Biology subjects

Ephrussi, A.

Publications and source records attributed to Ephrussi, A..

16 recordsLinked to original sources

bioRxiv2026

Granule microenvironment regulates the dual functions of FMR1

Fragile X Messenger Ribonucleoprotein 1 (FMR1) is an evolutionarily conserved RNA binding protein with important functions in cognition and female reproduction, and its disruption is associated with neurodevelopmental and reproductive disorders including the Fragile X syndrome. FMR1 is best known for its role as a translation repressor. However, several recent studies also suggest a role of FMR1 as a translation enhancer raising fundamental questions about the molecular regulation of these opposing functions. In this study, we identify FMR1 as part of the oskar mRNA-protein complex in the Drosophila oocyte and study the role of FMR1 as a translational enhancer of oskar. We provide the molecular mechanism for the dual functions of FMR1 and show that the two major RNA-binding domains of FMR1, the KH domains and the RGG box, play distinct separable roles in regulating translation. The KH domains enhance translation of mRNAs while the RGG box containing C-terminal domain (CTD) is required to repress translation. We further show that the condensation propensity of FMR1 containing granules regulates the two antagonistic functions, such that phase separation by FMR1-CTD creates the molecular microenvironment necessary for the repressive activity, whereas reduction in phase separation is associated with increased translation. Our findings highlight the importance of biomolecular condensates not just as a means of molecular compartmentalization but as a fundamental regulatory principle that dictates the functional output of modular protein domains.

molecular biology↗

bioRxiv2024

A direct interaction between the RNA-binding proteins Staufen and Tm1-I/C regulates oskar mRNP composition and transport

In the Drosophila female germline, oskar messenger RNA is transported on microtubules from the nurse cells to the posterior pole of the oocyte, where it is translated. Transport of oskar transcripts from the nurse cells into the oocyte requires dynein, while localization of the mRNAs within the oocyte to the posterior pole is dependent upon kinesin-1. Staufen, a dsRNA-binding protein, has been shown to bind the oskar mRNA transport complex in the oocyte and inactivate dynein; however, it remains unclear how kinesin is activated. Here, using surface plasmon resonance, nuclear magnetic resonance spectroscopy and RNA imaging within egg chambers, we demonstrate that Staufen directly interacts with Tm1, a non-canonical kinesin adaptor. This work provides a molecular explanation for the previously unclear role of Staufen in oskar mRNA localization.

molecular biology↗

bioRxiv2023

An architectural role of oskar mRNA in granule assembly

Ribonucleoprotein (RNP) granules are membraneless condensates that organize the intracellular space by compartmentalization of specific RNAs and proteins1. Studies have shown that RNA tunes the phase behavior of RNA binding proteins (RBPs)2-4, but the role of intermolecular RNA-RNA interactions in assembly of RNP granules in vivo remains less explored5-7. Here, we determine the role of a sequence-specific RNA-RNA kissing-loop interaction in assembly of mesoscale oskar RNP granules in the female Drosophila germline. A two-nucleotide mutation that disrupts kissing-loop-mediated oskar mRNA dimerization impairs condensate formation in vitro, oskar granule assembly in the developing oocyte - leading to defective posterior localization of the RNA, and abrogation of oskar-associated processing bodies (P-bodies) upon nutritional stress. This specific trans RNA-RNA interaction acts synergistically with the scaffold RBP, Bruno8, in driving condensate assembly. Our study highlights the architectural contribution of an mRNA and its specific secondary structure and tertiary interactions in formation of an RNP granule essential for embryonic development.

cell biology↗

bioRxiv2023

Nutritional stress-induced regulation of microtubule organization and mRNP transport by HDAC1 controlled α-tubulin acetylation

In response to nutritional stress, microtubules in cells of the Drosophila female germline are depleted from the cytoplasm and accumulate cortically. This triggers aggregation of mRNPs into large reticulated sponge bodies and oogenesis arrest. Here, we show that hyperacetylation of -tubulin at lysine 40 (K40) alters microtubule dynamics and sponge body formation. We found that depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress response, causing hyperacetylation of -tubulin, as well as accumulation of maternally deposited mRNPs in reticulated sponge bodies. Using in vitro and in vivo studies, we identify HDAC1 as a direct regulator of -tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of -tubulin acetylation, enabling the microtubule dynamics required for mRNP transport. Using quantitative phosphoproteomics we identify nutritional stress-induced changes in protein phosphorylation that act upstream of -tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate -tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.

developmental biology↗

bioRxiv2022

Tropomyosin 1-I/C co-ordinates kinesin-1 and dynein motors during oskar mRNA transport

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In Drosophila, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar mRNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. Here, through in vitro reconstitution, we show that Tm1-I/C, a Tropomyosin-1 isoform, links kinesin-1 in an inactive state to DDBE-associated oskar mRNA. NMR spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing a tug-of-war between the opposite polarity motors until kinesin-1 is activated in the oocyte. Our work reveals a novel strategy ensuring sequential activity of microtubule motors.

biochemistry↗

bioRxiv2022

Stably bound adaptor proteins modulate directionality of RNP transport

Kinesin-1 and cytoplasmic dynein are molecular motors that mediate long range transport of cargoes along the microtubule cytoskeleton. oskar RNA has been documented to switch between the motors during its localization in the Drosophila germline syncytium. oskar RNA undergoes dynein-mediated transport from the transcriptionally active nurse cells into the oocyte, following which the RNA translocates via kinesin to the posterior pole. Adaptor proteins link the RNA to its motors: the Egalitarian-Bicaudal-D complex links dynein to oskar RNA for the initial phase of transport, whereas atypical Tropomyosin 1 (aTm1) links kinesin-1 to oskar RNA for the latter phase. Components of the Exon Junction Complex (EJC) as well as the SOLE, a stem loop formed upon splicing of oskar RNA, have also been found to be necessary for kinesin-mediated transport of oskar RNA. In this study, to dissect the minimal elements required for kinesin-based transport, we tethered aTm1 or kinesin-1 to oskar RNA constructs lacking the SOLE. Our results suggest that stably bound aTm1 can indeed bypass the SOLE and EJC to mediate kinesin-1 activity, but the effects of tethered aTm1 are less potent than that of tethered kinesin-1. We also tethered Bicaudal-D to oskar RNA, to test whether this would affect kinesin-directed transport of oskar RNA, and found that tethered Bicaudal-D directs dynein mediated localization. Our results show that activated Bicaudal-D, along with the recruited dynein, is sufficient for dynein activity. We also show that stable binding of kinesin-1 to the RNA cargo is sufficient for strong kinesin-1 activity. Stably bound aTm1, meanwhile, can only mediate mild kinesin activity, suggesting that other factors may be required to stabilize the binding of kinesin-1 to the RNA cargo.

cell biology↗

bioRxiv2021

Subcellular spatial transcriptomics identifies three mechanistically different classes of localizing RNAs

Intracellular RNA localization is a widespread and dynamic phenomenon that compartmentalizes gene expression and contributes to the functional polarization of cells. Thus far, mechanisms of RNA localization identified in Drosophila have been based on a few RNAs in different tissues, and a comprehensive mechanistic analysis of RNA localization in a single tissue is lacking. Here, by subcellular spatial transcriptomics we identify RNAs localized in the apical and basal domains of the columnar follicular epithelium (FE) and we analyze the mechanisms mediating their localization. Whereas the dynein/BicD/Egl machinery controls apical RNA localization, basally-targeted RNAs require kinesin-1 to overcome a "default" dynein-mediated transport. Moreover, a non-canonical, translation- and dynein-dependent mechanism mediates apical localization of a subgroup of dynein-activating adaptor RNAs (BicD, Bsg25D, hook). Altogether, our study identifies at least three mechanisms underlying RNA localization in the FE, and suggests a possible link between RNA localization and dynein/dynactin/adaptor complex formation in vivo.

cell biology↗

bioRxiv2021

Egalitarian feeds forward to Staufen to inhibit Dynein during mRNP transport

Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). Here we show that orchestration of oskar RNP transport in the Drosophila germline relies on the interplay of two double-stranded RNA binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). We find that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. We additionally show that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. Our observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.

cell biology↗

bioRxiv2021

Liquid-to-solid phase transition of oskar RNP granules is essential for their function in the Drosophila germline

Asymmetric localization of oskar RNP granules to the oocyte posterior is crucial for abdominal patterning and germline formation of the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of specific proteins. Genetic modification of scaffold granule proteins, or tethering the intrinsically disordered region of human Fused in Sarcoma to oskar mRNA, allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate localization and expression of a maternal RNA that instructs germline formation.

cell biology↗

bioRxiv2021

Validation and classification of RNA binding proteins identified by mRNA interactome capture

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1s RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

biochemistry↗

bioRxiv2021

Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before. SummaryRonchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.

cell biology↗

bioRxiv2019

Induction of Chk2 signaling in the germarium is sufficient to cause oogenesis arrest in Drosophila

The conserved RNA helicase Vasa is required for germ cell development in many organisms. It is established that in Drosophila loss of piRNA pathway components, including Vasa, causes Chk2-dependent oogenesis arrest, however the stage at which Chk2-signaling is triggered was unknown. We found that absence of Vasa during the germarial stages arrests oogenesis due to Chk2 activation. Importantly, once induced in the germarium, Chk2-mediated arrest of oogenesis cannot be overcome by restoration of Vasa to the arrested egg-chambers. We conclude that Vasa activity specifically in the germarium is essential for germ cell lineage development.

developmental biology↗

bioRxiv2018

The transcriptome-wide landscape and modalities of EJC binding in adult Drosophila

Splicing-dependent assembly of the exon junction complex (EJC) at canonical sites -20 to -24 nucleotides upstream of exon-exon junctions in mRNAs occurs in all higher eukaryotes and affects most major regulatory events in the life of a transcript. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila the most essential cytoplasmic EJC function is in localization of oskar mRNA. Here we developed a method for isolation of protein complexes and associated RNA-targets (ipaRt), which provides a transcriptome-wide view of RNA binding sites of the fully assembled EJC in adult Drosophila. We find that EJC binds at canonical positions, with highest occupancy on mRNAs from genes comprising multiple splice sites and long introns. Moreover, the occupancy is highest at junctions adjacent to strong splice sites, CG-rich hexamers and RNA structures. These modalities have not been identified by previous studies in mammals, where more binding was seen at non-canonical positions. The most highly occupied transcripts in Drosophila have increased tendency to be maternally localized, and are more likely to derive from genes involved in differentiation or development. Taken together, we identify the RNA modalities that specify EJC assembly in Drosophila on a biologically coherent set of transcripts.

biochemistry↗

bioRxiv2018

All four double-stranded RNA binding domains of Staufen2 contribute to efficient mRNA recognition and transcript localization

Throughout metazoans, Staufen (Stau) proteins are core factors of mRNA localization particles. They consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2) like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that only dsRBDs 3-4 are necessary and sufficient for mRNA binding. Here, we show that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient, all four dsRBDs recognize their target RNAs with high stability. Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization. In contrast, a rescue with mStau2 bearing RNA-binding mutations in dsRBD1-2 fails, confirming the physiological relevance of our findings. In summary, our data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau- family proteins of different species.

biochemistry↗

bioRxiv2018

Transposon silencing in the Drosophila female germline ensures genome stability in progeny embryos

The piRNA pathway functions in transposon control in the germ line of metazoans. The conserved RNA helicase Vasa is an essential piRNA pathway component, but has additional important developmental functions. Here we address the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. We find that transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. We show that elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. We find that suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. Our findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development.

developmental biology↗

bioRxiv2017

Enzymatic production of single molecule FISH and RNA capture probes

Arrays of singly-labelled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single molecule microscopy analysis and high efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that 1) all steps of the oligonucleotide labelling - including conjugation, enzymatic synthesis and product purification - can be performed in a standard biology laboratory, 2) the process yields >90 %, often >95 % labeled product with minimal carry-over of impurities and 3) the oligonucleotides can be labeled with different dyes or biotin, allowing single molecule FISH or RNA affinity purification to be performed.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC=\"FIGDIR/small/107599_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@a26162org.highwire.dtl.DTLVardef@a780d9org.highwire.dtl.DTLVardef@fc3a62org.highwire.dtl.DTLVardef@15d183c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗