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).<'em><+p
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