CE12 - Génétique, génomique et ARN

Regulation of ribosome activity by rRNA 2'-O-methylation: Toward rRNA epitranscriptomic control of translation – ActiMeth

Translational control by methylation of ribosomal RNA: towards a specialized ribosome.

Decipher the variable elements of the ribosome contributing to regulation of translation.

Deciphering the role of methylation of ribosomal RNA in the translation process

Deciphering the mechanisms of protein synthesis regulation is crucial in our understanding of gene expression in both healthy and pathological tissues. Historically, the genetic sequences present in messenger RNAs (mRNAs) and proteins that bind to mRNAs were considered to be the sole regulators of protein synthesis.<br />In this context, the ribosome has long been regarded as a mere effector of protein synthesis, playing no role on the choice of mRNAs to be translated and on the efficiency of translation. This view has been called into question in recent years by various studies showing that there is a great variability of composition within the population of ribosomes of the cell, and that this contributes to the regulation of translation. Changes in the composition of ribosomes have been demonstrated during embryogenesis and in pathologies, in particular cancer.<br />The objective of the ActiMeth project is to characterize the role of variations in ribosomal RNAs, and specifically their methylation, in the control of protein synthesis, by combining molecular and cellular biology with biophysical «single molecule« approaches to analyze translation ribosome by ribosome.<br />One of the challenges of the ActiMeth project is to provide mechanistic data to understand the role of these ribosomes in physiological mechanisms and in the emergence of pathologies.

This project is based on the inhibition of preselected methylation sites by targeting the corresponding snoRNAs with antisense oligonucleotides, and methylation mapping by a technology called RiboMethSeq. ActiMeth uses complementary approaches from the three partners, which will make it possible to analyze at the molecular level, ribosome by ribosome the dynamics of translation («single molecule« approach), at the cellular level the regulatory mechanisms, and to identify the cellular pathways of which regulation is based on the methylation of ribosomal RNAs by large-scale approaches (“riboSeq” approach).

The main parts of the project have been started and are in operation.
RiboSeq data analysis software called RiboDoc was made available to the community.
RiboMethSeq technology and all bioinformatics and statistical analysis tools have been implemented within the CRCL.

This project will provide a better understanding of how ribosomes participate in the mechanisms regulating protein synthesis. It will provide for the first time a detailed description of the relationship between methylation of ribosomal RNAs and translational regulations. This project will lay the foundations for a molecular exploration to link the composition of ribosomal RNAs with the key translation events, namely the initiation, elongation and termination phases.

Francois P. et al. RiboDoc: A Docker-based package for ribosome profiling analysis. Comput Struct Biotechnol J. 2021 May 7;19:2851-2860..

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.

Project coordination

Jean-Jacques DIAZ (Centre de Recherche en Cancérologie de Lyon)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

I2BC Institut de Biologie Intégrative de la Cellule
LPQM Laboratoire de Photonique Quantique Moléculaire
CRCL Centre de Recherche en Cancérologie de Lyon

Help of the ANR 533,414 euros
Beginning and duration of the scientific project: December 2019 - 48 Months

Useful links

Explorez notre base de projets financés

 

 

ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter