ribosome, RNA, switchSENSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome, initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE((R)) technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome. This study provides a proof of concept for the application of this method on large complexes.
Velazquez-Campoy A, Claro B, Abian O, Horing J, Bourlon L, Claveria-Gimeno R, Ennifar E, England P, Chaires J B, Wu D, Piszczek G, Brautigam C, Tso S C, Zhao H, Schuck P, Keller S, Bastos M
A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca(2+) and Mg(2+) binding to EDTA Journal Article
In: Eur Biophys J, vol. 50, no. 3-4, pp. 429-451, 2021, ISBN: 33864101, (1432-1017 (Electronic) 0175-7571 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Benchmark study, Data treatment, ENNIFAR, Isothermal Titration Calorimetry (ITC), Ligand-binding, Sample preparation, Standard reaction, Unité ARN
@article{,
title = {A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca(2+) and Mg(2+) binding to EDTA},
author = {A Velazquez-Campoy and B Claro and O Abian and J Horing and L Bourlon and R Claveria-Gimeno and E Ennifar and P England and J B Chaires and D Wu and G Piszczek and C Brautigam and S C Tso and H Zhao and P Schuck and S Keller and M Bastos},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33864101},
doi = {10.1007/s00249-021-01523-7},
isbn = {33864101},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Eur Biophys J},
volume = {50},
number = {3-4},
pages = {429-451},
abstract = {A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (DeltaH), and the so-called "stoichiometry" (n), a concentration-correction factor.},
note = {1432-1017 (Electronic)
0175-7571 (Linking)
Journal Article},
keywords = {Benchmark study, Data treatment, ENNIFAR, Isothermal Titration Calorimetry (ITC), Ligand-binding, Sample preparation, Standard reaction, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (DeltaH), and the so-called "stoichiometry" (n), a concentration-correction factor.
Ramos-Morales E, Bayam E, Del-Pozo-Rodriguez J, Salinas-Giege T, Marek M, Tilly P, Wolff P, Troesch E, Ennifar E, Drouard L, Godin J D, Romier C
The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination Journal Article
In: Nucleic Acids Res, vol. 49, no. 11, pp. 6529-6548, 2021, ISBN: 34057470, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Unité ARN
@article{,
title = {The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination},
author = {E Ramos-Morales and E Bayam and J Del-Pozo-Rodriguez and T Salinas-Giege and M Marek and P Tilly and P Wolff and E Troesch and E Ennifar and L Drouard and J D Godin and C Romier},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34057470},
doi = {10.1093/nar/gkab436},
isbn = {34057470},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nucleic Acids Res},
volume = {49},
number = {11},
pages = {6529-6548},
abstract = {Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.
Himmelstoss M, Erharter K, Renard E, Ennifar E, Kreutz C, Micura R
2'-O-Trifluoromethylated RNA - a powerful modification for RNA chemistry and NMR spectroscopy Journal Article
In: Chem Sci, vol. 11, no. 41, pp. 11322-11330, 2021, ISBN: 34094374, (2041-6520 (Print) 2041-6520 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {2'-O-Trifluoromethylated RNA - a powerful modification for RNA chemistry and NMR spectroscopy},
author = {M Himmelstoss and K Erharter and E Renard and E Ennifar and C Kreutz and R Micura},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34094374},
doi = {10.1039/d0sc04520a},
isbn = {34094374},
year = {2021},
date = {2021-01-01},
journal = {Chem Sci},
volume = {11},
number = {41},
pages = {11322-11330},
abstract = {New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA-ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with (19)F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2'-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2'-SCF3 modification being the most prominent one. A major drawback of the 2'-SCF3 group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2'-OCF3 modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2'-OCF3 labeled RNA and to investigate the impact of this modification. We present the syntheses of 2'-OCF3 adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2'-OCF3 group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3'-endo conformation of the 2'-OCF3 ribose as reflected in the (3) J (H1'-H2') coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in (19)F-NMR analysis of RNA structure equilibria and of RNA-small molecule interactions. The study is complemented by a crystal structure at 0.9 A resolution of a 27 nt hairpin RNA containing a single 2'-OCF3 group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.},
note = {2041-6520 (Print)
2041-6520 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA-ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with (19)F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2'-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2'-SCF3 modification being the most prominent one. A major drawback of the 2'-SCF3 group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2'-OCF3 modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2'-OCF3 labeled RNA and to investigate the impact of this modification. We present the syntheses of 2'-OCF3 adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2'-OCF3 group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3'-endo conformation of the 2'-OCF3 ribose as reflected in the (3) J (H1'-H2') coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in (19)F-NMR analysis of RNA structure equilibria and of RNA-small molecule interactions. The study is complemented by a crystal structure at 0.9 A resolution of a 27 nt hairpin RNA containing a single 2'-OCF3 group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.
Thepaut M, Campos-Silva R, Renard E, Barloy-Hubler F, Ennifar E, Boujard D, Gillet R
Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria Journal Article
In: Rna, 2021, ISBN: 34353925, (1469-9001 (Electronic) 1355-8382 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria},
author = {M Thepaut and R Campos-Silva and E Renard and F Barloy-Hubler and E Ennifar and D Boujard and R Gillet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34353925},
doi = {10.1261/rna.078773.121},
isbn = {34353925},
year = {2021},
date = {2021-01-01},
journal = {Rna},
abstract = {In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.},
note = {1469-9001 (Electronic)
1355-8382 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.
Andre C, Veillard F, Wolff P, Lobstein A M, Compain G, Monsarrat C, Reichhart J M, Burnouf D Y, Guichard G, Wagner J E
Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome Journal Article
In: RSC Chem Biol, vol. 2, no. 4, pp. 1296, 2021, ISBN: 34459830, (2633-0679 (Electronic) 2633-0679 (Linking) Published Erratum).
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, reichhart, Unité ARN
@article{,
title = {Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome},
author = {C Andre and F Veillard and P Wolff and A M Lobstein and G Compain and C Monsarrat and J M Reichhart and D Y Burnouf and G Guichard and J E Wagner},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34459830},
doi = {10.1039/d1cb90020j},
isbn = {34459830},
year = {2021},
date = {2021-01-01},
journal = {RSC Chem Biol},
volume = {2},
number = {4},
pages = {1296},
abstract = {[This corrects the article DOI: 10.1039/D0CB00060D.].},
note = {2633-0679 (Electronic)
2633-0679 (Linking)
Published Erratum},
keywords = {ARN-MS, ENNIFAR, reichhart, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
[This corrects the article DOI: 10.1039/D0CB00060D.].
Mrazikova K., Sponer J., Mlynsky V., Auffinger P., Kruse H.
Short-Range Imbalances in the AMBER Lennard-Jones Potential for (Deoxy)Ribose.Nucleobase Lone-Pair.pi Contacts in Nucleic Acids Journal Article
In: J Chem Inf Model, vol. 61, no. 11, pp. 5644-5657, 2021, ISBN: 34738826, (1549-960X (Electronic) 1549-9596 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{nokey,
title = {Short-Range Imbalances in the AMBER Lennard-Jones Potential for (Deoxy)Ribose.Nucleobase Lone-Pair.pi Contacts in Nucleic Acids},
author = {K. Mrazikova and J. Sponer and V. Mlynsky and P. Auffinger and H. Kruse},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34738826},
doi = {10.1021/acs.jcim.1c01047},
isbn = {34738826},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {J Chem Inf Model},
volume = {61},
number = {11},
pages = {5644-5657},
abstract = {The lone-pair.pi (lp.pi) (deoxy)ribose.nucleobase stacking is a recurring interaction in Z-DNA and RNAs that is characterized by sub-van der Waals lp.pi contacts (<3.0 A). It is a part of the structural signature of CpG Z-step motifs in Z-DNA and r(UNCG) tetraloops that are known to behave poorly in molecular dynamics (MD) simulations. Although the exact origin of the MD simulation issues remains unclear, a significant part of the problem might be due to an imbalanced description of nonbonded interactions, including the characteristic lp.pi stacking. To gain insights into the links between lp.pi stacking and MD, we present an in-depth comparison between accurate large-basis-set double-hybrid Kohn-Sham density functional theory calculations DSD-BLYP-D3/ma-def2-QZVPP (DHDF-D3) and data obtained with the nonbonded potential of the AMBER force field (AFF) for NpN Z-steps (N = G, A, C, and U). Among other differences, we found that the AFF overestimates the DHDF-D3 lp.pi distances by approximately 0.1-0.2 A, while the deviation between the DHDF-D3 and AFF descriptions sharply increases in the short-range region of the interaction. Based on atom-in-molecule polarizabilities and symmetry-adapted perturbation theory analysis, we inferred that the DHDF-D3 versus AFF differences partly originate in identical nucleobase carbon atom Lennard-Jones (LJ) parameters despite the presence/absence of connected electron-withdrawing groups that lead to different effective volumes or vdW radii. Thus, to precisely model the very short CpG lp.pi contact distances, we recommend revision of the nucleobase atom LJ parameters. Additionally, we suggest that the large discrepancy between DHDF-D3 and AFF short-range repulsive part of the interaction energy potential may significantly contribute to the poor performances of MD simulations of nucleic acid systems containing Z-steps. Understanding where, and if possible why, the point-charge-type effective potentials reach their limits is vital for developing next-generation FFs and for addressing specific issues in contemporary MD simulations.},
note = {1549-960X (Electronic)
1549-9596 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The lone-pair.pi (lp.pi) (deoxy)ribose.nucleobase stacking is a recurring interaction in Z-DNA and RNAs that is characterized by sub-van der Waals lp.pi contacts (<3.0 A). It is a part of the structural signature of CpG Z-step motifs in Z-DNA and r(UNCG) tetraloops that are known to behave poorly in molecular dynamics (MD) simulations. Although the exact origin of the MD simulation issues remains unclear, a significant part of the problem might be due to an imbalanced description of nonbonded interactions, including the characteristic lp.pi stacking. To gain insights into the links between lp.pi stacking and MD, we present an in-depth comparison between accurate large-basis-set double-hybrid Kohn-Sham density functional theory calculations DSD-BLYP-D3/ma-def2-QZVPP (DHDF-D3) and data obtained with the nonbonded potential of the AMBER force field (AFF) for NpN Z-steps (N = G, A, C, and U). Among other differences, we found that the AFF overestimates the DHDF-D3 lp.pi distances by approximately 0.1-0.2 A, while the deviation between the DHDF-D3 and AFF descriptions sharply increases in the short-range region of the interaction. Based on atom-in-molecule polarizabilities and symmetry-adapted perturbation theory analysis, we inferred that the DHDF-D3 versus AFF differences partly originate in identical nucleobase carbon atom Lennard-Jones (LJ) parameters despite the presence/absence of connected electron-withdrawing groups that lead to different effective volumes or vdW radii. Thus, to precisely model the very short CpG lp.pi contact distances, we recommend revision of the nucleobase atom LJ parameters. Additionally, we suggest that the large discrepancy between DHDF-D3 and AFF short-range repulsive part of the interaction energy potential may significantly contribute to the poor performances of MD simulations of nucleic acid systems containing Z-steps. Understanding where, and if possible why, the point-charge-type effective potentials reach their limits is vital for developing next-generation FFs and for addressing specific issues in contemporary MD simulations.
Monsarrat C., Compain G., Andre C., Engilberge S., Martiel I., Olieric V., Wolff P., Brillet K., Landolfo M., da Veiga C. Silva, Wagner J., Guichard G., Burnouf D. Y.
Iterative Structure-Based Optimization of Short Peptides Targeting the Bacterial Sliding Clamp Journal Article
In: J Med Chem, 2021, ISBN: 34806883, (1520-4804 (Electronic) 0022-2623 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Unité ARN
@article{nokey,
title = {Iterative Structure-Based Optimization of Short Peptides Targeting the Bacterial Sliding Clamp},
author = {C. Monsarrat and G. Compain and C. Andre and S. Engilberge and I. Martiel and V. Olieric and P. Wolff and K. Brillet and M. Landolfo and C. Silva da Veiga and J. Wagner and G. Guichard and D. Y. Burnouf},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34806883},
doi = {10.1021/acs.jmedchem.1c00918},
isbn = {34806883},
year = {2021},
date = {2021-01-01},
journal = {J Med Chem},
abstract = {The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the Escherichia coli SC ((Ec)SC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus. In each category, single modifications were identified that increased affinity to (Ec)SC. A combination of such modifications yielded in several cases to a substantially increased affinity compared to the parent peptides with Kd in the range of 30-80 nM. X-ray structure analysis of 11 peptide/(Ec)SC co-crystals revealed new interactions at the peptide-protein interface (i.e., stacking interactions, hydrogen bonds, and hydrophobic contacts) that can account for the improved binding. Several compounds among the best binders were also found to be more effective in inhibiting SC-dependent DNA synthesis.},
note = {1520-4804 (Electronic)
0022-2623 (Linking)
Journal Article},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the Escherichia coli SC ((Ec)SC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus. In each category, single modifications were identified that increased affinity to (Ec)SC. A combination of such modifications yielded in several cases to a substantially increased affinity compared to the parent peptides with Kd in the range of 30-80 nM. X-ray structure analysis of 11 peptide/(Ec)SC co-crystals revealed new interactions at the peptide-protein interface (i.e., stacking interactions, hydrogen bonds, and hydrophobic contacts) that can account for the improved binding. Several compounds among the best binders were also found to be more effective in inhibiting SC-dependent DNA synthesis.
2020
Abaeva I S, Vicens Q, Bochler A, Soufari H, Simonetti A, Pestova T V, Hashem Y, Hellen C U T
The Halastavi árva Virus Intergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism Journal Article
In: Cell Reports, vol. 33, no. 10, pp. 108476, 2020.
Abstract | Links | BibTeX | Tags: Cricket paralysis virus, Dicistrovirus, ENNIFAR, Halastavi árva virus; IRES, intergenic region, internal ribosomal entry site, pseudoknot, Ribosome, SERBP1, SERPINE1 mRNA binding protein 1, Unité ARN
@article{Abaeva2020,
title = {The Halastavi árva Virus Intergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism },
author = {I S Abaeva and Q Vicens and A Bochler and H Soufari and A Simonetti and T V Pestova and Y Hashem and C U T Hellen},
url = {https://pubmed.ncbi.nlm.nih.gov/33296660/},
doi = {10.1016/j.celrep.2020.108476 },
year = {2020},
date = {2020-12-08},
journal = {Cell Reports},
volume = {33},
number = {10},
pages = {108476},
abstract = {Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRESs) do not require initiator tRNA, an AUG codon, or initiation factors and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A sites to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By using in vitro reconstitution and cryoelectron microscopy (cryo-EM), we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI. },
keywords = {Cricket paralysis virus, Dicistrovirus, ENNIFAR, Halastavi árva virus; IRES, intergenic region, internal ribosomal entry site, pseudoknot, Ribosome, SERBP1, SERPINE1 mRNA binding protein 1, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRESs) do not require initiator tRNA, an AUG codon, or initiation factors and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A sites to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By using in vitro reconstitution and cryoelectron microscopy (cryo-EM), we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.
Auffinger P, Ennifar E, D'Ascenzo L
Deflating the RNA Mg 2+ bubble. Stereochemistry to the rescue! Journal Article
In: RNA, vol. 27, no. 3, pp. 243-252, 2020.
Abstract | Links | BibTeX | Tags: cryo-EM, ENNIFAR, ionic atmosphere, solvent, Unité ARN, validation, X-Ray
@article{P.2020,
title = {Deflating the RNA Mg 2+ bubble. Stereochemistry to the rescue! },
author = {P Auffinger and E Ennifar and L D'Ascenzo},
url = {https://doi.org/10.1261/rna.076067.120},
doi = {10.1261/rna.076067.120 },
year = {2020},
date = {2020-12-02},
journal = {RNA},
volume = {27},
number = {3},
pages = {243-252},
abstract = {Proper evaluation of the ionic structure of biomolecular systems remains challenging in X ray and cryo-EM techniques but is essential for advancing our understanding of complex structure/activity/solvent relationships. However, numerous studies overestimate the number of MgProper evaluation of the ionic structure of biomolecular systems remains challenging in X ray and cryo-EM techniques but is essential for advancing our understanding of complex structure/activity/solvent relationships. However, numerous studies overestimate the number of Mg2+ in the deposited structures and underrate the importance of stereochemical rules to correctly assign these ions. Herein, we re-evaluate the PDBid 6QNR and 6SJ6 models of the ribosome ionic structure and establish that stereochemical principles need consideration when evaluating ion binding features, even when K+ anomalous signals are available as it is the case for 6QNR. Assignment errors can result in misleading conceptions of the solvent structure of ribosomes and other RNA systems and should therefore be avoided. Our analysis resulted in a significant decrease of the number of bound Mg2+ in the 6QNR structure, suggesting that K+ and not Mg2+ is the prevalent ion in the ribosome 1st solvation shell. We stress that the use of proper stereochemical guidelines is critical for deflating the current Mg2+ bubble witnessed in many ribosome and other RNA structures. Herewith, we would like to draw the attention of the researchers interested in the ionic structure of biomolecular systems on the importance and complementarity of stereochemistry and other ion identification techniques such as those pertaining to the detection of anomalous signals of transition metals and K+ We also stress that for the identification of lighter ions such as Mg2+, Na+, , stereochemistry coupled with high resolution structures remain the best currently available option. in the deposited structures and underrate the importance of stereochemical rules to correctly assign these ions. Herein, we re-evaluate the PDBid 6QNR and 6SJ6 models of the ribosome ionic structure and establish that stereochemical principles need consideration when evaluating ion binding features, even when K+ anomalous signals are available as it is the case for 6QNR. Assignment errors can result in misleading conceptions of the solvent structure of ribosomes and other RNA systems and should therefore be avoided. Our analysis resulted in a significant decrease of the number of bound Mg2+ in the 6QNR structure, suggesting that K+ and not Mg2+ is the prevalent ion in the ribosome 1st solvation shell. We stress that the use of proper stereochemical guidelines is critical for deflating the current Mg2+ bubble witnessed in many ribosome and other RNA structures. Herewith, we would like to draw the attention of the researchers interested in the ionic structure of biomolecular systems on the importance and complementarity of stereochemistry and other ion identification techniques such as those pertaining to the detection of anomalous signals of transition metals and K+ We also stress that for the identification of lighter ions such as Mg2+, Na+, , stereochemistry coupled with high resolution structures remain the best currently available option. },
keywords = {cryo-EM, ENNIFAR, ionic atmosphere, solvent, Unité ARN, validation, X-Ray},
pubstate = {published},
tppubtype = {article}
}
Proper evaluation of the ionic structure of biomolecular systems remains challenging in X ray and cryo-EM techniques but is essential for advancing our understanding of complex structure/activity/solvent relationships. However, numerous studies overestimate the number of MgProper evaluation of the ionic structure of biomolecular systems remains challenging in X ray and cryo-EM techniques but is essential for advancing our understanding of complex structure/activity/solvent relationships. However, numerous studies overestimate the number of Mg2+ in the deposited structures and underrate the importance of stereochemical rules to correctly assign these ions. Herein, we re-evaluate the PDBid 6QNR and 6SJ6 models of the ribosome ionic structure and establish that stereochemical principles need consideration when evaluating ion binding features, even when K+ anomalous signals are available as it is the case for 6QNR. Assignment errors can result in misleading conceptions of the solvent structure of ribosomes and other RNA systems and should therefore be avoided. Our analysis resulted in a significant decrease of the number of bound Mg2+ in the 6QNR structure, suggesting that K+ and not Mg2+ is the prevalent ion in the ribosome 1st solvation shell. We stress that the use of proper stereochemical guidelines is critical for deflating the current Mg2+ bubble witnessed in many ribosome and other RNA structures. Herewith, we would like to draw the attention of the researchers interested in the ionic structure of biomolecular systems on the importance and complementarity of stereochemistry and other ion identification techniques such as those pertaining to the detection of anomalous signals of transition metals and K+ We also stress that for the identification of lighter ions such as Mg2+, Na+, , stereochemistry coupled with high resolution structures remain the best currently available option. in the deposited structures and underrate the importance of stereochemical rules to correctly assign these ions. Herein, we re-evaluate the PDBid 6QNR and 6SJ6 models of the ribosome ionic structure and establish that stereochemical principles need consideration when evaluating ion binding features, even when K+ anomalous signals are available as it is the case for 6QNR. Assignment errors can result in misleading conceptions of the solvent structure of ribosomes and other RNA systems and should therefore be avoided. Our analysis resulted in a significant decrease of the number of bound Mg2+ in the 6QNR structure, suggesting that K+ and not Mg2+ is the prevalent ion in the ribosome 1st solvation shell. We stress that the use of proper stereochemical guidelines is critical for deflating the current Mg2+ bubble witnessed in many ribosome and other RNA structures. Herewith, we would like to draw the attention of the researchers interested in the ionic structure of biomolecular systems on the importance and complementarity of stereochemistry and other ion identification techniques such as those pertaining to the detection of anomalous signals of transition metals and K+ We also stress that for the identification of lighter ions such as Mg2+, Na+, , stereochemistry coupled with high resolution structures remain the best currently available option.
Abel S, Marchi M, Solier J, Finet S, Brillet K, Bonneté F
In: Biochim Biophys Acta Biomembr, vol. in press, 2020.
Abstract | Links | BibTeX | Tags: DDM, ENNIFAR, Membrane transporter ShuA, Molecular dynamics simulations, Outer membrane model, SEC-MALLS, SEC-SAXS, Unité ARN
@article{Abel2020,
title = {Structural insights into the membrane receptor ShuA in DDM micelles and in a model of gram-negative bacteria outer membrane as seen by SAXS and MD simulations },
author = {S Abel and M Marchi and J Solier and S Finet and K Brillet and F Bonneté
},
url = {https://pubmed.ncbi.nlm.nih.gov/33157097/},
doi = {10.1016/j.bbamem.2020.183504 },
year = {2020},
date = {2020-11-01},
journal = {Biochim Biophys Acta Biomembr},
volume = {in press},
abstract = {Successful crystallization of membrane proteins in detergent micelles depends on key factors such as conformational stability of the protein in micellar assemblies, the protein-detergent complex (PDC) monodispersity and favorable protein crystal contacts by suitable shielding of the protein hydrophobic surface by the detergent belt. With the aim of studying the influence of amphiphilic environment on membrane protein structure, stability and crystallizability, we combine molecular dynamics (MD) simulations with SEC-MALLS and SEC-SAXS (Size Exclusion Chromatography in line with Multi Angle Laser Light Scattering or Small Angle X-ray Scattering) experiments to describe the protein-detergent interactions that could help to rationalize PDC crystallization. In this context, we compare the protein-detergent interactions of ShuA from Shigella dysenteriae in n-Dodecyl-β-D-Maltopyranoside (DDM) with ShuA inserted in a realistic model of gram-negative bacteria outer membrane (OM) containing a mixture of bacterial lipopolysaccharide and phospholipids. To evaluate the quality of the PDC models, we compute the corresponding SAXS curves from the MD trajectories and compare with the experimental ones. We show that computed SAXS curves obtained from the MD trajectories reproduce better the SAXS obtained from the SEC-SAXS experiments for ShuA surrounded by 268 DDM molecules. The MD results show that the DDM molecules form around ShuA a closed belt whose the hydrophobic thickness appears slightly smaller (~22 Å) than the hydrophobic transmembrane domain of the protein (24.6 Å) suggested by Orientations of Proteins in Membranes (OPM) database. The simulations also show that ShuA transmembrane domain is remarkably stable in all the systems except for the extracellular and periplasmic loops that exhibit larger movements due to specific molecular interactions with lipopolysaccharides (LPS). We finally point out that this detergent behavior may lead to the occlusion of the periplasmic hydrophilic surface and poor crystal contacts leading to difficulties in crystallization of ShuA in DDM. },
keywords = {DDM, ENNIFAR, Membrane transporter ShuA, Molecular dynamics simulations, Outer membrane model, SEC-MALLS, SEC-SAXS, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Successful crystallization of membrane proteins in detergent micelles depends on key factors such as conformational stability of the protein in micellar assemblies, the protein-detergent complex (PDC) monodispersity and favorable protein crystal contacts by suitable shielding of the protein hydrophobic surface by the detergent belt. With the aim of studying the influence of amphiphilic environment on membrane protein structure, stability and crystallizability, we combine molecular dynamics (MD) simulations with SEC-MALLS and SEC-SAXS (Size Exclusion Chromatography in line with Multi Angle Laser Light Scattering or Small Angle X-ray Scattering) experiments to describe the protein-detergent interactions that could help to rationalize PDC crystallization. In this context, we compare the protein-detergent interactions of ShuA from Shigella dysenteriae in n-Dodecyl-β-D-Maltopyranoside (DDM) with ShuA inserted in a realistic model of gram-negative bacteria outer membrane (OM) containing a mixture of bacterial lipopolysaccharide and phospholipids. To evaluate the quality of the PDC models, we compute the corresponding SAXS curves from the MD trajectories and compare with the experimental ones. We show that computed SAXS curves obtained from the MD trajectories reproduce better the SAXS obtained from the SEC-SAXS experiments for ShuA surrounded by 268 DDM molecules. The MD results show that the DDM molecules form around ShuA a closed belt whose the hydrophobic thickness appears slightly smaller (~22 Å) than the hydrophobic transmembrane domain of the protein (24.6 Å) suggested by Orientations of Proteins in Membranes (OPM) database. The simulations also show that ShuA transmembrane domain is remarkably stable in all the systems except for the extracellular and periplasmic loops that exhibit larger movements due to specific molecular interactions with lipopolysaccharides (LPS). We finally point out that this detergent behavior may lead to the occlusion of the periplasmic hydrophilic surface and poor crystal contacts leading to difficulties in crystallization of ShuA in DDM.
Wolff P, Villette C, Zumsteg J, Heintz D, Antoine L, Chane-Woon-Ming B, Droogmans L, Grosjean H, Westhof E
Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea Journal Article
In: RNA, vol. 26, no. 12, pp. 1957-1975, 2020.
Abstract | Links | BibTeX | Tags: (hyper)thermophiles Archaea mass spectrometry modifications tRNA, ARN-MS, ENNIFAR, Unité ARN, WESTHOF
@article{,
title = {Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea},
author = {P Wolff and C Villette and J Zumsteg and D Heintz and L Antoine and B Chane-Woon-Ming and L Droogmans and H Grosjean and E Westhof},
url = {https://pubmed.ncbi.nlm.nih.gov/32994183/},
doi = {10.1261/rna.077537.120},
year = {2020},
date = {2020-09-29},
journal = {RNA},
volume = {26},
number = {12},
pages = {1957-1975},
abstract = {To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale) and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.},
keywords = {(hyper)thermophiles Archaea mass spectrometry modifications tRNA, ARN-MS, ENNIFAR, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale) and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.
Simonetti A, Guca E, Bochler A, Kuhn L, Hashem Y
Structural Insights Into the Mammalian Late-Stage Initiation Complexes Journal Article
In: Cell Rep, vol. 31, no. 1, pp. 107497, 2020, ISBN: 32268096.
Abstract | Links | BibTeX | Tags: ENNIFAR, PPSE, Unité ARN
@article{,
title = {Structural Insights Into the Mammalian Late-Stage Initiation Complexes},
author = {A Simonetti and E Guca and A Bochler and L Kuhn and Y Hashem},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32268096?dopt=Abstract},
doi = {10.1016/j.celrep.2020.03.061},
isbn = {32268096},
year = {2020},
date = {2020-01-01},
journal = {Cell Rep},
volume = {31},
number = {1},
pages = {107497},
abstract = {In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.},
keywords = {ENNIFAR, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.
Werner S, Schmidt L, Marchand V, Kemmer T, Falschlunger C, Sednev M V, Bec G, Ennifar E, Höbartner C, Micura R, Motorin Y, Hildebrandt A, Helm M
In: Nucleic Acids Res, vol. 48, no. 7, pp. 3734-3746, 2020, ISBN: 32095818.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Machine Learning of Reverse Transcription Signatures of Variegated Polymerases Allows Mapping and Discrimination of Methylated Purines in Limited Transcriptomes},
author = {S Werner and L Schmidt and V Marchand and T Kemmer and C Falschlunger and M V Sednev and G Bec and E Ennifar and C Höbartner and R Micura and Y Motorin and A Hildebrandt and M Helm},
url = {https://pubmed.ncbi.nlm.nih.gov/32095818},
doi = {10.1093/nar/gkaa113},
isbn = {32095818},
year = {2020},
date = {2020-01-01},
journal = {Nucleic Acids Res},
volume = {48},
number = {7},
pages = {3734-3746},
abstract = {Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.
Vigouroux A, Aumont-Nicaise M, Boussac A, Marty L, Bello L Lo, Legrand P, Brillet K, Schalk I J, Moréra S
A unique ferrous iron binding mode is associated to large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa Journal Article
In: FEBS J, vol. 287, no. 2, pp. 295-309, 2020, ISBN: 31318478.
Abstract | Links | BibTeX | Tags: ENNIFAR, Pseudomonas aeruginosa iron pyoverdine siderophore solute-binding protein, Unité ARN
@article{,
title = {A unique ferrous iron binding mode is associated to large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa},
author = {A Vigouroux and M Aumont-Nicaise and A Boussac and L Marty and L Lo Bello and P Legrand and K Brillet and I J Schalk and S Moréra},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31318478},
doi = {10.1111/febs.15004},
isbn = {31318478},
year = {2020},
date = {2020-01-01},
journal = {FEBS J},
volume = {287},
number = {2},
pages = {295-309},
abstract = {Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Iron release from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore binding protein, respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P. aeruginosa. This article is protected by copyright. All rights reserved.},
keywords = {ENNIFAR, Pseudomonas aeruginosa iron pyoverdine siderophore solute-binding protein, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Iron release from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore binding protein, respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P. aeruginosa. This article is protected by copyright. All rights reserved.
Gasser C, Delazer I, Neuner E, Pascher K, Brillet K, Klotz S, Trixl L, Himmelstoß M, Ennifar E, Rieder D, Lusser A, Micura R
Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL) Journal Article
In: Angew Chem Int Ed Engl, vol. 59, no. 17, pp. 6881-6886, 2020, ISBN: 31999864.
Abstract | Links | BibTeX | Tags: ENNIFAR, RNA sequencing RNA structures gene expression nucleoside modifications oligonucleotides, Unité ARN
@article{,
title = {Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL)},
author = {C Gasser and I Delazer and E Neuner and K Pascher and K Brillet and S Klotz and L Trixl and M Himmelstoß and E Ennifar and D Rieder and A Lusser and R Micura},
url = {https://pubmed.ncbi.nlm.nih.gov/31999864/},
doi = {10.1002/anie.201916272},
isbn = {31999864},
year = {2020},
date = {2020-01-01},
journal = {Angew Chem Int Ed Engl},
volume = {59},
number = {17},
pages = {6881-6886},
abstract = {Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.},
keywords = {ENNIFAR, RNA sequencing RNA structures gene expression nucleoside modifications oligonucleotides, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.
Brillet K, Martinez-Zapien D, Bec G, Ennifar E, Dock-Bregeon A C, Lebars I
Different views of the dynamic landscape covered by the 5'-hairpin of the 7SK small nuclear RNA Journal Article
In: RNA, vol. 26, no. 9, pp. 1184-1197, 2020, ISBN: 32430362.
Abstract | Links | BibTeX | Tags: 7SK HEXIM RNA Tat structure, ENNIFAR, Unité ARN
@article{,
title = {Different views of the dynamic landscape covered by the 5'-hairpin of the 7SK small nuclear RNA},
author = {K Brillet and D Martinez-Zapien and G Bec and E Ennifar and A C Dock-Bregeon and I Lebars},
url = {https://pubmed.ncbi.nlm.nih.gov/32430362/},
doi = {10.1261/rna.074955.120},
isbn = {32430362},
year = {2020},
date = {2020-01-01},
journal = {RNA},
volume = {26},
number = {9},
pages = {1184-1197},
abstract = {The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral RNA in the infection cycle of the human immunodeficiency virus (HIV-1). Tat promotes the release of P-TEFb from the 7SKsnRNP and subsequent activation of transcription, by displacing HEXIM from the 5'-hairpin of the 7SKsnRNA. This hairpin (HP1), comprising the signature sequence of the 7SKsnRNA, has been the subject of three independent structural studies aiming at identifying the structural features that could drive the recognition by the two proteins, both depending on Arginine Rich Motifs (ARM). Interestingly, four distinct structures were determined. In an attempt to provide a comprehensive view of the structure-function relationship of this versatile RNA, we present here a structural analysis of the models, highlighting how HP1 is able to adopt distinct conformations with significant impact on the compactness of the molecule. Since these models are solved under different conditions by NMR and crystallography, the impact of the buffer composition on the conformational variation was investigated by complementary biophysical approaches. Finally, using Isothermal Titration Calorimetry, we determined the thermodynamic signatures of the Tat-ARM and HEXIM-ARM peptide interactions with the RNA, showing that they are associated with distinct binding},
keywords = {7SK HEXIM RNA Tat structure, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral RNA in the infection cycle of the human immunodeficiency virus (HIV-1). Tat promotes the release of P-TEFb from the 7SKsnRNP and subsequent activation of transcription, by displacing HEXIM from the 5'-hairpin of the 7SKsnRNA. This hairpin (HP1), comprising the signature sequence of the 7SKsnRNA, has been the subject of three independent structural studies aiming at identifying the structural features that could drive the recognition by the two proteins, both depending on Arginine Rich Motifs (ARM). Interestingly, four distinct structures were determined. In an attempt to provide a comprehensive view of the structure-function relationship of this versatile RNA, we present here a structural analysis of the models, highlighting how HP1 is able to adopt distinct conformations with significant impact on the compactness of the molecule. Since these models are solved under different conditions by NMR and crystallography, the impact of the buffer composition on the conformational variation was investigated by complementary biophysical approaches. Finally, using Isothermal Titration Calorimetry, we determined the thermodynamic signatures of the Tat-ARM and HEXIM-ARM peptide interactions with the RNA, showing that they are associated with distinct binding
Wolff P, Ennifar E
Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes Book Chapter
In: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, pp. 111-118, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006311.
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Native mass spectrometry RNA, Unité ARN
@inbook{,
title = {Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes},
author = {P Wolff and E Ennifar},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006311},
doi = {10.1007/978-1-0716-0278-2_9},
isbn = {32006311},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {111-118},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.},
keywords = {ARN-MS, ENNIFAR, Native mass spectrometry RNA, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.
Lechner A, Wolff P, Leize-Wagner E, François Y N
Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry Journal Article
In: Anal Chem, vol. 92, no. 10, pp. 7363-7370, 2020, ISBN: 32343557.
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Unité ARN
@article{,
title = {Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry},
author = {A Lechner and P Wolff and E Leize-Wagner and Y N François},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32343557?dopt=Abstract},
doi = {10.1021/acs.analchem.0c01345},
isbn = {32343557},
year = {2020},
date = {2020-01-01},
journal = {Anal Chem},
volume = {92},
number = {10},
pages = {7363-7370},
abstract = {Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.
Antoine L, Wolff P
Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry Book Chapter
In: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, pp. 101-110, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006310.
Abstract | Links | BibTeX | Tags: 2D Gel isolation Nano-LC-MS/MS Posttranscriptional tRNA modifications, ARN-MS, ENNIFAR, ROMBY, Unité ARN
@inbook{,
title = {Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry},
author = {L Antoine and P Wolff},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006310},
doi = {10.1007/978-1-0716-0278-2_8},
isbn = {32006310},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {101-110},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.},
keywords = {2D Gel isolation Nano-LC-MS/MS Posttranscriptional tRNA modifications, ARN-MS, ENNIFAR, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.
Bernacchi S, Ennifar E
In: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, pp. 237-250, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006318.
Abstract | Links | BibTeX | Tags: Aminoglycosides, dimerization, drug interaction, ENNIFAR, HIV-1, initiation site, ITC, MARQUET, PAILLART, RNA, Thermodynamics RNA, Unité ARN, Viral
@inbook{,
title = {Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry},
author = {S Bernacchi and E Ennifar},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006318},
doi = {10.1007/978-1-0716-0278-2_16},
isbn = {32006318},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {237-250},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity.},
keywords = {Aminoglycosides, dimerization, drug interaction, ENNIFAR, HIV-1, initiation site, ITC, MARQUET, PAILLART, RNA, Thermodynamics RNA, Unité ARN, Viral},
pubstate = {published},
tppubtype = {inbook}
}
Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity.
Vileno B, Lebars I
Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies Book Chapter
In: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, pp. 217-235, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006317.
Abstract | Links | BibTeX | Tags: ENNIFAR, RNA NMR EPR Paramagnetic Spin-label, Unité ARN
@inbook{,
title = {Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies},
author = {B Vileno and I Lebars},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006317},
doi = {10.1007/978-1-0716-0278-2_15},
isbn = {32006317},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {217-235},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {Many RNA architectures were discovered to be involved in essential biological pathways acting as catalysts and/or regulators of gene expression, transcription, translation, splicing, or viral infection. The key to understand their diverse biological functions is to investigate their structure and dynamic. Nuclear Magnetic Resonance (NMR) is a powerful method to gain insight into these properties. However, the study of high-molecular-weight RNAs by NMR remains challenging. Advances in biochemical and NMR methods over the recent years allow to overcome the limitation of NMR. In particular, the incorporation of paramagnetic probes, coupled to the measurement of the induced effects on nuclear spins, has become an efficient tool providing long-range distance restraints and information on dynamic in solution. At the same time, the use of spin label enabled the application of Electron Paramagnetic Resonance (EPR) to study biological macromolecules. Combining NMR and EPR is emerging as a new approach to investigate the architecture of biological systems.
Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies.},
keywords = {ENNIFAR, RNA NMR EPR Paramagnetic Spin-label, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Many RNA architectures were discovered to be involved in essential biological pathways acting as catalysts and/or regulators of gene expression, transcription, translation, splicing, or viral infection. The key to understand their diverse biological functions is to investigate their structure and dynamic. Nuclear Magnetic Resonance (NMR) is a powerful method to gain insight into these properties. However, the study of high-molecular-weight RNAs by NMR remains challenging. Advances in biochemical and NMR methods over the recent years allow to overcome the limitation of NMR. In particular, the incorporation of paramagnetic probes, coupled to the measurement of the induced effects on nuclear spins, has become an efficient tool providing long-range distance restraints and information on dynamic in solution. At the same time, the use of spin label enabled the application of Electron Paramagnetic Resonance (EPR) to study biological macromolecules. Combining NMR and EPR is emerging as a new approach to investigate the architecture of biological systems.
Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies.
Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies.
Kruse H, Mrazikova K, D'Ascenzo L, Sponer J, Auffinger P
Short but Weak! The Z-DNA lone-pair···π Conundrum Challenges Standard Carbon Van Der Waals Radii Journal Article
In: Angew Chem Int Ed Engl, pp. in press, 2020, ISBN: 32516461.
Abstract | Links | BibTeX | Tags: ENNIFAR, lp···pi interactions molecular recognition non-covalent interactions van der Waals radii Z-DNA, Unité ARN
@article{,
title = {Short but Weak! The Z-DNA lone-pair···π Conundrum Challenges Standard Carbon Van Der Waals Radii},
author = {H Kruse and K Mrazikova and L D'Ascenzo and J Sponer and P Auffinger},
url = {https://pubmed.ncbi.nlm.nih.gov/32516461/},
doi = {10.1002/anie.202004201},
isbn = {32516461},
year = {2020},
date = {2020-01-01},
journal = {Angew Chem Int Ed Engl},
pages = {in press},
abstract = {Current interest for lone-pair···π (lp···π) non-covalent interactions is gaining momentum in biochemistry and (supramolecular)chemistry. However, the physico-chemical origin of the exceptionally short (≈2.8 Å) oxygen to nucleobase plane distances observed in prototypical Z-DNA CpG steps remains unclear. Accurate quantum mechanical calculations including SAPT2+3 interaction energy decompositions demonstrate that lp···π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatics interactions combined with stereochemical effects imposed by the structural context. They also suggest that the carbon van der Waals (vdW) radii originally derived for sp3carbons should not be used for the smaller sp2 atoms. Using a more adapted carbon vdW radius results in lp···π contacts being no longer of the sub-vdW type. Overall, our findings challenge the whole lp···π concept that refers to elusive orbital interactions that cannot explain short contact distances.},
keywords = {ENNIFAR, lp···pi interactions molecular recognition non-covalent interactions van der Waals radii Z-DNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Current interest for lone-pair···π (lp···π) non-covalent interactions is gaining momentum in biochemistry and (supramolecular)chemistry. However, the physico-chemical origin of the exceptionally short (≈2.8 Å) oxygen to nucleobase plane distances observed in prototypical Z-DNA CpG steps remains unclear. Accurate quantum mechanical calculations including SAPT2+3 interaction energy decompositions demonstrate that lp···π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatics interactions combined with stereochemical effects imposed by the structural context. They also suggest that the carbon van der Waals (vdW) radii originally derived for sp3carbons should not be used for the smaller sp2 atoms. Using a more adapted carbon vdW radius results in lp···π contacts being no longer of the sub-vdW type. Overall, our findings challenge the whole lp···π concept that refers to elusive orbital interactions that cannot explain short contact distances.
Bochler A, Querido J B, Prilepskaja T, Soufari H, Simonetti A, Cistia M L Del, Kuhn L, Ribeiro A R, Valášek L S, Hashem Y
Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts Journal Article
In: Cell Rep, vol. 33, no. 12, pp. 108534, 2020, ISBN: 33357443.
Abstract | Links | BibTeX | Tags: ENNIFAR, ES6(S) ES7(S) ES9(S) Trypanosoma cruzi cryo-EM eIF1 eIF2 eIF3 eIF5-CTD k-DDX60 the 43S pre-initiation complex translation initiation, HASCHEM, PPSE, Unité ARN
@article{,
title = {Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts},
author = {A Bochler and J B Querido and T Prilepskaja and H Soufari and A Simonetti and M L Del Cistia and L Kuhn and A R Ribeiro and L S Valášek and Y Hashem},
url = {https://pubmed.ncbi.nlm.nih.gov/33357443/},
doi = {10.1016/j.celrep.2020.108534},
isbn = {33357443},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Cell Rep},
volume = {33},
number = {12},
pages = {108534},
abstract = {Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.},
keywords = {ENNIFAR, ES6(S) ES7(S) ES9(S) Trypanosoma cruzi cryo-EM eIF1 eIF2 eIF3 eIF5-CTD k-DDX60 the 43S pre-initiation complex translation initiation, HASCHEM, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.
Rol-Moreno J, Kuhn L, Marzi S, Simonetti A
In: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, pp. 329-339, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006323.