Tropomyosin 1-I/C coordinates 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 the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. We show through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance 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 competition with dynein until kinesin-1 is activated in the oocyte. Our work reveals a new strategy for ensuring sequential activity of microtubule motors.

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 processing bodies (P-bodies) and oogenesis arrest. Here, we show that hyperacetylation of α-tubulin at lysine 40 (K40) alters microtubule dynamics and P-body formation. We found that depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress response, causing α-tubulin hyperacetylation and accumulation of maternally deposited mRNPs in P-bodies. Through in vitro and in vivo studies, we identify HDAC1 as a direct regulator of α-tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of α-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 α-tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate α-tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.

An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA 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 interplay between 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.

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-encoding 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.

High-Resolution Live Imaging of Axonal RNP Granules in Drosophila Pupal Brain Explants.

Dynamic and local adjustments of the axonal proteome are observed in response to extracellular cues and achieved via translation of axonally localized mRNAs. To be localized, these mRNAs must be recognized by RNA binding proteins and packaged into higher-order ribonucleoprotein (RNP) granules transported along axonal microtubules via molecular motors. Axonal recruitment of RNP granules is not constitutive, but rather regulated by external signals such as developmental cues, through pathways yet to be identified. The Drosophila brain represents an excellent model system where to study the transport of RNP granules as it is triggered in specific populations of neurons undergoing remodeling during metamorphosis. Here, we describe a protocol enabling live imaging of axonal RNP granule transport with high spatiotemporal resolution in Drosophila maturing brains. In this protocol, pupal brains expressing endogenous or ectopic fluorescent RNP components are dissected, mounted in a customized imaging chamber, and imaged with an inverted confocal microscope equipped with sensitive detectors. Axonal RNP granules are then individually tracked for further analysis of their trajectories. This protocol is rapid (less than 1 hour to prepare brains for imaging) and is easy to handle and to implement.

Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development.

Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in 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 client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) 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 the localization and expression of a maternal RNA that instructs germline formation.

Live-Imaging of Axonal Cargoes in Drosophila Brain Explants Using Confocal Microscopy.

Live-imaging of axonal cargoes within central nervous system has been a long-lasting interest for neurobiologists as axonal transport plays critical roles in neuronal growth, function, and survival. Many kinds of cargoes are transported within axons, including synaptic vesicles and a variety of membrane-bound and membrane-less organelles. Imaging these cargoes at high spatial and temporal resolution, and within living brains, is technically very challenging. Here, we describe a quantitative method, based on customized mounting chambers, allowing live-imaging of axonal cargoes transported within the maturing brain of the fruit fly, Drosophila melanogaster. With this method, we could visualize in real time, using confocal microscopy, cargoes transported along axons. Our protocol is simple and easy to set up, as brains are mounted in our imaging chambers and ready to be imaged in about 1 h. Another advantage of our method is that it can be combined with pharmacological treatments or super-resolution microscopy.

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 used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed 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 EB1's 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 nonspecific, 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.

High-precision targeting workflow for volume electron microscopy.

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse 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.

Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex.

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.

Transcript specific mRNP capture from Drosophila egg-chambers for proteomic analysis.

mRNA binding proteins (RBPs) play a major role in post-transcriptional control of gene expression. To understand the complex regulatory processes regulating a specific mRNA during its life-time, a comprehensive view of the bound RBPs is essential. Here, we describe a method for transcript-specific isolation of endogenous ribonucleoprotein complexes (RNPs) from Drosophila egg-chambers. The method, which is based on in-solution hybridization of short biotinylated antisense DNA oligonucleotide probes to multiple segments of a transcript of interest allows unbiased identification of associated proteins by quantitative proteomics.

Nuclear Pores Assemble from Nucleoporin Condensates During Oogenesis.

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

Germ Cell Lineage Homeostasis in Drosophila Requires the Vasa RNA Helicase.

The conserved RNA helicase Vasa is required for germ cell development in many organisms. In Drosophila melanogaster loss of PIWI-interacting RNA pathway components, including Vasa, causes Chk2-dependent oogenesis arrest. However, whether the arrest is due to Chk2 signaling at a specific stage and whether continuous Chk2 signaling is required for the arrest is unknown. Here, we show that absence of Vasa during the germarial stages causes Chk2-dependent oogenesis arrest. Additionally, we report the age-dependent decline of the ovariole number both in flies lacking Vasa expression only in the germarium and in loss-of-function vasa mutant flies. We show that Chk2 activation exclusively in the germarium is sufficient to interrupt oogenesis and to reduce ovariole number in aging flies. Once induced in the germarium, Chk2-mediated arrest of germ cell development cannot be overcome by restoration of Vasa or by downregulation of Chk2 in the arrested egg chambers. These findings, together with the identity of Vasa-associated proteins identified in this study, demonstrate an essential role of the helicase in the germ cell lineage maintenance and indicate a function of Vasa in germline stem cell homeostasis.

The Transcriptome-wide Landscape and Modalities of EJC Binding in Adult Drosophila.

Exon junction complex (EJC) assembles after splicing at specific positions upstream of exon-exon junctions in mRNAs of all higher eukaryotes, affecting major regulatory events. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila, EJC is essential for localization of oskar mRNA. Here we developed a method for isolation of protein complexes and associated RNA targets (ipaRt) to explore the EJC RNA-binding landscape in a transcriptome-wide manner in adult Drosophila. We find the EJC at canonical positions, preferably on mRNAs from genes comprising multiple splice sites and long introns. Moreover, EJC occupancy is highest at junctions adjacent to strong splice sites, CG-rich hexamers, and RNA structures. Highly occupied mRNAs tend to be maternally localized and derive from genes involved in differentiation or development. These modalities, which have not been reported in mammals, specify EJC assembly on a biologically coherent set of transcripts in Drosophila.

Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains.

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.

Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos.

The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA 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.

Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization.

Fluorogenic oligonucleotide probes facilitate the detection and localization of RNA targets within cells. However, quantitative measurements of mRNA abundance are difficult when fluorescence signaling is based on intensity changes because a high concentration of unbound probes cannot be distinguished from a low concentration of target-bound probes. Here, we introduce qFIT (quantitative forced intercalation) probes that allow the detection both of probe-target complexes and of unbound probes on separate, independent channels. A surrogate nucleobase based on thiazole orange (TO) probes the hybridization status. The second channel involves a nonresponsive near-IR dye, which serves as a reporter of concentration. We show that the undesirable perturbation of the hybridization reporter TO is avoided when the near-IR dye Cy7 is connected by means of short triazole linkages in an ≥18 nucleotides distance. We used the qFIT probes to localize and quantify oskar mRNA in fixed egg chambers of wild-type and mutant Drosophila melanogaster by wash-free fluorescence in situ hybridization. The measurements revealed a relative 400-fold enrichment of oskar within a 3000 µm3 large volume at the posterior pole of stage 8-9 oocytes, which peaked at a remarkably high 1.8 µM local concentration inside 0.075 µm3 volume units. We discuss detection limits and show that the number of oskar mRNA molecules per oocyte is independent of the oocyte size, which suggests that the final levels are attained already during the onset of oskar localization at stage 8.

Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture.

Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.

In Vivo Visualization and Function Probing of Transport mRNPs Using Injected FIT Probes.

Fluorogenic hybridization methods, such as the use of FIT probes, enable the in vivo detection of specific mRNAs transcribed from their endogenous, genetically nonmodified loci. Here, we describe the design, synthesis and injection of nuclease resistant FIT probes into developing Drosophila oocytes to detect endogenous localizing mRNAs as wells as to probe function of structural RNA elements.