In all living organisms, translation of mRNA into protein is carried out by ribosomes, which ensure the decoding of the mRNA and peptide-bond formation. The ribosomes also guarantee the fidelity of translation through proofreading activities watching frameshift, amino-acid misincorporation and stop codon read-through. Ribosome composition was recently shown to vary at the protein and ribosomal RNA (rRNA) level according to physiological and pathological contexts in several organisms including mammals. Recent breakthrough studies established the proof of concept that ribosome composition variation results in changes in translational activity of the cell and of the ribosome. These observations unexpectedly shed light on the ribosome as a new regulator of mRNA translation. Today, the molecular mechanisms by which ribosome constituents may regulate the efficiency of the various steps of translation remains to be deciphered. rRNAs play a central role in the translation process, by ensuring ribosomal subunits joining, decoding of mRNA sequence, and catalyzing the peptide-bond formation through the 28S rRNA ribozyme activity. rRNA chemical modifications, which are mainly 2'-O-methylation (2'-O-Me) and pseudouridylation, are clustered within and around functional domains of the ribosomes, such as the A-site, the decoding center, the peptidyl-transferase-center and the inter-subunits interface, and are believed to modulate local interactions and structures. Several groups, including ACTIMETH partners, showed that 2'-O-Me variations induce changes in translational programs and modulates ribosomes functionality. However, there is no mechanistic knowledge as to how rRNAs and their modifications regulate the functioning of the ribosome. We propose that rRNA 2'-O-Me directly controls the ribosome functional properties, and contribute to translation efficiency and regulation, at the level of initiation and elongation. The aim of the present proposal is to decipher how rRNA 2'-O-Me modulates efficiency of ribosome functions, in order to provide mechanistic insight into the regulation of mRNA translation by variation in human ribosome composition. In ACTIMETH, we will specifically target 2'-O-Me sites and determine at the molecular level the steps of translation initiation and elongation that are impacted. Specifically, we will explore the role of 2'-O-Me (1) on cellular translation, (2) on the assembly of translating ribosomes on various mRNAs, (3) on the decoding accuracy of ribosome and (4) on dynamics of translation initiation and elongation. To ensure the success of ACTIMETH programs, the consortium gathers three partners with international reputation in the fields of ribosome biology, translation regulation, fidelity and recoding, and established expertise in cutting-edge multidisciplinary technics including hybrid in vitro translation, ribosome profiling and biophysical methods to analyze ribosome composition and activity, translation control and translation dynamics by single-molecule techniques. ACTIMETH is built on a strong set of published and preliminary data that were obtained by the three partners during existing collaborations. ACTIMETH is a fully original program as it aims at deciphering new molecular mechanisms governing the translation process, and in particular the contribution of rRNA modifications to the main steps of protein synthesis. ACTIMETH will contribute novel molecular description of the role of 2'-O-Me sites on translation efficiency of specific cellular mRNAs that are 2'-O-Me sensitive. This data will also provide the ground for structural analysis of differentially methylated ribosomes, and their interaction with associated factors and mRNAs. Finally, ACTIMETH will provide molecular information relevant to understanding and targeting disease-specific ribosomes that are being described in cancer and genetic diseases such as ribosomopathies and non-sense associated diseases.

Resonant waveguide gratings (RWG) induce strong local electromagnetic fields on their top surface, which are useful in many applications. In this proposal, we will develop a high-sensitivity wide-field optical microscopy by using RWG-enhanced upconversion fluorescence (UCF) of infrared-excited rare-earth nanoparticles, as well as a new RWG-based phase-shift dark-field sensing technique based on a polarimetric approach. First, a RWG UCF microscopy (RWG-UCFM) will be developed by using rare-earth upconversion nanoparticles (UCNPs) as biomarkers and the RWG as a substrate. The proposed RWG-UCFM can provide high contrast images because the RWG substrate can greatly enhance UCF efficiency of labelled-UCNPs thanks to the excitation resonance effect, which a strong local electromagnetic field will be built on the surface of the RWG. Comparing with other available fluorescence microscopies, RWG-UCFM provides advantages like higher sampling speed, higher signal/noise ratio, and higher spatial resolution. Especially, since the RWG-UCF excitation wavelength is at 980 nm, drawbacks like photobleaching of fluorophores, autofluorescence and large scattering from bio-tissue, which are typically penalties of traditional fluorescence microscopy, are no longer a problem for the RWG-UCFM. The RWG-based microscopy will provide very good axial resolution (< 200 nm) thanks to evanescent wave property of local electromagnetic field built on the top of the RWG. The lateral resolution of the RWG-based microscopy is determined the lateral propagation of the guided mode resonance (GMR) mode and also the numerical aperture (NA) of the objective lens. Its lateral resolution will be typically 2 micrometres, if an objective lens of NA=0.5 is used. To improve its lateral resolution, we propose to use a structured illumination excitation, which is produced either by the interference of two excitation beams (non resonant excitation) or by using one beam resonant excited the RWG structure. With the unique multi-photon nonlinear absorption property of UCNPs, the lateral spatial resolution of the RWG-UCFM will be greatly enhanced (lateral resolution < 75 nm is expected to be obtained). Second, a label-free wide-field polarimetric microscopy will be developed using the RWG as a substrate. The RWG will be very sensitive to the change of refractive index on its top surface as GMR occurs. We will explore the change of polarization state of an incident beam transmitting through the RWG caused by the change of refractive index. The RWG substrate will be combined with a polarization state sensing setup and a good spatial resolution optical imaging system to form a label-free wide-field RWG polarimetric microscopy (RWG-PM). The RWG-PM can provide high spatial resolution images of the interfacial refractive index distribution of a specimen on the surface of the RWG due to narrow GMR spectra linewidth, restricted propagation and evanescent field enhancement on the RWG surface. To demonstrate the advantages of the two new proposed microscopy methods, bio-specimens will be imaged. We therefore aim to visualize chromatin structures using antibody against methylated histone H3K9me3 in OML1-P and OML1-R cells, and compare the structure images performed by our methods with those obtained by a conventional confocal microscope.