UMR8261 Expression Génétique Microbienne

CNRS / Université Paris Diderot Paris 7

Directeur : Harald Putzer, Directeur-adjoint : Ciarán Condon

  • the helicases
  • their importance
  • our project
  • members
  • collaborations
  • funding
  • publications
  • V. Heurgué-Hamard


RNA helicases: their structure, function and properties


Resp: N. Kyle Tanner Ph.D., DR-CDI CNRS

Tél: (33) 1 58 41 52 37
FAX: (33) 1 58 41 50 25
Addresse: Pièce123
Institut de Biologie Physico-Chimique (IBPC)
13 rue Pierre et Marie Curie
75005 Paris France

  News: Our laboratory is currently hosting Valérie Heurgué-Hamard. Her project is the methylation of the translational apparatus

RNA helicases: What are they?

  RNA helicase is a generic term for families of proteins that belong to larger superfamilies (SF) of NTP-dependent proteins involved in the processing, replication, repair, expression and remodeling of nucleic acids complexes. There are a least seven superfamilies of these proteins that use DNA or RNA as substrates. They are characterized by conserved nucleotide binding motifs and core structures consisting of parallel beta-sheets and surrounding helixes that are similar to the solved crystal structure of the bacterial RecA protein, which is involved in homologous recombination. For that reason, the core structures are called RecA-like domains. The majority of the superfamilies contain a single RecA-like domain while SF1 and 2 have two linked domains. The vast majority of the RNA helicases belong to SF2, and to a lesser extent SF1.


Fig. 1. Modeled structure of the two RecA-like domains of Ded1 (residues 117-529) based on the solved crystal structure of the related Vasa protein (pdb coordinates 2db3, Sengoku et al, 2006, Cell 125:287-300). The bound ligands (AMP-PNP, Mg2+ and RNA U2–U6) are from the Vasa structure.

RNA helicases: What do they do?

   RNA helicases use the energy of nucleotide triphosphate binding and hydrolysis to do work. The vast majority use ATP or dATP as an energy source, but others can use UTP, CTP and GTP as well. Despite being called “helicases,” a number of different activities have been attributed to these proteins. For example, retinoic acid-inducible protein 1 (RIG-I) and the similar melanoma differentiation-associated protein 5 (MDA5) recognize the double-stranded RNA produced during viral infections and activate antiviral signaling pathways. SecA is a cofactor of the bacterial SecYEG complex involved in polypeptide translocation across the membrane. Other RNA helicases function as RNA chaperones to facilitate the formation of functional RNAs, as RNPases to dissociate proteins bound to RNAs and as assembly proteins to facilitate the formation of functional ribonucleoprotein complexes. Of course, some of these proteins have been shown to unwind RNA-RNA and RNA-DNA duplexes as well.


RNA helicases: Why are they important?

   RNA helicases are ubiquitous to all life forms, from bacteria to higher eukaryotes. Even viruses, such as HCV, encode their own RNA helicases, although viruses exploit host-encoded RNA helicases as well. RNA helicases are associated with all processes involving RNA from transcription, splicing, transport, quality control, translation and degradation. Many of the proteins are essential, and they are rarely interchangeable, which indicates a high degree of specialization in the cell. As an indication of their importance, RNA helicases are implicated in cell cycle and developmental regulation, in cancer, aging, neurological disorders and in other maladies. RNA helicases are potential targets for prophylactic and therapeutic agents.

RNA helicases: What are DEAD-box proteins?

   DEAD-box proteins are ATP-dependent RNA binding proteins and RNA-dependent ATPase. They constitute the largest family of RNA helicases, and they belong in SF2, which is often called the DExD/H-box protein superfamily. They are ubiquitous to virtually all organisms. The yeast Saccharomyces cerevisiae has 25 different DEAD-box proteins and the bacterium Escherichia coli has five. In yeast, for the most part the proteins are essential and are not interchangeable. In E. coli, the proteins are not essential individually under laboratory conditions. DEAD-box proteins are implicated in all the processes indicated above, however they are rather poor helicases. In general, the proteins are only able to disrupt relatively short duplexes (a dozen base pairs), typically at very large excesses of protein to substrate ratios in vitro, and in general these proteins lack directionality, which means they are nonprocessive. However, protein partners could confer both directionality and processivity in vivo.
   DEAD-box proteins are characterized by nine conserved motifs that are involved in ligand binding and in conferring the enzymatic activity. They are named after the characteristic residues of motif II (Asp-Glu-Ala-Asp), which translates to D-E-A-D in the single letter nomenclature. However, there are DEAD-box proteins with alternative sequences for motif II and moreover proteins from other families that encode DEAD for motif II. Thus, DEAD-box proteins are classified by a number of different features, and particularly by the characteristic Q motif that we discovered and characterized.

Fig. 2. Schematic representation of the D-E-A-D box core showing the nine conserved motifs. Also shown are the positions of those residues that interact directly with the two substrates, RNA and ATP, as indicated in several independent structures. Arrows indicate the N- and C- terminal extensions that frequently flank the core on both sides. The underlined P indicates an interaction with a phosphate.


RNA helicases: What do we do?

  We are interested in understanding how RNA helicases work, how their specificity and regulation are conferred, and why they play such important roles in the cell. We concentrate on the DEAD-box proteins, and we typically use yeast and bacteria as model systems.

  We are interested in characterizing how the conserved “helicase” core works. We use a triad of approaches. First, we use sequence alignments and molecular modeling to identify regions of interest. Then we use bacterial and yeast genetics to assay the effects of deletions and site-specific mutations in vivo. Finally, we use purified recombinant proteins, expressed in bacteria, to characterize their enzymatic properties. These include their RNA-dependent ATPase activity, their RNA binding affinities and their capacity to displace RNA-RNA and RNA-DNA duplexes in vitro. We use various DEAD-box proteins in order to elucidate common features, but we concentrate on Ded1 from yeast because it is easy to work with and because it has one of the highest activities for a DEAD-box protein. It can easily dissociate a 50-fold excess of an 18 base pair RNA-DNA duplex in under 5 minutes, with a rate of ATP hydrolysis of 350 molecules ATP per Ded1 per minute.

  We are studying the molecular mechanisms in finer detail using single-molecule approaches. In association with Dr. Ulrich Bockelmann at ESCPI, we are studying bacterial helicases with single-molecule optical tweezers and single-molecule FRET. In collaboration with Dr. Vincent Croquette, ENS, we are studying the properties of Ded1 using single-molecule magnetic tweezers. However, in vitro Ded1 lacks substrate specificity and enzymatic regulation that must exist in vivo.

  Thus, we also are interested in identifying the protein partners and RNA substrates of Ded1. We use pull-down experiments of cellular extracts to identify factors interacting with Ded1 in vivo that we subsequently characterize in vitro with purified recombinant proteins. We use fusion proteins with fluorescent tags to colocalize Ded1 with its partners in living cells under different conditions in collaboration with Dr. Naïma Belgareh-Touzé, IBPC.

  Finally, we are collaborating with Dr. Ikram Guizani, Pasteur Institute Tunis, to screen for biologically active agents specific for the DEAD-box protein LeIF from the parasitic trypanosomatid protozoa Leishmania. Dr. Michael Nilges, Pasteur Institute Paris, heads the unit modeling the three-dimensional structure of LeIF and identifying potential targets for therapeutic agents, while Dr. Hélène Munier-Lehmann, Pasteur Institute Paris, is involved in large-scale screening of potential agents.




N. Kyle Tanner Ph.D, DR-CDI CNRS
Josette Banroques Ph.D, CR1 CNRS, VAC
Thierry Bizebard Ph. D, CR1 CNRS
Valérie Heurgué-Hamard Ph. D, CR1 CNRS
Caroline_Lacoux Post-Doc ANR
Emmeline Huvelle T CNRS
Molka Mokdadi Etudiante en thèse (Institut Pasteur, Tunis) cotutelle : Kyle Tanner, Ikraim Guizani (Institut Pasteur, Tunis)
Hilal Yeter Etudiante en thèse
Yosser Zina Abdelkrim-Guediche étudiante en thèse (Institut Pasteur Tunis) co-direction : Kyle Tanner et Ikraim Guizani (Institut Pasteur Tunis)
  Marine Pasquier 2eme année BTS, ESTBA, Paris


Emeline Coleno, IE CDD

Samar Hodeib, étudiante en thèse, en co-direction avec Vincent Croquette

Oznur Ozturk, Etudiante Licence L3, Université Paris 13

Robin Liset, 1ere année BTS, ESTBA, Paris

Guillaume Sevin, stage 2nde année BTS

Amar Baahmed, stage 2nde année BTS

Bruna Frota de Carvalho, MD, étudiante stagiaire

Agnès Le Saux, IR

Jinane Ait Benkhali Post-Doc

Yosser Zina ABDELKRIM Ep GUEDICHE, stagiaire, étudiante en thèse Institut Pasteur Tunis

Olivier Cordin, Post-Doc

Diogo Mendonça, M2 Erasmus

Lucas Rousselet, BTS 1ere année

Richard H. Buckingham, Ph.D., DR1 CNRS directeur émérite

Marc Dreyfus, Ph.D., DR1 CNRS, émérite associé à l' UMR7141 depuis jan 2014

Meriem Senissar, étudiante en thèse

Céline Adam, Assistant Ingénieur

Viriya OK, étudiante stagiaire IUT

Mathilde Bercy, étudiante en M2, en association avec Dr. Ulrich Bockelmann.

Hala Bounihi, Assistant Ingénieur

Gwendoline Cartier, étudiante en thèse


Funding & Support:

  Besides CNRS, which strongly supports the Laboratory (UPR9073) and of which J. Banroques, K. Tanner, V. Heurgué-Hamard, E. Huvelle and T. Bizebard are employees, and the University Paris VII, the team is supported by the following grants:

- ANR "programme blanc": Grant HelicaRN (Ref. 2010 BLAN 1503 01). Coordinator Marc Dreyfus with Ulrich Bockelmann (2010-2014)

- ANR grant TrMTase (n°ANR-14-CE09-0016-01). Coordinator: Valérie Heurgué-Hamard, partner Marc Graille (2015-2017)

- Ecole Doctorale GGC, Paris 11, to Meriem Senissar (2011-2013)

- Grant from Pierre-Gilles de Gennes Fondation for Research (2012-2013).
Title of the project: “RNA helicases at work in single-molecule assays.”
Coordinator Vincent Croquette with N. Kyle Tanner, Hervé Le Hir, and Jean-Baptiste Boulé.

- Initiative d’Excellence DYNAMO (ANR-11-LABX-0011-01)

- ANR "programme blanc": Grant HeliDEAD (ANR-13-BSV8-0009-01). Coordinator N. Kyle Tanner with Vincent Croquette and Naïma Belgareh-Touzé (2014-2016)











Main publications since 2000


Methylation of the translational apparatus


  Our work focuses on the methylation of the translational machinery. Our model organism is S. cerevisiae, which is an extraordinarily powerful genetic system with a well-defined translation system. The main tools we are using are molecular biology, yeast genetics, biochemistry and protein structure determination (in collaboration with structural biologists).

  The translational apparatus (ribosome, translation factors and transfer RNAs) is the target of multiple post-transcriptional and post-translational modifications. Methylation is one of the most common modifications, and a significant percentage of proteins, across all organisms, catalyze the transfer of a methyl group from S-adenosylmethionine to a substrate. In humans, one third of these methyltransferases (MTase) have been linked to cancer and mental disorders. However, little is known about the function of these modifications.

  The aim of our project is to improve our understanding of the biological functions of protein and RNA methylation. We are interested in Trm112, a yeast protein placed at the interface between ribosome synthesis and translation, which functions as an activating platform of four methyltransferases (MTases) with different substrates. Our goal is to gain insights into the function of the MTases interacting with Trm112.

  We showed that class 1 translation termination factors were methylated in bacteria (E. coli, C. trachomatis) and eukaryotes (S. cerevisiae, Homo sapiens, Mus musculus). These proteins are essential for releasing a functional protein when the ribosome encounters a stop codon on the mRNA. Methylation occurs on the glutamine from the conserved Gly-Gly-Gln motif, which is involved in catalyzing peptide release at the ribosomal peptidyl-transferase center. We identified the methyltransferases (MTases) involved in this process, characterized their function, and determined their structure (for a review, see Graille et al., 2012).


Protein complex purification
In vitro methylation : kinetics and SAM binding measurements
3D structure of eukaryotic termination factor eRF1 MTase: Mtq2p-Trm112p complex (collaboration with Marc Graille, Ecole Polytechnique, Palaiseau)

  By using genetic and biochemical approaches, we characterized the molecular mechanism of these MTases and the activating function of Trm112 (Heurgué-Hamard et al., 2006, Liger et al., 2011).

  Trm112 interacts with three MTases involved in translational apparatus modification (Mtq2 methylates translation termination eRF1; Trm9 and Trm11 methylate tRNAs). In collaboration with Denis Lafontaine (Université Libre de Bruxelles, Charleroi, Belgique), we recently identified a new function of Trm112 in ribosome biogenesis via its interaction with Bud23, which is an MTase specific for 18S rRNA methylation at position G1575. Trm112 interacts directly with Bud23 in vitro, and it is required for its stability in vivo. Consequently, in the absence of Trm112, Bud23 is no longer able to bind nascent pre-ribosomes, which are rapidly degraded by the nucleolar surveillance pathway involving the TRAMP complexes (Figaro et al, 2012).

bla bla
Yeast polysome profile Trm112 interactions

  Trm112 is thus active in rRNA, tRNA and translation factor modification, placing it strategically at the interface between ribosome synthesis and function in translation.

  Currently, our goal is to improve, at the molecular level and through structural and functional studies, our understanding of the role of the methylation in eukaryotic translation, in cell biology and also in the molecular basis of diseases.


  • Marc Graille, CNRS UMR7654, Ecole Polytechnique, Palaiseau
  • Denis Lafontaine, FRS, Université Libre de Bruxelles, Charleroi-Gosselies, Belgique
  • Måns Ehrenberg, Department of Cell and Molecular Biology, Uppsala University, Suède

Main Publications











AccueilImprimerContactPlan du siteCredits