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Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). Here we show that orchestration ofoskarRNP transport in theDrosophilagermline 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 ofoskarmRNA by dynein bothin vitroandin vivo. Following delivery of nurse cell-synthesizedoskarmRNA 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 withstaufen(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 ofstaumRNA, and thus protein, in the oocyte enables motor switching onoskarRNPs by downregulating dynein activity.

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. InDrosophila, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliveroskarmRNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. Here, throughin vitroreconstitution, we show that Tm1-I/C, a Tropomyosin-1 isoform, links kinesin-1 in an inactive state to DDBE-associatedoskarmRNA. 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.

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.

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.

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.

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.

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.

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 ofoskarmRNA to the posterior pole of theDrosophilaoocyte is mediated byDrosophilakinesin-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 oneaTm1 chain, in antiparallel orientation. We show thataTm1 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 withaTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show thataTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishesaTm1 from classical motor adaptors.

SummaryAsymmetric localization ofoskarRNP granules to the oocyte posterior is crucial for abdominal patterning and germline formation of theDrosophilaembryo. We show thatoskarRNP granules in the oocyte are condensates with solid-like physical properties. Using purifiedoskarRNA and scaffold proteins Bruno and Hrp48, we confirmin vitrothatoskargranules 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 tooskarmRNA, allowed modulation of granule material propertiesin vivo. The resulting liquid-like properties impairedoskarlocalization 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.

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 inDrosophila melanogasterlarvae and ovarian follicular cells in adultDrosophila, 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.

Ronchi et al. present a workflow to facilitate precise targeting for volume EM (vEM) acquisitions. This method allows ultrastructural visualization of single cells within a millimeter-range specimen based on molecular identity characterized by fluorescence., 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.

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 μm

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved.

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.

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.

We are pleased to welcome several new JCB editorial board members.

Summary 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., Graphical Abstract, Highlights • Nucleoporins condense into precursor granules that mature into nuclear pore complexes (NPCs) • Microtubule-dependent transport and RanGTP promote NPC biogenesis in vivo • nup358 mRNA localizes to sites of NPC biogenesis in a translation-dependent manner • Nup358 protein condensation is Ran regulated and controls NPC assembly beyond nuclei, Nuclear pores form through progressive condensation events during oogenesis

Suppression of transposons by the Piwi-interacting RNA biogenesis factor Vasa in the supporting nurse cells is essential to prevent their accumulation in the developing oocyte, ensuring proper Drosophila embryonic development., 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.

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., The Staufen family of RNA-binding proteins are conserved microtubule dependent mRNA transporter factors. Here the authors use biochemical and functional approaches to characterize the RNA-binding properties of mouse Staufen 2 and study the mRNA binding capacity of its two domains dsRBDs 1 and 2.

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 theDrosophilafemale germline and early embryos. We find that transient loss ofvasaexpression 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 invasamutant 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.

The simultaneous imaging of different RNA molecules in homogeneous solution is a challenge and requires optimisation to enable unambiguous staining of intracellular RNA targets. Our approach relies on single dye forced intercalation (FIT) probes, in which a visco-sensitive reporter of the thiazole orange (TO) family serves as a surrogate nucleobase and provides enhancements of fluorescence upon hybridisation. Previous FIT probes spanned the cyan and green emission range. Herein, we report for the first time chromophores for FIT probes that emit in the red range (above 600 nm). Such probes are valuable to overcome cellular auto-fluorescent background and enable multiplexed detection. In order to find suitable chromophores, we developed a submonomer approach that facilitated the rapid analysis of different TO family dyes in varied sequence positions. A carboxymethylated 4,4′-methine linked cyanine, which we named quinoline blue (QB), provided exceptional response characteristics at the 605 nm emission maximum. Exceeding previously reported base surrogates, the emission of the QB nucleotide intensified by up to 195-fold upon binding of complementary RNA. Owing to large extinction coefficients and quantum yields (up to e = 129.000 L mol−1 cm−1 and Φ = 0.47, respectively) QB-FIT probes enable imaging of intracellular mRNA. A mixture of BO-, TO- and QB-containing FIT probes allowed the simultaneous detection of three different RNA targets in homogenous solution. TO- and QB-FIT probes were used to localize oskar mRNA and other polyadenylated mRNA molecules in developing oocytes from Drosphila melanogaster by means of wash-free fluorescent in situ hybridisation and super resolution microscopy (STED).

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.

Gene expression is a fundamental process that underlies development, homeostasis, and behavior of organisms. The fact that it relies on nucleic acid intermediates, which can specifically interact with complementary probes, provides an excellent opportunity for studying the multiple steps—transcription, RNA processing, transport, translation, degradation, and so forth—through which gene function manifests. Over the past three decades, the toolbox of nucleic acid science has expanded tremendously, making high-precision in situ detection of DNA and RNA possible. This has revealed that many—probably the vast majority of—transcripts are distributed within the cytoplasm or the nucleus in a nonrandom fashion. With the development of microscopy techniques we have learned not only about the qualitative localization of these molecules but also about their absolute numbers with great precision. Single-molecule techniques for nucleic acid detection have been transforming our views of biology with elementary power: cells are not average members of their population but are highly distinct individuals with greatly and suddenly changing gene expression, and this behavior of theirs can be measured, modeled, and thus predicted and, finally, comprehended. WIREs Dev Biol 2015, 4:135–150. doi: 10.1002/wdev.170 For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

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.

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.

Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.

When temperature fluctuation threatens the fidelity of Drosophila oogenesis, Klar restrains posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the capacity of the anchoring machinery., Communication usually applies feedback loop–based filters and amplifiers to ensure undistorted delivery of messages. Such an amplifier acts during Drosophila melanogaster midoogenesis, when oskar messenger ribonucleic acid (mRNA) anchoring depends on its own locally translated protein product. We find that the motor regulator Klar β mediates a gain-control process that prevents saturation-based distortions in this positive feedback loop. We demonstrate that, like oskar mRNA, Klar β localizes to the posterior pole of oocytes in a kinesin-1–dependent manner. By live imaging and semiquantitative fluorescent in situ hybridization, we show that Klar β restrains oskar ribonucleoprotein motility and decreases the posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the output of the anchoring machinery. This negative regulatory effect of Klar is particularly important for overriding temperature-induced changes in motility. We conclude that by preventing defects in oskar anchoring, this mechanism contributes to the developmental robustness of a poikilothermic organism living in a variable temperature environment.

International audience; : Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues [1-4]. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) γ neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches [5]. Here, we identify the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting [6-12]. We show that whereas Imp is dispensable for the initial growth of MB γ neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, we demonstrate that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. Our study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation.

mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem–loop in the oskar 3′ UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.

Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL mol(-1) cm(-1) at λex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification.

Arrays of singly labeled 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 (i) all steps of the oligonucleotide labeling—including conjugation, enzymatic synthesis, and product purification—can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.

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.Graphical abstract

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport‐competent mRNPs. We show that the posterior‐targeting kinesin‐1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein‐dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin‐1 recruitment requires the DmTropomyosin1‐I/C isoform, an atypical RNA‐binding tropomyosin that binds directly to dimerizing oskar 3′UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin‐1 and that the motor is activated during mid‐oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo‐dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo‐transport processes and between the cargo‐associated dynein and kinesin‐1.