RNA molecules are constantly forming intramolecular and intermolecular base pairs. The role of RNA helicases, which are found in all organisms, is to catalyze their rearrangements. However, the mode of action of these enzymes has remained mysterious. The HelicaRN project proposes to elucidate these molecular properties using single molecule approaches.
This research project proposes to study the DEAD-box family of helicases, which are enzymes that are essential for the biology of RNA in nearly all organisms. Indeed, the inappropriate expression of these enzymes can provoke cancer in humans; this reflects the capacity of these enzymes to modify or stabilize RNA structures. However, the mode of action of these proteins is largely unknown, in part because the enzymatic activity is transient and because the proteins leave no marks on the substrate. We propose to use single-molecule techniques, in real time, to clarify the mode of action and to avoid the difficulties inherent in studying heterogeneous populations of molecules. We will combine the single-molecule techniques with more classic in vitro and in vivo approaches that we have mastered in the laboratory; these include enzymatic assays, mutagenesis, molecular modeling, and phylogenetic comparisons. By using these approaches, we hope to address three fundamental questions on the biology of these proteins: 1) how do these proteins use ATP to modify RNA structures; 2) how is the substrate specificity determined in vivo; and 3) what is their exact mode of action on the substrates.
The single-molecule measurements used here (force needed to unwind a duplex in the presence or absence of a helicase, the distance between extremities of duplex strands measured by Förster resonance energy transfer (FRET)) focus on the RNA substrate and permit us to compare the conformation of the RNA in the presence or absence of the helicase. We hope to detect reaction intermediates that would be invisible with classical approaches.
RNA helicases use the energy derived from the hydrolysis of ATP to modify the conformations of RNAs or ribonucleoprotein complexes within the cell, and thereby they participate in regulating cellular activity. Clearly the activities of the helicases must be highly specific and highly regulated to prevent extraneous modifications, as might occur with cancer. By using certain helicases as models, the results obtained in this project clarify how the correct RNA is recognized, how the RNA is modified and how the enzymes use the energy of ATP to carry out these actions.
The most spectacular result of using these very sensitive methods is that we are now able to measure the force necessary to modify the conformation of individual RNA molecules, and we can determine how the bound proteins stabilize these conformations. The forces used are extremely weak, and they correspond to the order of 10 pN, which is equivalent to the weight of only one microgram of material!
This project is aimed at advancing our fundamental understanding of biological molecules through a synergistic collaboration between biologists and physicists.
The objectives of this project are to increase our fundamental knowledge of the biology of RNA. The practical applications of this work are not immediate, even though this is the long term objective of the researchers. Indeed, N.K. Tanner has deposited a patent (in association with others) on using DEAD-box helicases to induce the production of cytokines (2008), and the team of U. Bockelmann has a patent on a system for stabilizing a dual optical trap (2010). We can hope for further innovations will emerge from the HelicaRN grant.
1- Banroques, J., Cordin, O., Doere, M., Linder, P., &Tanner, N. K. (2011) J Mol Biol, 413,451-72.
Although the core structures are highly conserved, the RNA helicases differ by their amino- and carboxyl-terminal extensions. This article analyzes th
This is a joint proposal consisting of two laboratories working in Paris in neighboring institutions; one laboratory consists of biochemists with expertise in RNA and the other consists of physicists specializing in single molecule measurements. The objective of this proposal is to study the DEAD-box proteins, which are a family of proteins that are essential for nearly all processes involving RNA.
Discovered in 1989, these proteins are present in nearly all organisms. They are RNA-dependent ATPases that are characterized by nine conserved motifs, including the D-E-A-D motif that conferred their name. Many of them are capable of displacing duplex RNA in vitro in an ATP-dependent manner (helicase activity) or to promote the formation of duplexes in the absence of ATP (annealing activity). However, the molecular mechanism by which it accomplishes this is poorly understood, and three major issues remain unresolved. The first concerns the helicase activity. These enzymes resemble the helicases involved in DNA replication and repair, but the mode of action appears quite different. The DNA helicases are processive translocases that move unidirectionally on the single-stranded DNA while continuously displacing the complementary strand. In contrast, DEAD-box proteins do not show polarity (directionality) on the single-stranded RNA, and they only are able to displace very short duplexes (< 20 base pairs). According to the current model, these proteins interact on or near the double-stranded RNA in the presence of ATP and disrupt a few base pairs, which subsequently would destabilize the duplex. ATP hydrolysis would cause the enzyme to release the RNA, and the duplex would reanneal if an insufficient number of the base pairs were disrupted. This model proposes that the duplex oscillates between two states, consisting of an intact and partially unwound helix, during the ATPase cycle of the protein. The existence of these states has not yet been demonstrated, and we plan to address this question by using single molecule technologies either with RNA hairpins under tension (optical tweezers) or with the transfer of fluorescence between the two ends of oligonucleotides in a duplex (Förster resonance energy transfer). The second question asks how these proteins achieve their specific roles in vivo when they show little or no RNA specificity in vitro. This is most likely accomplished through the interactions of specific cofactors, but these are unknown for a majority of the DEAD-box proteins. We plan to isolate and identify the cofactors of the yeast Ded1 protein, which we have extensively studied in the laboratory. DEAD-box proteins also are probably regulated by modifications, and Ded1 is most likely a phosphoprotein. Moreover the highly conserved “helicase core” is flanked by highly variable amino- and carboxyl-terminal sequences that could modulate the activity or specificity of the protein. We will use a wide range of biochemical and genetic techniques to address these issues. The third question asks, “what is the exact role of these proteins in vivo?” What RNA structures are substrates for the proteins? How do they work? Are they RNA chaperones? RNPases? Or are they simply ATP-dependent RNA clamps? We will address these questions with SrmB, which is an Escherichia coli protein involved in ribosome assembly. We have identified the probable site of action of SrmB on the 23S rRNA, and we have evidence that it assists in the folding of this subdomain (RNA chaperone activity). We will use optical tweezers to measure the force necessary to fold the subdomain in the presence and absence of SrmB.
This project was first proposed in 2009, and it was placed on a supplementary funding list. In the interim, we have obtained many results that demonstrate the feasibility of the proposed interdisciplinary approaches. This proposal focuses on these approaches.
Monsieur Marc DREYFUS (CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CNRS CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
ESPCI - CNRS CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS A
Help of the ANR 415,000 euros
Beginning and duration of the scientific project: - 36 Months