The dynamics of fluorescently labeled endogenous gurken mRNA in Drosophila

Howard Hughes Medical Institute, Princeton University, NJ 08544, USA.
Journal of Cell Science (Impact Factor: 5.43). 04/2008; 121(Pt 6):887-94. DOI: 10.1242/jcs.019091
Source: PubMed


During Drosophila oogenesis, the targeted localization of gurken (grk) mRNA leads to the establishment of the axis polarity of the egg. In early stages of oogenesis, grk mRNA is found at the posterior of the oocyte, whereas in the later stages grk mRNA is positioned at the dorsal anterior corner of the oocyte. In order to visualize the real-time localization and anchorage of endogenous grk mRNA in living oocytes, we have utilized the MS2-MCP system. We show that MCP-GFP-tagged endogenous grk mRNA localizes properly within wild-type oocytes and behaves aberrantly in mutant backgrounds. Fluorescence recovery after photobleaching (FRAP) experiments of localized grk mRNA in egg chambers reveal a difference in the dynamics of grk mRNA between young and older egg chambers. grk mRNA particles, as a population, are highly dynamic molecules that steadily lose their dynamic nature as oogenesis progresses. This difference in dynamics is attenuated in K10 and sqd(1) mutants such that mislocalized grk mRNA in older stages is much more dynamic compared with that in wild-type controls. By contrast, in flies with compromised dynein activity, properly localized grk mRNA is much more static. Taken together, we have observed the nature of localized grk mRNA in live oocytes and propose that its maintenance changes from a dynamic to a static process as oogenesis progresses.

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Available from: Trudi Schüpbach
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    • "Subsequently, grk mRNA moves toward the region surrounding the oocyte nucleus and eventually accumulates in a crescent between the nucleus and the neighboring cortex by becoming tightly associated with electron-dense structures called sponge bodies. This localization and anchoring process requires a heterogeneous nuclear ribonucleoprotein (hnRNP) Sqd, and promotes the local activation of Grk translation (Delanoue et al., 2007; Jaramillo et al., 2008; Weil et al., 2012). The localized Grk protein is secreted and activates the EGFR in lateral follicle cells to establish the DV axis of the egg and future embryo (Neuman-Silberberg and Schüpbach, 1993). "
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    ABSTRACT: In Drosophila melanogaster, the anteroposterior (AP) and dorsoventral (DV) axes of the oocyte and future embryo are established through the localization and translational regulation of gurken (grk) mRNA. This process involves binding of specific factors to the RNA during transport and a dynamic remodeling of the grk-containing ribonucleoprotein (RNP) complexes once they have reached their destination within the oocyte. In ovaries of spindle-class females, an activated DNA damage checkpoint causes inefficient Grk translation and ventralization of the oocyte. In a screen for modifiers of the oocyte DV patterning defects, we identified a mutation in the eIF1A gene as a dominant suppressor. We show that reducing the function of eIF1A in spnB ovaries suppresses the ventralized eggshell phenotype by restoring Grk expression. This suppression is not the result of more efficient DNA damage repair or of disrupted checkpoint activation, but is coupled to an increase in the amount of grk mRNA associated with polysomes. In spnB ovaries, the activated meiotic checkpoint blocks Grk translation by disrupting the accumulation of grk mRNA in a translationally competent RNP complex that contains the translational activator Oo18 RNA-binding protein (Orb); this regulation involves the translational repressor Squid (Sqd). We further propose that reduction of eIF1A allows more efficient Grk translation possibly because of the presence of specific structural features in the grk 5'UTR.
    Full-text · Article · Sep 2014 · Development
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    • "Another motor that might contribute to the targeting of RNPs is Dynein. The role of Dynein in the subcellular localization of RNPs has mainly been studied in oocytes of D. melanogaster, [43], however, a number of results suggest that it could also be involved in polarized targeting of RNPs in neurons. For instance, the parallel alignment of microtubules in a plus end distal configuration [44] suggests that RNPs carried by Dynein would be unable to enter the axon, and thus would be localized specifically to the dendrites. "
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    ABSTRACT: The localization of mRNAs within axons and dendrites allows neurons to manipulate protein levels in a time and location dependent manner and is essential for processes such as synaptic plasticity and axon guidance. However, an essential step in the process of mRNA localization, the decision to traffic to dendrites and/or axons, remains poorly understood. Here we show that Myosin Va and actin filaments are necessary for the dendritic localization of the mRNA binding protein Staufen 1 and of mRNA encoding the microtubule binding protein Map2. Blocking the function or expression of Myosin Va or depolymerizing actin filaments leads to localization of Staufen 1 and of Map2 mRNA in both axons and dendrites. Furthermore, interaction with Myosin Va plays an instructive role in the dendritic localization of Hermes 1, an RNA binding protein. Wild-type Hermes 1 localizes to both axons and dendrites, whereas Hermes 1 fused with a Myosin Va binding peptide, localizes specifically to dendrites. Thus, our results suggest that targeting of mRNAs to the dendrites is mediated by a mechanism that is dependent on actin and Myosin Va.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "To address if Slimb is a stable component of the pole plasm or continually turns over at the posterior, we performed fluorescence recovery after photobleaching (FRAP) experiments with GFP-Slimb-D-F-box (Figure S1 available online). The GFP- Slimb-D-F-box signal returns to around 60% of its prebleaching level in 10 min, which is comparable to other dynamically localized components within the oocyte, such as grk and bcd mRNAs (Jaramillo et al., 2008; Weil et al., 2006). Thus, Slimb is constantly recruited to the posterior, consistent with the hypothesis that it recognizes a substrate that is constantly produced at the posterior. "
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    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.
    Full-text · Article · Aug 2013 · Developmental Cell
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