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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.

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.

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.

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.

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

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.

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.

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.

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.

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.

RNAs are known to regulate diverse biological processes, either as protein-encoding molecules or as non-coding RNAs. However, a third class that comprises RNAs endowed with both protein coding and non-coding functions has recently emerged. Such bi-functional ‘coding and non-coding RNAs’ (cncRNAs) have been shown to play important roles in distinct developmental processes in plants and animals. Here, we discuss key examples of cncRNAs and review their roles, regulation and mechanisms of action during development.

Initially assumed to be a special feature of highly polarized eukaryotic cells, recent evidence suggests that mRNA localization coupled with local translation is a widespread strategy for spatial restriction of protein synthesis within cells. Genome-wide analyses and live imaging approaches have shed new light on the prevalence and the mechanistic details of this phenomenon. Here we review some of the recent findings that have emerged from research from the RNA localization field, from the birth of mRNAs in the nucleus, to their delivery at specific sites within the cytoplasm.

oskar mRNA localization at the oocyte posterior pole is essential for correct patterning of the Drosophila embryo. Here we show at the ultrastructural level that endogenous oskar ribonucleoprotein complexes (RNPs) assemble sequentially with initial recruitment of Hrp48 and the exon junction complex (EJC) to oskar transcripts in the nurse cell nuclei, and subsequent recruitment of Staufen and microtubule motors, following transport to the cytoplasm. oskar particles are non-membrane-bound structures that coalesce as they move from the oocyte anterior to the posterior pole. Our analysis uncovers a role for the EJC component Barentsz in recruiting Tropomyosin II (TmII) to oskar particles in the ooplasm and reveals that TmII is required for kinesin binding to the RNPs. Finally, we show that both kinesin and dynein associate with oskar particles and are the primary microtubule motors responsible for transport of the RNPs within the oocyte.

The localization of mRNAs to subcellular compartments provides a mechanism for regulating gene expression with exquisite temporal and spatial control and recent studies suggest that a large fraction of mRNAs localize to distinct cytoplasmic domains. In this review, we focus on cis-acting RNA localization elements, RNA-binding proteins, and the assembly of mRNAs into granules that are transported by molecular motors along cytoskeletal elements to their final destination in the cell.

SummaryEnsconsin is a conserved microtubule-associated protein (MAP) that interacts dynamically with microtubules, but its cellular function has remained elusive. We show that Drosophila ensconsin is required for all known kinesin-1-dependent processes in the polarized oocyte without detectable effects on microtubules. ensconsin is also required in neurons. Using a single molecule assay for kinesin-1 motility in Drosophila ovary extract, we show that recruitment to microtubules and subsequent motility is severely impaired without ensconsin. Ensconsin protein is enriched at the oocyte anterior and apically in polarized epithelial cells, although required for localization of posterior determinants. Par-1 is required for ensconsin localization and directly phosphorylates it at conserved sites. Our results reveal an unexpected function of a MAP, promoting productive recruitment of a specific motor to microtubules, and an additional level of kinesin regulation. Furthermore, spatial control of motor recruitment can provide additional regulatory control in Par-1 and microtubule-dependent cell polarity.

The Drosophila melanogaster body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. Here, we investigate the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. We show that exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. We show that, in vivo, Rab6 is found in a complex with Bicaudal-D, and that Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila.

The Drosophila oskar (osk) mRNA is unusual in that it has both coding and noncoding functions. As an mRNA, osk encodes a protein required for embryonic patterning and germ cell formation. Independent of that function, the absence of osk mRNA disrupts formation of the karyosome and blocks progression through oogenesis. Here we show that loss of osk mRNA also affects the distribution of regulatory proteins, relaxing their association with large RNPs within the germline, and allowing them to accumulate in the somatic follicle cells. This and other noncoding functions of the osk mRNA are mediated by multiple sequence elements with distinct roles. One role, provided by numerous binding sites in two distinct regions of the osk 3′ UTR, is to sequester the translational regulator Bruno (Bru), which itself controls translation of osk mRNA. This defines a novel regulatory circuit, with Bru restricting the activity of osk, and osk in turn restricting the activity of Bru. Other functional elements, which do not bind Bru and are positioned close to the 3′ end of the RNA, act in the oocyte and are essential. Despite the different roles played by the different types of elements contributing to RNA function, mutation of any leads to accumulation of the germline regulatory factors in the follicle cells.

International audience; Axonal transport is essential for the initial growth, maintenance and synaptic plasticity of axons, and altered axonal transport has been observed in different models of neurodegenerative pathologies. Dissecting the mechanisms underlying axonal transport in developing or degenerating brains requires dynamic imaging of axonal cargo movement in living samples. Whereas methods exist to image axonal transport in Drosophila larval neurons, they are not suitable to follow this process during metamorphosis, when brains undergo extensive remodeling. Here we present a simple method that enables confocal imaging of both fast and slow axonal transport in Drosophila pupal brain explants. We describe how to prepare chambers adapted for live imaging, how to maintain brain explants under physiological conditions and how to monitor and quantitatively analyze the movement of fluorescently labeled cargoes. This protocol requires minimal equipment and is ideally suited for experiments that combine genetics, optogenetics and pharmacological approaches. The brains can be prepared for image acquisition in 1.5 h, and the protocol can be performed easily in any fly laboratory.

Measuring translation in space and time The ribosome translates the information contained within messenger RNAs (mRNAs) into proteins. When and where ribosomes encounter mRNAs can regulate gene expression. Halstead et al. developed an RNA biosensor that allows single molecules of mRNAs that have never been translated to be distinguished from ones that have undergone translation by the ribosome in living cells (see the Perspective by Popp and Maquat). The authors demonstrated the utility of their technique by examining the spatial and temporal regulation of translation in single cells and in Drosophila oocytes during development. Science , this issue p. 1367 ; see also p. 1316

The Drosophila maternal effect gene oskar encodes the posterior determinant responsible for the formation of the posterior pole plasm in the egg, and thus of the abdomen and germline of the future fly. Previously identified oskar mutants give rise to offspring that lack both abdominal segments and a germline, thus defining the `posterior group phenotype'. Common to these classical oskar alleles is that they all produce significant amounts of oskar mRNA. By contrast, two new oskar mutants in which oskar RNA levels are strongly reduced or undetectable are sterile, because of an early arrest of oogenesis. This egg-less phenotype is complemented by oskar nonsense mutant alleles,as well as by oskar transgenes, the protein-coding capacities of which have been annulled. Moreover, we show that expression of the oskar 3′ untranslated region (3′UTR) is sufficient to rescue the egg-less defect of the RNA null mutant. Our analysis thus reveals an unexpected role for oskar RNA during early oogenesis, independent of Oskar protein. These findings indicate that oskar RNA acts as a scaffold or regulatory RNA essential for development of the oocyte.

Establishment of the Drosophila embryonic axes provides a striking example of RNA localization as an efficient mechanism for protein targeting within a cell. oskar mRNA encodes the posterior determinant and is essential for germline and abdominal development in the embryo. Tight restriction of Oskar activity to the posterior is achieved by mRNA localization-dependent translational control, whereby unlocalized mRNA is translationally repressed and repression is overcome upon mRNA localization. Here we identify the previously reported oskar RNA binding protein p50 as Hrp48, an abundant Drosophila hnRNP. Analysis of three hrp48 mutant alleles reveals that Hrp48 levels are crucial for polarization of the oocyte during mid-oogenesis. Our data also show that Hrp48, which binds to the 5' and 3' regions of oskar mRNA, plays an important role in restricting Oskar activity to the posterior of the oocyte, by repressing oskar mRNA translation during transport.

Targeting proteins to specific domains within the cell is central to the generation of polarity, which underlies many processes including cell fate specification and pattern formation during development. The anteroposterior and dorsoventral axes of the Drosophila melanogaster embryo are determined by the activities of localized maternal gene products. At the posterior pole of the oocyte, Oskar directs the assembly of the pole plasm,and is thus responsible for formation of abdomen and germline in the embryo. Tight restriction of oskar activity is achieved by mRNA localization,localization-dependent translation, anchoring of the RNA and protein, and stabilization of Oskar at the posterior pole. Here we report that the type 1 regulatory subunit of cAMP-dependent protein kinase (Pka-R1)is crucial for the restriction of Oskar protein to the oocyte posterior. Mutations in PKA-R1 cause premature and ectopic accumulation of Oskar protein throughout the oocyte. This phenotype is due to misregulation of PKA catalytic subunit activity and is suppressed by reducing catalytic subunit gene dosage. These data demonstrate that PKA mediates the spatial restriction of Oskar for anteroposterior patterning of the Drosophila embryo and that control of PKA activity by PKA-R1 is crucial in this process.

Many cells store neutral lipids, as triacylglycerol and sterol esters, in droplets. PAT-domain proteins form a conserved family of proteins that are localized at the surface of neutral lipid droplets. Two mammalian members of this family, Perilipin and adipose differentiation-related protein, are involved in lipid storage and regulate lipolysis. Here, we describe the Drosophila PAT-family member Lsd2. We showed that Lsd2 is predominantly expressed in tissues engaged in high levels of lipid metabolism, the fat body and the germ line of females. Ultrastructural analysis in the germ line showed that Lsd2 localizes to the surface of lipid droplets. We have generated an Lsd2 mutant and described its phenotype. Mutant adults have a reduced level of neutral lipid content compared to wild type, showing that Lsd2 is required for normal lipid storage. In addition, ovaries from Lsd2 mutant females exhibit an abnormal pattern of accumulation of neutral lipids from mid-oogenesis, which results in reduced deposition of lipids in the egg. Consistent with its expression in the female germ line, we showed that Lsd2 is a maternal effect gene that is required for normal embryogenesis. This work demonstrates that Lsd2 has an evolutionarily conserved function in lipid metabolism and establishes Drosophila melanogaster as a new in vivo model for studies on the PAT-family of proteins.

Translational regulation of localized transcripts is a powerful mechanism to control the precise timing and localization of protein expression within a cell. In the Drosophila germline, oskar transcript must be translationally repressed until its localization at the posterior pole of the oocyte, as ectopic production of Oskar causes severe patterning defects. Translational repression of oskar mRNA is mediated by the RNA-binding protein Bruno, which binds to specific motifs in the oskar 3'UTR. Here we show that Bruno over-expression causes defects in antero-posterior and dorso-ventral patterning, consistent with a role of Bruno in both oskar and gurken mRNA regulation. We also show that Bruno and gurken interact genetically. Finally, we show that Bruno binds specifically to the gurken 3'UTR and that the dorso-ventral defects caused by Bruno over-expression are due to a reduction of Gurken levels in the oocyte. We conclude that Bruno plays similar roles in translational regulation of gurken and oskar.

mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem–loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.

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.

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome. DOI: http://dx.doi.org/10.7554/eLife.03058.001

In eukaryotes, RNA processing events in the nucleus influence the fate of transcripts in the cytoplasm. The multi-protein exon junction complex (EJC) associates with mRNAs concomitant with splicing in the nucleus and plays important roles in export, translation, surveillance and localization of mRNAs in the cytoplasm. In mammalian cells, the ribosome associated protein PYM (HsPYM) binds the Y14-Mago heterodimer moiety of the EJC core, and disassembles EJCs, presumably during the pioneer round of translation. However, the significance of the association of the EJC with mRNAs in a physiological context has not been tested and the function of PYM in vivo remains unknown. Here we address PYM function in Drosophila, where the EJC core proteins are genetically required for oskar mRNA localization during oogenesis. We provide evidence that the EJC binds oskar mRNA in vivo. Using an in vivo transgenic approach, we show that elevated amounts of the Drosophila PYM (DmPYM) N-terminus during oogenesis cause dissociation of EJCs from oskar RNA, resulting in its mislocalization and consequent female sterility. We find that, in contrast to HsPYM, DmPYM does not interact with the small ribosomal subunit and dismantles EJCs in a translation-independent manner upon over-expression. Biochemical analysis shows that formation of the PYM-Y14-Mago ternary complex is modulated by the PYM C-terminus revealing that DmPYM function is regulated in vivo. Furthermore, we find that whereas under normal conditions DmPYM is dispensable, its loss of function is lethal to flies with reduced y14 or mago gene dosage. Our analysis demonstrates that the amount of DmPYM relative to the EJC proteins is critical for viability and fertility. This, together with the fact that the EJC-disassembly activity of DmPYM is regulated, implicates PYM as an effector of EJC homeostasis in vivo., Author Summary The multi-protein exon junction complex (EJC) is deposited at exon-exon junctions on mRNAs upon splicing. EJCs, with Y14, Mago, eIF4AIII and Barentsz proteins at their core, are landmarks of the nuclear history of RNAs and play important roles in their post-transcriptional regulation. In mammalian cells, the Y14-Mago interacting protein PYM associates with ribosomes and disassembles EJCs in the cytoplasm. However, the physiological function of PYM and its regulation in vivo remains unknown. We have analysed PYM function during Drosophila oogenesis, where the EJC is essential for oskar mRNA localization in the oocyte, a prerequisite for embryonic patterning and germline formation. We find that Drosophila PYM interacts with Y14-Mago but, in contrast to mammalian PYM, does not bind ribosomes. We demonstrate that EJCs associated with oskar mRNA in vivo are disassembled by PYM over-expression in a translation-independent manner, causing oskar mislocalization. Our in vivo analysis shows that the Drosophila PYM C-terminal domain modulates PYM-Y14-Mago interaction, revealing that PYM is regulated in Drosophila. Furthermore, PYM is essential for viability of flies lacking one functional copy of y14 or mago, supporting a role of PYM in EJC homeostasis. Our results highlight a distinct mode of regulation of the EJC-dissociating protein PYM in Drosophila.

Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe-target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.

Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5′ sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons. Refereed/Peer-reviewed

Background: mRNA localization is a powerful and widely employed mechanism for generating cell asymmetry. In Drosophila , localization of mRNAs in the oocyte determines the axes of the future embryo. oskar mRNA localization at the posterior pole is essential and sufficient for the specification of the germline and the abdomen. Its posterior transport along the microtubules is mediated by Kinesin I and several proteins, such as Mago-nashi, which, together with oskar mRNA, form a posterior localization complex. It was recently shown that human Y14, a nuclear protein that associates with mRNAs upon splicing and shuttles to the cytoplasm, interacts with MAGOH, the human homolog of Mago-nashi. Results: Here, we show that Drosophila Y14 interacts with Mago-nashi in vivo. Immunohistochemistry reveals that Y14 is predominantly nuclear and colocalizes with oskar mRNA at the posterior pole. We show that, in y14 mutant oocytes, oskar mRNA localization to the posterior pole is specifically affected, while the cytoskeleton appears to be intact. Conclusions: Our findings indicate that Y14 is part of the oskar mRNA localization complex and that the nuclear shuttling protein Y14 has a specific and direct role in oskar mRNA cytoplasmic localization.

Background: The oskar (osk) gene encodes a determinant of posterior identity in Drosophila , and the localization of osk RNA to the pole plasm at the posterior pole of the oocyte is essential for development of the embryo. The mechanisms by which osk RNA is localized are unknown. Results: To study the mechanisms underlying localization of osk RNA, we have injected fluorescently labelled RNA into oocytes at stages 9, 10 and 11. Injected osk RNA localizes to the pole plasm, reproducing localization of the endogenous RNA. In oocytes at stages 10 and 11, the long-range movement of injected osk RNA is promoted by a vigorous, microtubule-dependent cytoplasmic flow, or ooplasmic streaming. Treatment with colchicine, a microtubule-destabilizing drug, inhibits ooplasmic streaming and prevents localization of the RNA from an injection site distal to the posterior pole. If the RNA is injected close to the posterior pole, however, it localizes even in the presence of colchicine. Similarly, in small oocytes, such as stage 9 oocytes, localization of injected osk RNA is insensitive to colchicine. Conclusions: These results reveal that microtubule-dependent cytoplasmic flows could contribute to the long-range transport of osk RNA, whereas microtubule-independent processes could mediate short-range transport. These results also highlight the role of the osk RNA anchor in the localization process.

The posterior pole plasm of the Drosophila egg contains the determinants of abdominal and germ-cell fates of the embryo. Pole plasm assembly is induced by oskar RNA localized to the posterior pole of the oocyte. Genetics has revealed three additional genes, staufen, vasa, and tudor, that are also essential for pole plasm formation. Staufen protein is required for both oskar RNA localization and translation. Vasa and Tudor are localized dependent on Oskar protein and are required to accumulate Oskar protein stably at the posterior pole. We have explored interactions between these gene products at the molecular level and find that Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in Oskar that abolish pole plasm formation in vivo. Finally, we show that in the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. These results suggest that the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. In addition, we discuss the possible biological role of the Oskar-Staufen interaction.

A novel protein required for RNA interference in Drosophila, Armitage, was identified in a screen for genes involved in embryonic axis formation. In armitage mutants, oocyte polarity and the regulation of oskar mRNA translation are impaired, suggesting that RNA silencing regulates the first steps of Drosophila development.

A cell-free system using cytoplasmic extract from single–Drosophila syncytial embryos reveals that the spacing between nuclei is preprogrammed and relies on microtubule aster migration., In the early embryo of many species, comparatively small spindles are positioned near the cell center for subsequent cytokinesis. In most insects, however, rapid nuclear divisions occur in the absence of cytokinesis, and nuclei distribute rapidly throughout the large syncytial embryo. Even distribution and anchoring of nuclei at the embryo cortex are crucial for cellularization of the blastoderm embryo. The principles underlying nuclear dispersal in a syncytium are unclear. We established a cell-free system from individual Drosophila melanogaster embryos that supports successive nuclear division cycles with native characteristics. This allowed us to investigate nuclear separation in predefined volumes. Encapsulating nuclei in microchambers revealed that the early cytoplasm is programmed to separate nuclei a distinct distance. Laser microsurgery revealed an important role of microtubule aster migration through cytoplasmic space, which depended on F-actin and cooperated with anaphase spindle elongation. These activities define a characteristic separation length scale that appears to be a conserved property of developing insect embryos.

oskar RNA localization to the posterior pole of the Drosophila melanogaster oocyte requires splicing of the first intron and the exon junction complex (EJC) core proteins. The functional link between splicing, EJC deposition and oskar localization has been unclear. Here we demonstrate that the EJC associates with oskar mRNA upon splicing in vitro and that Drosophila EJC deposition is constitutive and conserved. Our in vivo analysis reveals that splicing creates the spliced oskar localization element (SOLE), whose structural integrity is crucial for ribonucleoprotein motility and localization in the oocyte. Splicing thus has a dual role in oskar mRNA localization: assembling the SOLE and depositing the EJC required for mRNA transport. The SOLE complements the EJC in formation of a functional unit that, together with the oskar 3' UTR, maintains proper kinesin-based motility of oskar mRNPs and posterior mRNA targeting.

mRNA localization coupled with translational control is a highly conserved and widespread mechanism for restricting protein expression to specific sites within eukaryotic cells. In Drosophila, patterning of the embryo requires oskar mRNA transport to the posterior pole of the oocyte and translational repression prior to localization. oskar RNA splicing and the 3′ untranslated region (UTR) are required for posterior enrichment of the mRNA. However, reporter RNAs harboring the oskar 3′ UTR can localize by hitchhiking with endogenous oskar transcripts. Here we show that the oskar 3′ UTR contains a stem–loop structure that promotes RNA dimerization in vitro and hitchhiking in vivo. Mutations in the loop that abolish in vitro dimerization interfere with reporter RNA localization, and restoring loop complementarity restores hitchhiking. Our analysis provides insight into the molecular basis of RNA hitchhiking, whereby localization-incompetent RNA molecules can become locally enriched in the cytoplasm, by virtue of their association with transport-competent RNAs.

mRNA localization coupled with translational control is a widespread and conserved strategy that allows the localized production of proteins within eukaryotic cells. In Drosophila, oskar (osk) mRNA localization and translation at the posterior pole of the oocyte are essential for proper patterning of the embryo. Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs. In cultured mammalian cells, Ge-1 protein is required for P body formation. Combining genetic, biochemical and immunohistochemical approaches, we show that, in vivo, Drosophila Ge-1 (dGe-1) is an essential gene encoding a P body component that promotes formation of these structures in the germline. dGe-1 partially colocalizes with osk mRNA and is required for osk RNP integrity. Our analysis reveals that although under normal conditions dGe-1 function is not essential for osk mRNA localization, it becomes critical when other components of the localization machinery, such as staufen, Drosophila decapping protein 1 and barentsz are limiting. Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.

Local translation of asymmetrically enriched mRNAs is a powerful mechanism for functional polarization of the cell. In Drosophila, exclusive accumulation of Oskar protein at the posterior pole of the oocyte is essential for development of the future embryo. This is achieved by the formation of a dynamic oskar ribonucleoprotein (RNP) complex regulating the transport of oskar mRNA, its translational repression while unlocalized, and its translational activation upon arrival at the posterior pole. We identified the nucleo–cytoplasmic shuttling protein PTB (polypyrimidine tract-binding protein)/hnRNP I as a new factor associating with the oskar RNP in vivo. While PTB function is largely dispensable for oskar mRNA transport, it is necessary for translational repression of the localizing mRNA. Unexpectedly, a cytoplasmic form of PTB can associate with oskar mRNA and repress its translation, suggesting that nuclear recruitment of PTB to oskar complexes is not required for its regulatory function. Furthermore, PTB binds directly to multiple sites along the oskar 3′ untranslated region and mediates assembly of high-order complexes containing multiple oskar RNA molecules in vivo. Thus, PTB is a key structural component of oskar RNP complexes that dually controls formation of high-order RNP particles and translational silencing.

During Drosophila oogenesis, oskar mRNA is transported to the posterior pole of the oocyte, where it is locally translated and induces germ-plasm assembly. Oskar protein recruits all of the components necessary for the establishment of posterior embryonic structures and of the germline. Tight localization of Oskar is essential, as its ectopic expression causes severe patterning defects. Here, we show that the Drosophila homolog of mammalian Lasp1 protein, an actin-binding protein previously implicated in cell migration in vertebrate cell culture, contributes to the accumulation of Oskar protein at the posterior pole of the embryo. The reduced number of primordial germ cells in embryos derived from lasp mutant females can be rescued only with a form of Lasp that is capable of interacting with Oskar,revealing the physiological importance of the Lasp-Oskar interaction.

Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine methyltransferases(PRMTs) in specific biological processes remain to be clarified. In this study we characterize the Drosophila PRMT Capsuléen, homologous to human PRMT5. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In particular, we show that the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are abolished in csul mutant germ cells. We identify the spliceosomal Sm proteins as in vivo substrates of Capsuléen and demonstrate that Capsuléen,together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues in Sm proteins. Finally, we show that Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating that Tudor localization and Sm methylation are separate processes. Our results thus reveal the role of a PRMT in protein localization in germ cells.

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation., Synopsis Muscle pattern formation during embryogenesis requires the activity of a distinct network of genes. In the model organism Drosophila, this process involves the determination of stem-cell-like muscle founder cells, their differentiation, and their attraction to tendon-like epidermal cells, termed apodemes, to which the muscles attach. In order to systematically identify genes involved in these processes, a collection of fruit fly strains was generated that can be used for the ectopic expression of more than 3,700 individual fruit fly genes in a spatiotemporally restricted manner. In order to address muscle pattern formation, the collection was used to express the genes in the developing apodemes and in a series of distinct epidermal cells that serve as migration substrate for developing muscles towards the apodemes. In addition to already known factors, some 60 novel gene activities were found to interfere under these circumstances with the formation of the muscle pattern. In addition to providing a most valuable tool for the Drosophila community of researchers, the results provide a framework for a detailed analysis of the gene network and insight into molecular mechanisms underlying embryonic muscle pattern formation.