Biology subjects

Dufourt, J.

Publications and source records attributed to Dufourt, J..

10 recordsLinked to original sources

bioRxiv2026

SARS-CoV-2 nucleocapsid protein engages with viral RNA and ERGIC lipids to drive viral core assembly

Severe acute respiratory syndrome coronavirus 2 assembles at the ER-Golgi intermediate compartment (ERGIC), yet the molecular basis of nucleocapsid (N) protein interactions with host membranes remains unclear. Using in vitro reconstituted lipid membranes and viral RNA- N complexes, we show that full-length N binds phosphatidylinositol (PI)- and phosphatidylserine (PS)-containing membranes and induces lipid clustering, an effect amplified by viral RNA and ERGIC-like membrane lipid composition. The isolated N-terminal domain lacks this activity, whereas the C-terminal domain retains membrane-associated multimerization. Although, lipid bilayers promote co-clustering of N and PI lipids, facilitating ribonucleoprotein (RNP) assembly, even on simple membranes. Importantly, ERGIC- mimicking membranes enhances this co-clustering further stabilizing RNPs of dimensions matching the viral core. In cells, viral RNA enhances N clustering without altering particle production. These findings reveal cooperative interactions between N, viral RNA, and ERGIC lipids as key drivers of lipid-dependent viral core formation, providing a mechanistic framework for the early steps of viral assembly.

biophysics↗

bioRxiv2026

Real-time single-molecule imaging in zebrafish embryos uncovers non-canonical translation

Precise spatiotemporal control of protein synthesis is essential during embryogenesis, yet directly measuring translation kinetics in vivo remains challenging in vertebrates. In particular, it remains unclear how translation efficiency is determined for key developmental regulators, and which kinetic steps limit their production. Here, we used ALFA array-based nascent chain labeling combined with lattice light-sheet microscopy to visualize bmp2b translation in real time and at single-molecule resolution in early zebrafish embryos. When combined with MS2/MCP labeling to visualize all bmp2b mRNAs, we found that only some of them are being actively translated, suggesting that limited mRNA translation competence contributes to overall translation efficiency. We found that bmp2b translation operates below a maximal initiation regime, as replacement of its UTRs with viral UTRs increases ribosome loading. Furthermore, the bmp2b, but not actb2, 5'UTR supports cap-independent translation with ribosome loading comparable to cap-dependent initiation. Altogether, this approach provides a quantitative in vivo framework to dissect translation kinetics during early vertebrate development.

developmental biology↗

bioRxiv2023

Repressor sequestration activates translation of germ granule localized mRNA

Biomolecular condensates organize and compartmentalize biochemical processes within cells1. Among these, ribonucleoprotein (RNP) granules are characterized as storage depots for translationally repressed mRNA2-8. Whether RNP granules can also activate translation and how such translation is achieved remains unclear9. This question is particularly relevant for embryonic germ granules, whose activity has been linked to germ cell fate10-13. Here, we use single-molecule imaging to show that embryonic germ cell RNP granules in Drosophila are the sites of active translation for nanos mRNA. Translating nanos mRNA is oriented with the 5end preferentially at the germ granule surface while the 3UTR is buried within the granule. Untranslated nanos mRNAs remain internal within the granule. Quantitative analysis of translational kinetics demonstrates that germ granules activate translation by antagonizing translational repression rather than changing the rate or efficiency of translation. We generated separation-of-function mutations in the disordered linker region of the scaffold protein Oskar that specifically impede nanos translation without affecting germ granule morphology or RNA localization. These mutations reveal that nanos translation is dependent on the sequestration of translational repressors within germ granules. Together, our findings show that RNP granules regulate localized protein synthesis through compartmentalized relief of translational repression raising the possibility that similar repressor-activator switches control translation in other condensates.

cell biology↗

bioRxiv2023

Unraveling human, rodent and snake Kolmioviridae replication to anticipate cross-species transmission

The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake deltavirus (SDeV) in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of infected cells revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of infectious clones showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.

microbiology↗

bioRxiv2023

Boosting the toolbox for live imaging of translation

Live imaging of translation based on tag recognition by a single chain antibody is a powerful technique to assess translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.

developmental biology↗

bioRxiv2021

The control of transcriptional memory by stable mitotic bookmarking

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is thought to be mediated, in part, by mitotic bookmarking by transcription factors (TF). However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that the pioneer-like transcription factor GAF acts as stable mitotic bookmarker during zygotic genome activation. We report that GAF remains associated to a large fraction of its interphase targets including at cis-regulatory sequences of key developmental genes, with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we provide evidence that GAF binding establishes competence for rapid activation upon mitotic exit.

developmental biology↗

bioRxiv2020

Imaging translation dynamics in live embryos reveals spatial heterogeneities

The translation of individual mRNA molecules is a key biological process, yet this multi-step process has never been imaged in living multicellular organisms. Here we deploy the recently developed Suntag method to visualize and quantify translation dynamics of single mRNAs in living Drosophila embryos. By focusing on the translation of the conserved major epithelial-mesenchymal transition (EMT)-inducing transcription factor Twist, we identified spatial heterogeneity in mRNA translation efficiency and reveal the existence of translation factories, where clustered mRNAs are co-translated preferentially at basal perinuclear regions. Simultaneous visualization of transcription and translation dynamics in a living multicellular organism opens exciting new avenues for understanding of gene regulation during development.

developmental biology↗

bioRxiv2019

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base-pairing, leading to both their destabilization in the soma, and stabilization in the germ plasm. Here, we report a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A) binding protein PABP and the translation initiation factor eIF3. Polysome gradient profiling identifies Aub role at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results reveal a new mode of mRNA regulation by Aub, highlighting PIWI protein versatility in mRNA regulation.

developmental biology↗

bioRxiv2018

Temporal Control of Transcription by Zelda in living Drosophila embryos

Abstract/introPioneer factors have the exquisite ability to engage their target sites at nucleosomal DNA, which leads to a local remodeling of chromatin and the establishment of a transcriptional competence. However, the direct impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains elusive. In Drosophila embryos, the maternally deposited activator Zelda (Zld) exhibits key pioneer factor properties and indeed regulates the awakening of the zygotic genome. The analysis of thousands of endogenous Zld bound regions in various genetic contexts, as well as the study of isolated synthetic enhancers with static approaches, led to the proposal that Zld could act as a quantitative developmental timer. Here we employ quantitative live imaging methods and mathematical modeling to directly test the effect of Zld on temporal coordination in gene activation and on mitotic memory. Using an automatic tracking software, we quantified the timing of activation in hundreds of nuclei and their progeny in Drosophila embryos. We demonstrate that increasing the number of Zld binding sites accelerates the kinetics of transcriptional activation regardless of their past transcriptional state. In spite of its known pioneering activities, we show that Zld is not a mitotic bookmarker and is neither necessary nor sufficient to foster mitotic memory. Fluorescent recovery after photo-bleaching and fluorescent correlation spectroscopy experiments reveal that, Zld is highly dynamic and exhibits transient binding to chromatin. We propose that Zld low binding rates could be compensated for by local accumulation of Zld in nuclear microenvironments in vivo, thus allowing rapid and coordinated gene activation.

developmental biology↗

bioRxiv2017

Translational Repression Of The Drosophila nanos mRNA Involves The RNA Helicase Belle And RNA Coating By Me31B And Trailer hitch

Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3 UTR. In a comprehensive mass-spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that coating of the RNA by a Me31B*Tral complex may be at the core of repression.

biochemistry↗