Spatio-temporal coupling between transcription and translation dynamics during development

Informazioni relative al progetto

LightRNA2Prot

ID dell’accordo di sovvenzione: 101044609

DOI

10.3030/101044609

Data della firma CE 26 Ottobre 2022

Data di avvio 1 Gennaio 2023

Data di completamento 31 Dicembre 2028

Finanziato da

• European Research Council (ERC)

Costo totale

€ 2 000 000,00

Contributo UE

€ 2 000 000,00

Coordinato da

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 1 - LightRNA2Prot (Spatio-temporal coupling between transcription and translation dynamics during development)

Periodo di rendicontazione: 2023-01-01 al 2025-06-30

Sintesi del contesto e degli obiettivi generali del progetto

During development, precise control of gene expression allows the reproducible establishment of patterns, leading to the adoption of cellular identities at the right time and place. What are the mechanisms behind such precision? To date, this question has been primarily examined from the focal point of transcription, with the quantification of the precise timing, location and levels of gene activation. However, precision in mRNA production is functionally relevant only if it leads to precision in protein expression. While the linear correlation between the levels of a given mRNA and the amount of protein it encodes has been assumed for the last six decades within the central dogma of molecular biology, many examples challenge this view. The control of translation, particularly in distinct subcellular compartments where mRNA are targeted, may lie at the heart of this mRNA to protein disconnect. In the context of a reproducible developmental program with accurate cell fate decisions, such as patterning of the Drosophila blastoderm, we hypothesize that spatio-temporal control of translation allows the fine-tuning of expression of pivotal developmental proteins. LightRNA2Prot aims to unravel the mechanisms underlying the lack of correlation between mRNA and proteins levels to elucidate the sources of reproducible cell fate decisions during development. Importantly, it considers both the layers of regulation constituted by transcription and translation respectively, as well as their potential coupling. We will use quantitative imaging methods that simultaneously monitor mRNA, nascent peptides, and protein in Drosophila embryos with unprecedented spatio-temporal resolution, combined with genetic/optogenetic manipulations to dissect the mechanisms at play. LightRNA2Prot focuses on two key developmental questions: 1-How do the translation dynamics of key localized developmental transcripts modulate precise patterning? 2-How are (co)-transcriptional nuclear events coordinated with translational control in space and time? Combined with mathematical modeling and phenotypic characterization, our quantitative approaches will provide a dynamic multiscale view of gene expression control in vivo. The novel integration of translational control has the potential to dramatically change our view of stereotypic pattern formation and reproducible cell fate decisions during development.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

Research and technological achievements
WP1: Unmasking the translation dynamics of localized patterning genes.
Task 1. Mapping expression dynamics of localized patterning genes
We focused on a set of transcription factor encoding genes, that are essential for driving early patterning. In addition to these zygotic model genes, we also examined, through collaborative work, a few maternal mRNA, such as bicoid, or nanos.
Currently, we have very exciting data which is revealing unprecedented insight into when and where translation occurs in the early embryo. We were also able to reliably infer the translation kinetics of these model mRNA and how these evolve in space and time.
Task 2. Unravelling the supports of translation heterogeneities
This aspect of the project was not yet prioritized.
Task 3: Elucidate the developmental functions of translation heterogeneities
The genetic tools have been obtained, notably the delocalization of model mRNA to the apical cytoplasm, using specific 3’UTR sequences. The phenotypic characterization of these alleles is in progress.This project was led by a research engineer, who is currently on parental leave.
The modelling aspects of the project were supposed to be performed in collaboration with Pr. John Reinitz (University of Chicago), but he sadly and suddenly passed away in January 2025. For these two reasons, this aspect of lightRNA2Prot project is currently on hold.

WP2: Coordination between transcription and translation dynamics
Task 1: Dissecting the impact of transcription kinetics on translation dynamics
Through a rigorous quantitative analysis of single molecule RNA FISH and IF, we observed an effect of the promoter sequence on translation efficiencies. This promising result now needs to be consolidated.

Task 2: Deciphering the molecular coordinators linking transcription and translation: m6A pathway.
This talk comprises two aspects, i) investigating how the m6A pathway affects translation in space and time and ii) how the exon junction complex, EJC affects translational control.
For the EJC, a new ERC post doc was just hired (in Feb 2025) and is currently building the various genetic constructs.
For the m6A pathway, I recruited Iryna Mohylyak, a senior post doc under ERC suppot, to lead this ambitious and innovative project. Iryna Mohylyak generated fluorescently tagged CRISPR alleles for members of the m6A pathway as well as optogenetically tagged alleles to perturb the localisation of key members of the m6A pathway.
We are currently validating these lines and investigation the resulting phenotypes in terms of gene expression control.


Novel methodologies, inter-disciplinary developments and knowledge transfer
• optimization of the genetic tools to image translation and mRNAs
• Gene editing with SEED technology
• Imaging set-up: lattice light sheet
• Data analysis and modelling (collaboration with Tim Saunders lab, University of Warwick, UK).

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

The core findings of the ERC consolidator grant results are not yet published. But I foresee at least one high impact paper on the discoveries made in WP1, a second technical paper on LLS imaging and image analysis. The development of WP2 is still preliminary but I anticipate unexpected discoveries for that part of the project as well. Here is the list of publications, with members of ERC team shown in bold.

Publications of 1 article:
Athilingam, Wilby, Bensidoun, Trullo, Verbrugghe, Lagha, Saunders# and Weil# (2024) Regulation of bicoid mRNA throughout oogenesis and early embryogenesis impacts protein gradient formation. Biorxiv. DOI : 10.1101/2024.11.11.622966 (under review).

Publication of a book chapter and 1 review:
Bensidoun, Verbrugghe and Lagha1#. (2024) "Imaging Translation in Early Embryo Development". Methods in Molecular Biology, In Press. biorxiv. DOI: 10.1101/2024.12.09.626398
Maillard, Bensidoun and Lagha1#. (2025) " Reshaping transcription and translation dynamics during the awakening of the zygotic genome". Current Opinions in Genes and Development, DOI.org/10.1016/j.gde.2025.102344