Oskar

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Anne Ephrussi - One of the best experts on this subject based on the ideXlab platform.

  • Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization
    2018
    Co-Authors: Imre Gaspar, Anne Ephrussi, Felix Hövelmann, Jasmine Chamiolo, Oliver Seitz
    Abstract:

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

  • The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage
    Genes & Development, 2017
    Co-Authors: Marcin Jeske, Christoph W. Müller, Anne Ephrussi
    Abstract:

    : 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.

  • An RNA-binding Atypical Tropomyosin Recruits kinesin-1 Dynamically to Oskar mRNPs
    The EMBO Journal, 2016
    Co-Authors: Imre Gaspar, Vasiliy O. Sysoev, Artem Komissarov, Anne Ephrussi
    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.

  • an rna binding tropomyosin recruits kinesin 1 dynamically to Oskar mrnps
    bioRxiv, 2016
    Co-Authors: Imre Gaspar, Artem Komissarov, Vasily Sysoev, Anne Ephrussi
    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 precise recruitment and regulation of motor proteins to form transport-competent mRNPs. Using high- and super-resolution imaging, we determine the steps in motor recruitment to Oskar mRNPs. We show that the posterior-targeting kinesin-1 is recruited upon nuclear export of Oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. We demonstrate that DmTropomyosin1-I/C is an atypical RNA-binding, nucleocytoplasmic shuttling Tropomyosin1 isoform that binds the Oskar 3′UTR through recognition of a supramolecular RNA motif created upon dimerization of Oskar molecules. Our data show that, in the oocyte, kinesin-1 is recruited by DmTropomyosin1-I/C to a dynamically changing, small subset of Oskar mRNPs and is activated by the functionalized spliced Oskar RNA localization element, revealing an ergonomic, coordinated mechanism of cargo transport.

  • The Crystal Structure of the Drosophila Germline Inducer Oskar Identifies Two Domains with Distinct Vasa Helicase- and RNA-Binding Activities
    Cell reports, 2015
    Co-Authors: Mandy Jeske, Christoph W. Müller, Matteo Bordi, Sebastian Glatt, Sandra Müller, Vladimir Rybin, Anne Ephrussi
    Abstract:

    In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. Here, we show that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, we present high-resolution crystal structures of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. Our findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function.

Paul M. Macdonald - One of the best experts on this subject based on the ideXlab platform.

  • knock down analysis reveals critical phases for specific Oskar noncoding rna functions during drosophila oogenesis
    G3: Genes Genomes Genetics, 2021
    Co-Authors: Andrew Kenny, Sabine Mohr, Miles B. Morgan, Paul M. Macdonald
    Abstract:

    The Oskar transcript, acting as a noncoding RNA, contributes to a diverse set of pathways in the Drosophila ovary, including karyosome formation, positioning of the microtubule organizing center, integrity of certain ribonucleoprotein particles, control of nurse cell divisions, restriction of several proteins to the germline, and progression through oogenesis. How Oskar mRNA acts to perform these functions remains unclear. Here we use a knock down approach to identify the critical phases when Oskar is required for three of these functions. The existing transgenic shRNA for removal of Oskar mRNA in the germline targets a sequence overlapping a regulatory site bound by Bruno1 protein to confer translational repression, and was ineffective during oogenesis. Novel transgenic shRNAs targeting other sites were effective at strongly reducing Oskar mRNA levels and reproducing phenotypes associated with the absence of the mRNA. Using GAL4 drivers active at different developmental stages of oogenesis, we found that early loss of Oskar mRNA reproduced defects in karyosome formation and positioning of the microtubule organizing center, but not arrest of oogenesis. Loss of Oskar mRNA at later stages was required to prevent progression through oogenesis. The noncoding function of Oskar mRNA is thus required for more than a single event.

  • Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of Oskar mRNA in the Drosophila oocyte
    PLOS Genetics, 2021
    Co-Authors: Sabine Mohr, Andrew Kenny, Simon T. Y. Lam, Miles B. Morgan, Craig A. Smibert, Howard D. Lipshitz, Paul M. Macdonald
    Abstract:

    Localization of Oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the Oskar 3’ UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the Oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of Oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the Oskar 3’ UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of Oskar mRNA.

  • Multiple cis-acting signals, some weak by necessity, collectively direct robust transport of Oskar mRNA to the oocyte
    Journal of cell science, 2017
    Co-Authors: Young Hee Ryu, Youme Gim, Mark J. Snee, Andrew Kenny, Paul M. Macdonald
    Abstract:

    ABSTRACT Localization of mRNAs can involve multiple steps, each with its own cis -acting localization signals and transport factors. How is the transition between different steps orchestrated? We show that the initial step in localization of Drosophila Oskar mRNA − transport from nurse cells to the oocyte − relies on multiple cis -acting signals. Some of these are binding sites for the translational control factor Bruno, suggesting that Bruno plays an additional role in mRNA transport. Although transport of Oskar mRNA is essential and robust, the localization activity of individual transport signals is weak. Notably, increasing the strength of individual transport signals, or adding a strong transport signal, disrupts the later stages of Oskar mRNA localization. We propose that the Oskar transport signals are weak by necessity; their weakness facilitates transfer of the Oskar mRNA from the oocyte transport machinery to the machinery for posterior localization.

  • BREs Mediate Both Repression and Activation of Oskar mRNA Translation and Act In trans
    Developmental cell, 2010
    Co-Authors: Brad Reveal, Nan Yan, Mark J. Snee, Chin I. Pai, Youme Gim, Paul M. Macdonald
    Abstract:

    Asymmetric positioning of proteins within cells is crucial for cell polarization and function. Deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of the Oskar mRNA, repression of its translation prior to localization, and finally activation of translation. Translational repression is mediated by BREs, regulatory elements positioned in two clusters near both ends of the Oskar mRNA 3' UTR. Here we show that some BREs are bifunctional: both clusters of BREs contribute to translational repression, and the 3' cluster has an additional role in release from BRE-dependent repression. Remarkably, both BRE functions can be provided in trans by an Oskar mRNA with wild-type BREs that is itself unable to encode Oskar protein. Regulation in trans is likely enabled by assembly of Oskar transcripts in cytoplasmic RNPs. Concentration of transcripts in such RNPs is common, and trans regulation of mRNAs may therefore be widespread.

  • Two distinct domains of Bruno bind specifically to the Oskar mRNA.
    RNA biology, 2008
    Co-Authors: Mark J. Snee, Dianne Benz, Judy Jen, Paul M. Macdonald
    Abstract:

    Selective deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of Oskar mRNA, combined with translational regulation to ensure that only the localized mRNA produces protein. The Bruno protein binds to Bruno Response Elements (BREs) in the Oskar mRNA, and prevents translation of unlocalized Oskar mRNA. Bruno contains three copies of the RNA Recognition Motif (RRM), a protein motif that often binds directly to RNA. Either of two nonoverlapping parts of Bruno--RRMs 1 and 2, and RRM 3 and 42 flanking amino acids--can bind specifically to BRE-containing RNA, but both domains are required for maximal binding. When expressed in Drosophila ovaries, Bruno proteins with a single RNA binding domain mutated have reduced repressive activity, while mutation of both binding domains largely eliminates this activity. Notably, the same proteins expressed as fusions to GFP accumulate in nuclei, with the most severe mislocalization occurring when both RNA binding domains are mutated. A similar mislocalization of endogenous Bruno occurs when mRNA export is blocked. Thus, Bruno shuttles between the nucleus and cytoplasm, and may first bind Oskar mRNA in the nucleus.

Daniel St Johnston - One of the best experts on this subject based on the ideXlab platform.

  • Localised Dynactin protects growing microtubules to deliver Oskar mRNA to the posterior cortex of the Drosophila oocyte
    2017
    Co-Authors: Ross Nieuwburg, Dmitry Nashchekin, Maximilian Ah Jakobs, Andrew P. Carter, Philipp Khuc Trong, Raymond E. Goldstein, Daniel St Johnston
    Abstract:

    The localisation of Oskar mRNA to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Kinesin 1 transports Oskar mRNA to the oocyte posterior along a polarised microtubule cytoskeleton that grows from non-centrosomal microtubule organising centres (ncMTOCs) along the anterior/lateral cortex. Here we show that the formation of this polarised microtubule network also requires the posterior regulation of microtubule growth. A mutation in the Dynactin Arp1 subunit causes most Oskar mRNA to localise in the posterior cytoplasm rather than cortically. Oskar mRNA transport and anchoring are normal in this mutant, but the microtubules fail to reach the posterior pole. Thus, Dynactin acts as an anti-catastrophe factor that extends microtubule growth posteriorly. Kinesin 1 transports Dynactin to the oocyte posterior, creating a positive feedback loop that increases the length and persistence of the posterior microtubules that deliver Oskar mRNA to the cortex.

  • Oskar Is Targeted for Degradation by the Sequential Action of Par-1, GSK-3, and the SCF-Slimb Ubiquitin Ligase
    Developmental cell, 2013
    Co-Authors: Eurico Morais-de-sá, Antonio Vega-rioja, Vítor Trovisco, Daniel St Johnston
    Abstract:

    Translation of Oskar messenger RNA (mRNA) is activated at the posterior of the Drosophila oocyte, producing Long Oskar, which anchors the RNA, and Short Oskar, which nucleates the pole plasm, containing the posterior and germline determinants. Here, we show that Oskar is phosphorylated by Par-1 and GSK-3/Shaggy to create a phosphodegron that recruits the SCF-Slimb ubiquitin ligase, which targets Short Oskar for degradation. Phosphorylation site mutations cause Oskar overaccumulation, leading to an increase in pole cell number and embryonic patterning defects. Furthermore, the nonphosphorylatable mutant produces bicaudal embryos when Oskar mRNA is mislocalized. Thus, the Par-1/GSK-3/Slimb pathway plays important roles in limiting the amount of pole plasm posteriorly and in degrading any mislocalized Oskar that results from leaky translational repression. These results reveal that Par-1 controls the timing of pole plasm assembly by promoting the localization of Oskar mRNA but inhibiting the accumulation of Short Oskar protein.

  • anterior posterior axis specification in drosophila oocytes identification of novel bicoid and Oskar mrna localization factors
    Genetics, 2011
    Co-Authors: Chin-wen Chang, Dmitry Nashchekin, Lucy Wheatley, Uwe Irion, Katja Dahlgaard, Tessa G. Montague, Jacqueline Hall, Daniel St Johnston
    Abstract:

    The Drosophila melanogaster anterior-posterior axis is established during oogenesis by the localization of bicoid and Oskar mRNAs to the anterior and posterior poles of the oocyte. Although genetic screens have identified some trans-acting factors required for the localization of these transcripts, other factors may have been missed because they also function at other stages of oogenesis. To circumvent this problem, we performed a screen for revertants and dominant suppressors of the bicaudal phenotype caused by expressing Miranda-GFP in the female germline. Miranda mislocalizes Oskar mRNA/Staufen complexes to the oocyte anterior by coupling them to the bicoid localization pathway, resulting in the formation of an anterior abdomen in place of the head. In one class of revertants, Miranda still binds Staufen/Oskar mRNA complexes, but does not localize to the anterior, identifying an anterior targeting domain at the N terminus of Miranda. This has an almost identical sequence to the N terminus of vertebrate RHAMM, which is also a large coiled-coil protein, suggesting that it may be a divergent Miranda ortholog. In addition, we recovered 30 dominant suppressors, including multiple alleles of the spectroplakin, short stop, a lethal complementation group that prevents Oskar mRNA anchoring, and a female sterile complementation group that disrupts the anterior localization of bicoid mRNA in late oogenesis. One of the single allele suppressors proved to be a mutation in the actin nucleator, Cappuccino, revealing a previously unrecognized function of Cappuccino in pole plasm anchoring and the induction of actin filaments by Long Oskar protein.

  • In Vivo Imaging of Oskar mRNA Transport Reveals the Mechanism of Posterior Localization
    Cell, 2008
    Co-Authors: Vitaly Zimyanin, Ilan Davis, Katsiaryna Belaya, Jacques Pécréaux, Michael J. Gilchrist, Alejandra Clark, Daniel St Johnston
    Abstract:

    Oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following Oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, Oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the Oskar mRNA complex plays a distinct role in particle formation and transport.

  • An Oskar-Dependent Positive Feedback Loop Maintains the Polarity of the Drosophila Oocyte
    Current biology : CB, 2007
    Co-Authors: Vitaly Zimyanin, Nick Lowe, Daniel St Johnston
    Abstract:

    Summary The localization of Oskar mRNA to the posterior of the Drosophila oocyte defines the site of assembly of the pole plasm, which contains the abdominal and germline determinants [1–3]. Oskar mRNA localization requires the polarization of the microtubule cytoskeleton, which depends on the recruitment of PAR-1 to the posterior cortex in response to a signal from the follicle cells, where it induces an enrichment of microtubule plus ends [4–7]. Here, we show that overexpressed Oskar mRNA localizes to the middle of the oocyte, as well as the posterior. This ectopic localization depends on the premature translation of Oskar protein, which recruits PAR-1 and microtubule-plus-end markers to the oocyte center instead of the posterior pole, indicating that Oskar regulates the polarity of the cytoskeleton. Oskar also plays a role in the normal polarization of the oocyte; mutants that disrupt Oskar mRNA localization or translation strongly reduce the posterior recruitment of microtubule plus ends. Thus, Oskar mRNA localization is required to stabilize and amplify microtubule polarity, generating a positive feedback loop in which Oskar recruits PAR-1 to the posterior to increase the microtubule cytoskeleton's polarization, which in turn directs the localization of more Oskar mRNA.

Ruth Lehmann - One of the best experts on this subject based on the ideXlab platform.

  • Phase transitioned nuclear Oskar promotes cell division of Drosophila primordial germ cells.
    eLife, 2018
    Co-Authors: Kathryn E. Kistler, Ruth Lehmann, Thomas R. Hurd, Tatjana Trcek, Ruoyu Chen, Feng Xia Liang, Joseph Sall, Masato Kato
    Abstract:

    Germ granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. Here we study two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. We show that cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, we find that Oskar drives their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar’s nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.

  • Long Oskar Controls Mitochondrial Inheritance in Drosophila melanogaster
    Developmental cell, 2016
    Co-Authors: Thomas R. Hurd, Beate Herrmann, Julia Sauerwald, Justina Sanny, Markus Grosch, Ruth Lehmann
    Abstract:

    Inherited mtDNA mutations cause severe human disease. In most species, mitochondria are inherited maternally through mechanisms that are poorly understood. Genes that specifically control the inheritance of mitochondria in the germline are unknown. Here, we show that the long isoform of the protein Oskar regulates the maternal inheritance of mitochondria in Drosophila melanogaster. We show that, during oogenesis, mitochondria accumulate at the oocyte posterior, concurrent with the bulk streaming and churning of the oocyte cytoplasm. Long Oskar traps and maintains mitochondria at the posterior at the site of primordial germ cell (PGC) formation through an actin-dependent mechanism. Mutating long Oskar strongly reduces the number of mtDNA molecules inherited by PGCs. Therefore, Long Oskar ensures germline transmission of mitochondria to the next generation. These results provide molecular insight into how mitochondria are passed from mother to offspring, as well as how they are positioned and asymmetrically partitioned within polarized cells.

  • Localization of Oskar RNA regulates Oskar translation and requires Oskar protein
    Development (Cambridge England), 1995
    Co-Authors: Christopher Rongo, Elizabeth Rose Gavis, Ruth Lehmann
    Abstract:

    The site of Oskar RNA and protein localization within the oocyte determines where in the embryo primordial germ cells form and where the abdomen develops. Initiation of Oskar RNA localization requires the activity of several genes. We show that ovaries mutant for any of these genes lack Oskar protein. Using various transgenic constructs we have determined that sequences required for Oskar RNA localization and translational repression map to the Oskar 3′UTR, while sequences involved in the correct temporal activation of translation reside outside the Oskar 3′UTR. Upon localization of Oskar RNA and protein at the posterior pole, Oskar protein is required to maintain localization of Oskar RNA throughout oogenesis. Stable anchoring of a transgenic reporter RNA at the posterior pole is disrupted by Oskar nonsense mutations. We propose that initially localization of Oskar RNA permits translation into Oskar protein and that subsequently Oskar protein regulates its own RNA localization through a positive feedback mechanism. SUMMARY

  • Induction of germ cell formation by Oskar.
    Nature, 1992
    Co-Authors: Anne Ephrussi, Ruth Lehmann
    Abstract:

    The Oskar gene directs germ plasm assembly and controls the number of germ cell precursors formed at the posterior pole of the Drosophila embryo. Mislocalization of Oskar RNA to the anterior pole leads to induction of germ cells at the anterior. Of the eight genes necessary for germ cell formation at the posterior, only three, Oskar, vasa and tudor, are essential at an ectopic site.

  • Oskar organizes the germ plasm and directs localization of the posterior determinant nanos
    Cell, 1991
    Co-Authors: Anne Ephrussi, Laura K. Dickinson, Ruth Lehmann
    Abstract:

    Oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. We have cloned Oskar and show that Oskar RNA is localized to the posterior pole of the oocyte when germ plasm forms. This polar distribution of Oskar RNA is established during oogenesis in three phases: accumulation in the oocyte, transport toward the posterior, and finally maintenance at the posterior pole of the oocyte. The colocalization of Oskar and nanos in wild-type and bicaudal embryos suggests that Oskar directs localization of the posterior determinant nanos. We propose that the pole plasm is assembled stepwise and that continued interaction among its components is required for germ cell determination.

Imre Gaspar - One of the best experts on this subject based on the ideXlab platform.

  • Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization
    2018
    Co-Authors: Imre Gaspar, Anne Ephrussi, Felix Hövelmann, Jasmine Chamiolo, Oliver Seitz
    Abstract:

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

  • An RNA-binding Atypical Tropomyosin Recruits kinesin-1 Dynamically to Oskar mRNPs
    The EMBO Journal, 2016
    Co-Authors: Imre Gaspar, Vasiliy O. Sysoev, Artem Komissarov, Anne Ephrussi
    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.

  • an rna binding tropomyosin recruits kinesin 1 dynamically to Oskar mrnps
    bioRxiv, 2016
    Co-Authors: Imre Gaspar, Artem Komissarov, Vasily Sysoev, Anne Ephrussi
    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 precise recruitment and regulation of motor proteins to form transport-competent mRNPs. Using high- and super-resolution imaging, we determine the steps in motor recruitment to Oskar mRNPs. We show that the posterior-targeting kinesin-1 is recruited upon nuclear export of Oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. We demonstrate that DmTropomyosin1-I/C is an atypical RNA-binding, nucleocytoplasmic shuttling Tropomyosin1 isoform that binds the Oskar 3′UTR through recognition of a supramolecular RNA motif created upon dimerization of Oskar molecules. Our data show that, in the oocyte, kinesin-1 is recruited by DmTropomyosin1-I/C to a dynamically changing, small subset of Oskar mRNPs and is activated by the functionalized spliced Oskar RNA localization element, revealing an ergonomic, coordinated mechanism of cargo transport.

  • Klar ensures thermal robustness of Oskar localization by restraining RNP motility
    The Journal of cell biology, 2014
    Co-Authors: Imre Gaspar, Anne Ephrussi, Sean L. Cotton, Dae-hwan Kim, Michael A. Welte
    Abstract:

    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.

  • Control of RNP motility and localization by a splicing-dependent structure in Oskar mRNA
    Nature Structural & Molecular Biology, 2012
    Co-Authors: Sanjay Ghosh, Virginie Marchand, Imre Gaspar, Anne Ephrussi
    Abstract:

    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.