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Structural differences in mRNA translation machineries between eukaryotic pathogens and their mammalian hosts

Periodic Reporting for period 3 - transtryp (Structural differences in mRNA translation machineries between eukaryotic pathogens and their mammalian hosts)

Reporting period: 2021-01-01 to 2022-06-30

Protein translation consists in translating the genetic information carried by the mRNA to amino acids. This process is performed by the ribosome that is essentially universally conserved in all cells. However, its structure and assembly present significant differences between bacteria and eukaryotes. Partly because of these differences, the bacterial ribosome can be targeted specifically by a number of antibiotics without hindering the translation process in the eukaryotic host cells. However, the relative conservation of the ribosome among eukaryotes complicates substantially the search for specific drugs against eukaryotic pathogens such as certain protozoa like Plasmodia and kinetoplastids.
Our previous work along with other studies demonstrates the existence of significant structural differences between ribosomes of certain protozoa and mammals. Using Cryogenic electron microscopy, we endeavor to investigate such ribosomal structural differences. It is anticipated that, because of their location on the ribosome, the structural differences will affect some of the vital steps of protein translation especially the initiation process. 1. We will focus on the structural differences on translation initiation (i) by characterizing for the first time initiation complexes from kinetoplastids as compared to their mammalian counterparts; (ii) by following up on our previous work on solving the structure of various mammalian initiation complexes. 2. We will focus on the structure of protozoa-specific ribosomal features and we will (i) draw an inventory of structures of such specific features and (ii) attempt to fish for any molecules they interact with. 3. We will investigate the structure of Plasmodial ribosomes at different stages of the parasite life cycle, as their content vary.
Our results will significantly advance our understanding of protein translation in protozoa and will represent a promising step in the research for more efficient treatments against eukaryotic pathogens.
Two manuscripts presenting high-resolution structures of the mRNA late-stage 48S initiation complex and the 34S pre-initiation complex in mammals and Trypanosma cruzi, respectively, are in preparation and will soon be submitted. Thanks to sample grid preparation optimizations we are able to routinely generate structures at near-atomic resolutions of most of our ribosomal complexes. The easy access to a last generation electron microscope (Talos Arctica, equipped with a Falcon III and now a K2 direct detection camera) provided a tremendous boost to the project’s progress and several new perspectives are in reach such as late-maturation ribosomal complexes and the mitochondrial mRNA translation machinery from kinetoplastids.
We have developed transfection assays of Leishmania Taretolae (a kinetoplastids), which will be required once we will start the functional studies of various initiation factors.
Our results show for the first time high-resolution mRNA translation (pre)initiation complexes from both mammals and pathogenic kinetoplastids. The comparison of these complexes allow the identification of several potential molecular targets that can be exploited in the future for the development of safer an more specific therapies against these dangerous parasites, infectious to numerous mammals including humans. Till the end of this project we will continue the investigation of such structural differences at many stages of the mRNA translation process, which will sum up in the establishment of an inventory of vital molecular targets.