Ribosomes play an essential role in living things: they are the central sources of protein production in all types of cells. In eukaryotes, the assembly and maturation of these gigantic protein factories are initiated in the cell nucleus by various factors, including Rea1, which is the largest and most complex yeast protein. Thanks to cryoelectron microscopy, Helgo Schmidt's team at the IGBMC (CNRS/Inserm/University of Strasbourg) unveils the fine structure of the maturation factor Rea1. Published on 21 November 2018 in the journal eLife, this work makes it possible to visualize at the atomic scale one of the major mechanisms of ribosome maturation.
Real molecular machines, ribosomes decipher the information contained in a copy of DNA, called messenger RNA, to synthesize proteins. But how are ribosomes themselves produced? Their assembly is initiated in the nucleus where the proteins and RNAs constituting the large ribosomal subunit interact with more than 200 assembly factors. Ultimately, the large mature subunit is found in the cellular cytoplasm where it joins the small ribosomal subunit to form functional ribosomes. In order to promote the export of the large subunit from the nucleus to the cytoplasm, the Rea1 protein eliminates some of the assembly factors.
Rea1 is a huge protein of 5000 amino acids, which consists of a ring and a tail. At the ring level, the hydrolysis of ATP occurs, which allows the release of energy. This mechanism triggers a remodeling process in the tail, which in turn generates a mechanical force to eliminate assembly factors. Despite the crucial importance of ribosome maturation, the fine structure and mechanism of Rea1 remained largely enigmatic.
Using cryoelectron microscopy, the researchers were able to construct a detailed atomic model of Rea1, which revealed that an "arm" associated with the center of the ring regulates the hydrolysis of ATP. The "arm" also controls the formation of a binding site that allows Rea1 to interact with the assembly factors it must eliminate. In addition, the model revealed large portions of the tail of Rea1 and provided insights into a key question, namely how hydrolysis of ATP in the ring could cause tail remodeling.
All these results provide important information on the molecular architecture of the largest ribosome maturation factor.
This study was funded by a LabEx Chair (LabEx INRT),an ATIP-Avenir grant as well as a Région Grand Est jeunes chercheurs fellowship.
The IMCBio (Integrative Molecular and Cellular Biology) Graduate School aims to train a new generation of researchers interested in interfaces in biological sciences. The official launch of this innovative training project will take place in the presence of Michel Deneken, the President of the University of Strasbourg, Jules Hoffmann, winner of the 2011 Nobel Prize in Physiology and Medicine, together with members of the management of the State Major Investment Programmes at the National Research Agency, on Wednesday 28 November at 10am at the University Palace in Strasbourg. During this meeting, the various stages of the implementation of this major project and its objectives of training excellence through research will be presented.
Learning research by doing
Through multiple internships in laboratories, complementary training modules at master's level, summer schools, and a high-level doctoral training program, IMCBio aims to train students with a strong motivation for research to become future researchers interested in interfaces in biological sciences. With the joint support of the University of Strasbourg (Faculty of Life Sciences and Doctoral School of Life and Health), the CNRS and the Inserm, this project, which is based on the graduate school model, has three objectives: to strongly link training to research, to enhance the strengths of the scientific themes of Strasbourg and Illkirch sites and to contribute to its international influence.
An attractive training program
In this context, 17 students in their first year of Master's degree were selected for their first term in September 2018. "A successful and remarkable start to the school year, says Bertrand Séraphin, Director of the Institute of Genetics, Molecular and Cellular Biology (IGBMC) and Project Director. We have received requests not only from French and foreign students who would like to join the course next academic year, but also from other institutions interested in this innovative teaching model.” Thanks to the support of the Faculty of Life Sciences of the University of Strasbourg, the first class of IMCBio students has already benefited from high-level scientific seminars in different disciplines. Nicolas Matt, co-director of the project and professor-researcher at the Institute of Molecular and Cellular Biology (IBMC, M3I), has planned to open the ethics seminar he organizes to all students in biology courses. "The budget of the graduate school allows us to invite researchers at the forefront of their field and we obviously want to make it available to the entire community," he asserts before adding: "There will also be training courses that will restricted to smaller numbers of students and will be reserved for IMCBio students.”
From 2019, the prestigious IMCBio programme will be open to doctoral students and should enable development of competitive research projects over three years.
The IMCBio Graduate School
Based on a strategic grouping of Strasbourg's molecular and cellular biology strengths from three laboratories of excellence (LabEx INRT, NetRNA and MitoCross) with the help of state-of-the-art technological tools, five National Infrastructures in Health Biology (CELPHEDIA/PHENOMIN, FRISBI, France Genomics, Ingestem and IFB at the IGBMC) as well as the new insectarium (Equipex I2MC at the IBMC), this innovative training project with a budget of €6,282,000 is one of the four graduate schools winners supported by the University of Strasbourg.
Project Director: Bertrand Séraphin, Research Director, CNRS
Co-director of the project: Nicolas Matt, Senior Lecturer, University of Strasbourg
Project Coordinator: Pauline Vorburger, firstname.lastname@example.org
LabEx representatives: INRT (Bertrand Séraphin), MitoCross (Ivan Tarassov), NetRNA (Pascale Romby)
Chirality is a property of asymmetry between an object and its mirror image; the etymology of the word "chiral", meaning "hand" in ancient Greek, refers to the fact that the right hand and the left hand, although images of each other in symmetry with respect to a plane, are not superposable. Most molecules, many types of cells and also drugs have this asymmetry of which some of their properties depend. Julien Vermot's team at the IGBMC (CNRS/Inserm/University of Strasbourg) with the help of the group of Willy Supatto (Ecole Polytechnique, Paris) and Andrej Vilfan (Max Planck Insitute, Göttingen) used advanced live imaging techniques to show that the cilia on the surface of the cells of the right-left organizer of the zebrafish have a chiral orientation between the right and left sides. This work shows that tissues, like molecules and cells, have chiral properties and could help to understand how axes of symmetry are broken during organ genesis. Results published in the journal Cell reports on November 20, 2018.
The asymmetric distribution of organs within the body cavity such as the heart is established very early in embryogenesis. Symmetry breaking occurs in the so called left-right organizer, where cells are dictated where left and right embryonic sides are. Left-right symmetry breaking requires motile organelles protruding from the cell surface called cilia. It is now well established that cilia generate a directional flow in the left-right organizer that breaks the embryonic symmetry by acting as propellers. However, many observations have suggested that the chirality of proteins or cells could participate in breaking the embryonic axis of symmetry. Using intravital imaging tools based on multiphoton microscopy and dedicated image analysis approaches, the researchers had the surprise to observe that the spatial orientation of cilia and show that they are oriented asymmetrically.
Considering that such asymmetry was never observed before, the researchers seeked to establish the origin of this chirality. They focused on a well-known property of cells: the planar polarity. The planar cell polarity is thought to act as a cellular compass allowing the cells to orient themselves in the planar axis of the tissues. Without it, the heart, lungs, skin and other organs would not develop properly. The researchers observed that the orientation of the cilia became symmetrical when the polarity of the cells was defective. The researchers concluded that the cellular polarity is essential to establish chirality.
While the planar polarity is partly responsible for the asymmetric orientation of lashes, it does not fully explain the mechanism by which cilia orient themselves asymmetrically. The researchers assume that other factors need to be taken into account: the asymmetric structure of the lash could affect their positioning as they beat, and the anchoring of the cilia and its alignment in the cell could also be involved.
By revealing these unexpected properties of asymmetric tissue organization, this work opens new perspectives on understanding the role of chirality on the shape and positioning of our organs.
This study was funded by the ANR, FRM, the EMBO Young Investigator Program, ERC and the Slovenian Research Agency.
Registrations are now open for the Instruct Biennial Structural Biology Conference to take place in Alcalá de Henares (near Madrid) 22-24 of May 2019. Confirmed speakers includes: Sjors Scheres, Julia Mahamid, Eva Pereiro, Andrej Sali, Teresa Carlomagno, Alex de Marco, Dave Stuart and H M Al-Hashimi. There are 10 fellowships available for graduate students which will cover travel and accommodation costs. 4 speakers will be selected from abstracts. Register now https://instruct-eric.eu/biennial2019
There will be also a talk by the third Bertini award winner. The award recognises a significant achievement in frontier research that utilises an integrative structural biology approach. The award is the first commemorating Ivano Bertini who develop powerful new methods in NMR and built up the world class Centre for Magnetic Resonance in Florence. The award of €15000 is endowed by Bruker BioSpin and you can submit a nomination here https://instruct-eric.eu/submit-call/instruct-ivano-bertini-award-2019
The Instruct Internship Programme funds research visits of 3-6 months duration to Instruct Centres in Europe. The aim is to facilitate valuable collaborations with Instruct research groups applying techniques that are not available in the applicant’s laboratory. Applications should specifically focus on the benefit to the applicant’s research. Internships may be hosted at any institution that hosts an Instruct Centre, providing the applicant is a resident of a different full Instruct member country at the time of making the application.
More info at https://instruct-eric.eu/internships
Visualize without disturbing structures and processes taking place in the living cell nucleus is now possible thanks to an innovative technique developed by Laszlo Tora’s team at the IGBMC (CNRS/Inserm/Unistra) in close collaboration with Etienne Weiss at the ESBS Research Institute (CNRS/Unistra). This method allows to introduce, efficiently and without damaging the cells, antibodies labelled with small fluorescent molecules. In the cytoplasm of cells, these fluorescent antibodies bind to their antigens or targets. When the target is a nuclear protein, antibodies are transported with them into the nucleus, allowing researchers to locate and track the movements of this nuclear protein with high accuracy and in real time. The results of this study were published on February 12th, 2018 in the Journal of Cell Biology.
Protein detection by immunofluorescence is a technique that has been widely used for many years, where proteins or their post translational modifications are identified with antibodies coupled to microscopically detectable fluorescent compounds. The disadvantage of this process is that the cells need to be fixed and permeabilized, which may damage them, to allow the entry of labelled antibodies. Furthermore, the need to fix cells does not allow the observation of the progression of a biological mechanism over time.
In this new study, researchers from Laszlo Tora’s and Etienne Weiss's teams have developed an ingenious method that allows them to observe chromatin-based mechanisms in the nucleus of living cells, without altering the observed processes. This technique allows the introduction of any antibody labelled with fluorescent compounds that bind specifically to nuclear factors into the cells by using an electric micro-shock that does not modify cells physiology. When these antibodies are in the cytoplasm, they bind to the nuclear factors produced in this cell compartment. Since these factors are naturally transported to the nucleus to exert their function, the nuclear factors linked antibodies are taken to the nucleus. "The antibodies, which bind to these newly synthesized factors, are transported in the nucleus of the cell like small backpacks and, as they are marked with fluorochromes of any color, they will send a signal which allows us to observe the nuclear factors to which they are linked", explains Laszlo Tora. In addition, this visualization of nuclear factors in living cells is very effective because each antibody can be labelled with five to seven fluorescent molecules. The treated cells were observed with a confocal microscope and, in collaboration with the Basel Imaging Centre, with a very high-resolution microscope, the 3D-SIM. Precise observation of proteins that have nuclear activity during the cell cycle allows to see their dynamics and motion in the nucleus of living cells. Researchers have also shown that this strategy makes it possible to detect in real time the appearance and management of DNA damages in the nucleus by detecting a post translational modifications. By using an antibody fragment that has the same properties as a complete antibody and targets a major actor in triggering DNA repair (gammaH2AX), researchers were able to reveal areas in the nucleus where cell DNA is damaged by a substance causing genome changes, called genotoxic agent.
Given the large number of antibodies available on the market, this simple and innovative method will undoubtedly provide new information about nuclear proteins and a better understanding of their behavior in transmitting and maintaining the integrity of genetic information.
This study was funded by the ANR, ERC and the Regional League Against Cancer.
Ribosomes play a vital role in living beings: they are protein production stations in all cell types. These large complexes are made up of two kinds of molecules: proteins and ribosomal RNA (rRNA). Through structural studies, Bruno Klaholz's team at the IGBMC revealed more than 130 chemical modifications of the rRNAs. Published in the journal Nature on November 15 2017, these results not only provide a better understanding of the structural and functional roles of human ribosome modifications, but also open up new therapeutic pathways for the treatment of pathologies such as cancer or certain genetic diseases in which dysregulation of protein synthesis is involved.
Genuine molecular machines, ribosomes decipher the information contained in a copy of DNA, called messenger RNA, to synthesize proteins. During ribosomes biogenesis, chemical modifications occur at precise locations of rRNAs. The alteration of these modifications is sometimes associated with disruptions in protein synthesis. Moreover, these modifications in the rRNA structure seem to modulate the activity of antibiotics, indicating that they can influence the ribosome interaction with this kind of drugs.
Chemical modifications of rRNA have been known for decades, particularly in microbes, but their observation remained difficult. In this study, Bruno Klaholz's team at the IGBMC determined the localization and three-dimensional structure of 136 various types of modifications (methylations, acetylations, etc.) of human ribosome rRNAs. The researchers have thus uncovered the role of these chemical modifications which consists in stabilizing the rRNA structure.
The researchers also analyzed the structure of three antibiotics-binding sites, revealing that these molecules are either in direct contact or very close to the rRNAs modifications sites, implying a role of these modifications in the mechanism of action and specificity of these inhibitors. Moreover, the unexpected presence of certain rRNA modifications suggests that rRNA modifications in human ribosomes could vary between different cell types, especially between normal cells and cancer cells, and could thus constitute a signature for cancer states.
This study represents a major result made possible by cryo electron microscopy, a revolutionary method for the observation of macromolecules coupled with image processing and 3D reconstruction, by which the researchers were able to construct a detailed atomic model of the human ribosome. Recognized recently by the Nobel Prize in Chemistry 2017, this technique provides the double advantage of being able to study biological samples without altering their properties and also allows to fix the orientation of the objects to enable a 3D reconstruction. This work was carried out using a high-resolution electron microscope (Titan Krios) within the French and European infrastructures, FRISBI and Instruct, hosted at the Centre for Integrative Biology at IGBMC.
Beyond mapping at the atomic level, the visualization of rRNA chemical modifications provides unprecedented insights into their role in the stabilization of rRNA structure, in the translation process and in diseases related to ribosome dysfunctions, paving the way for the development of new drug.
This study was supported by the INCa, the Ligue, the ANR, the CPER 2007-2013 (the Greater Region, the Eurometropole of Strasbourg, the General Council of the Bas-Rhin, the Ministry of National Education, higher education and research), FRM, Inserm, CNRS, FRISBI and Instruct-ERIC.
The Sweden Nobel Prize in Chemistry 2017 was awarded to Switzerland's Jacques Dubochet, the German-born American Joachim Frank and Britain's Richard Henderson for developing electronic cryo-microscopy, a revolutionary molecular observation method coupled with 3D reconstruction. This technique allows not only to study biological samples without altering their properties, as it is the case with dyes, but also to produce high-resolution three-dimensional structures of biomolecules which represents a revolution in the field of structural biology.
FRISBI open access to national and international scientific, academic and industrial scientific communities to cryo-microscopy devices of high technology: Polara and Titan Krios, see our platforms catalogue http://frisbi.eu/platforms-catalogue/
At a Royal Society awards ceremony celebrating the prestigious new European Commission-approved status for structural biology Instruct Research Infrastructure, UK Minister for Universities, Science, Research and Innovation Jo Johnson recognised the value and relevance of collaborative work between the UK and European scientists.
The defense of an organism depends on its ability to recognize danger signals. In mammals including humans, macrophages represent a predominant cellular system that acts against pathogens and removes cellular debris. Activation of macrophages represents a crucial immune defense mechanism and its deregulation contributes to uncontrolled inflammation and autoimmune diseases. Romeo Ricci’s team at the IGBMC has identified a new signaling pathway involved in the intracellular innate immune defense system. For the first time, they provided evidence that the Golgi apparatus, an important cellular organelle, is involved in the activation of the inflammasome, an intracellular multiprotein complex capable of sensing tissue damage and infectious agents. They also showed that pharmacologic interference with this newly identified mechanism can be used to mitigate inflammation. This study was published on 17 July 2017 in the Journal of Experimental Medecine (JEM).
The macrophage harbors two defense systems against dangers signals: on the one hand, specific receptors on their membrane surface and, on the other hand, intracellular complexes including the the various subsets of inflammasomes that are subjects of this study.
Romeo Ricci’s teamwas particularly interested in the NLRP3 inflammasome subunit activation mechanism. NLRP3 has been recently shown to directly bind to mitochondria-associated endoplasmic reticulum membranes (MAMs) allowing for inflammasome activation. This work showed for the first time that the Golgi apparatus activates Protein Kinase D (PKD) leading to NLRP3 phosphorylation. This phosphorylation was essential to release NLRP3 from MAMs resulting in recruitment of additional components and assembly of the mature NLRP3 inflammasome in the cytoplasm triggering an inflammatory response.
Patients affected by a rare genetic disease called CAPS, in which the gene encoding for the NLRP3 protein is mutated, the NLRP3 inflammasome is constitutively activated resulting in auto-inflammation. Using immune cells isolated from these patients, they demonstrated that inhibition of PKD blocked NLP3 inflammasome activation in vitro and mitigated release of pro-inflammatory factors providing a solid basis for new treatment options in NLRP3-related inflammatory diseases.
The study was funded by a USIAS fellow grant from the University of Strasbourg and the European Research Council (ERC).
The European Commission adopted the Instruct-ERIC decision on the 4th July 2017. Instruct is the 17th ERIC to be adopted.
Instruct was initiated in 2008 as a European-funded Preparatory Phase Project 211252 in the ESFRI Programme. In 2011, Instruct entered a transitional phase and in 2012 was operational, providing the first access to Instruct infrastructure through its Centres. Instruct has grown to provide new infrastructure, now modes of service delivery, excellent training courses and internships and has supported scientists through its R&D Pilot programme. Instruct-ERIC will provide the stability and sustainability for Instruct services to the structural biology community and to other life sciences communities within Europe.
Instruct was awarded an EU-funded project 'Instruct-ULTRA' in 2017 to further expand Instruct membership and services. Instruct-ULTRA will enable Instruct to work with new communities and extend and evolve the Instruct infrastructure to meet the demand of researchers wanting high quality structural technologies and methods.
In the nine years of Instruct development, there have been many people who have contributed and this success is thanks to them. Congratulations to all who played their part and we look forward to a bright and productive future as Instruct-ERIC.
See more at https://www.structuralbiology.eu
Chemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs.
This work was carried out using a high resolution electronic cryo-microscope (Titan Krios) within the French and European infrastructures, FRISBI and Instruc-ERIC