Ribosomes are molecular machines, which catalyze the translation of genetic information from an mRNA filament into protein during a 4 steps process. In eukaryotes, differently from prokariotes the start codon recognition occurs through multiple pathways depending on the mRNA signals, and requires a specific set of initiation factors (eIFs) that guide the dynamic assembly of multiple intermediate complexes. This project aims at understanding how specific mRNA structures dictate its corresponding translation initiation pathways (canonical and IRES-mediated), identifying the eIFs involved in these processes and unraveling their mechanism of action. Our lab set up a multidisciplinary strategy including purification of translation initiation complexes (ICs) directly from cell extracts, characterization of components by mass spectrometry analysis and structure analysis using single-particle cryo-electron microscopy. The use of cell extracts is instrumental as it allowed the discovery of poorly characterized complexes, inaccessible for in vitro complex reconstitution. Because defects on the initiation pathways can led to several human pathologies, the project will be primarily focused on the structure investigation of ribosomal complexes involving specific mRNAs known to be involved in various pathologies (cancer, viral diseases) and on the role of specific eIFs and/or regulatory factors in the recruitment and positioning of the mRNAs onto the ribosome.
Our lab is generally interested in the structural biology of RNAs that regulate translation initiation. Such RNAs are either internal to the translation machinery (e.g. ribosomal RNA expansion segments) or external (e.g. internal ribosome entry sites (IRES) among viral and eukaryotic messenger RNA). They are often over 200 nucleotides long and highly structured. In order to answer key biological questions regarding the mechanism of action of these regulatory RNA, we use an integrated approach that combines sequence analysis, chemical mapping, crystallography, and cryo-electron microscopy.
Our project consist of modelling the initiation of the translation complexes of multiple organisms. The modelling is the final step of the structural determination of any macromolecule. In our team, we use electronic density map made by cryo-electron microscopy. Modelling can be done by two different and complementing methods which are modelling by homology and ab initio, both of them use multiple and complementary tools.
Modelling by homology consists of the determination of one macromolecule 3D structure by using it structural similarities with a solved structure. This technique is called the threading and consists of deriving the structure of a macromolecule starting from it sequence. This sequence will be thread to a solved structure of the same macromolecule from an other genetically closely-related species in order to build it own 3D structure. Homology modelling can be done with the program Assemble2 for RNA and web services like SwissModel for proteins. The resulting structure need to be checked and corrected because it is possible that there is some gaps in the structure even with closely-related species. These gaps will be filled using the ab initio modelling method.
Ab initio modelling is the determination of unsolved structure. Unlike homology modelling, we don’t use any reference structure to build our model. It consists of building the model only by interpreting it density map therefore this one need to have a high resolution to distinct secondary structure like alpha helix or beta sheet in order to build our model correctly. There is several tools for ab initio modelling like Chimera which is a very complete program for modelling. We also use web services to predict secondary and tertiary structures of proteins which are I-tasser, SwissModel and Sympred for proteins and the program Assemble2 for RNA.
The final step of modelling is the refinement of the built structure. This refinement can be done with the Molecular Dynamics Flexible Fitting (MDFF) method which will flexibly fit the atomic structure in the density map. MDFF method consists of a molecular dynamics simulation of the atomic structure using external forces proportional to the gradient of the density map. MDFF is run with the programs VMD and NAMD
The refinement can also be done by Real Space Refinement in the program Coot. This method consists of the fitting of the atoms in the highest neighboring density with the lowest geometric distortion (angles and bond lenght for example).
Presently we investigate the mechanism of translation in two different eukaryotic pathogens : Kinetoplastids and Plasmodium.
Kinetoplastids are unicellular eukaryotic pathogens responsible for several human and animal diseases, such as Chagas disease (caused by Trypanosoma cruzi), African Trypanosomiasis (caused by Trypanosoma brucei ssp.) or Leishmaniasis (caused by Leishmania spp.), and the molecular aspects of translation initiation in these organisms remain poorly understood. Usefully for the discovery and development of specific and safer drugs against these organisms, cryo-electron microscopy (cryo-EM) provides a new path for deciphering the structures of their ribosomes and translation-related complexes.
In comparison with ribosomal structures from higher eukaryotes, the ribosomes from kinetoplastids (Figure 1) show the presence of extraordinarily large expansion segments (ESs) in the rRNA secondary structure, and contrarily to its higher eukaryotic version, the kinetoplastid large ribosomal subunit (LSU) rRNA has five cleavage sites.
Figure 1 : Presentation of atomic models of SSU (left) and LSU (right) from T. brucei ribosome. The regions colored in grey indicate the universally conserved ribosomal features, and eukaryote-specific conserved elements are shown in yellow. The red regions indicate the specific core elements of the Trypanosome ribosome (Hashem et al., 2013).
We believe that these kinetoplastid-specific components are involved in important regulatory mechanisms of translation in these organisms such as the initiation process, due to the localization of these elements on the 40S subunit1 (Hashem et al., 2013a). For example, the structure and position of mammalian eIF3―the largest and most complex initiation factor which is implicated in almost all steps of initiation― are complementary to some of these specific ESs of 40S subunits in kinetoplastids (Hashem et al., 2013b), namely, large expansion segments 6S and 7S suggesting a role in stabilizing the eIF3-40S subunit interaction in these organisms (Figure 2).
Figure 2 : Shape complementarity between eIF3 (red mesh) and the T. brucei expansion segments ES6 (hot pink) and ES7 (cyan) (Hashem et al., 2013b).
Plasmodium is a protozoan parasite transmitted by the bite of infected female Anopheles mosquitoes, causing several forms of malaria. The mRNA translation process in these parasites takes place in three intracellular compartments : the cytosol, mitochondria and apicoplast. The cytosolic ribosome ensures the translation of thousands of proteins encoded by the nuclear genome of Plasmodium. Several direct and indirect observations on the cytosolic ribosome indicate the uniqueness of the process of translation, such as the presence of inserts and extensions in some ribosomal proteins and translation factors, as well as the presence of large inserts of rRNA, called expansion segments, substantially different from those of other known eukaryotes. Moreover, Plasmodium has several types of ribosomal RNA sequences that are expressed at different stages of parasite development, giving several types of ribosomes. For example, in P. falciparum, which has a life cycle in two stages (mosquito and human), there are two types of ribosomal RNA sequences, types A and S, each having two variations, A1, A2 and S1, S2. A1 and A2 are expressed predominantly in the liver and blood stages. S1 is expressed predominantly in the gametocytes during the blood stage and S2 in the sporozoites that develop in the salivary glands of the mosquito.
Thanks to recent advances on cryo-electron microscopy (Cryo-EM), this powerful method has made possible the study of flexible macromolecular complexes, such as the ribosome or ribosomal subunits, enabling us to obtain structures at near-atomic resolution. A deep understanding of the translation mechanism in kinetoplastids and in Plasmodium could have a huge impact on the design of new drugs against these pathogens. So, presently we are studying protein translation in eukaryotic pathogens by cryo-electron microscopy.