A late phase of germ plasm accumulation during Drosophila oogenesis requires lost and rumpelstiltskin

Asymmetric mRNA localization is an effective mechanism for establishing cellular and developmental polarity. Posterior localization of oskar in the Drosophila oocyte targets the synthesis of Oskar to the posterior, where Oskar initiates the assembly of the germ plasm. In addition to harboring germline determinants, the germ plasm is required for localization and translation of the abdominal determinant nanos. Consequently, failure of oskar localization during oogenesis results in embryos lacking germ cells and abdominal segments. oskar accumulates at the oocyte posterior during mid-oogenesis through a well-studied process involving kinesin-mediated transport. Through live imaging of oskar mRNA, we have uncovered a second, mechanistically distinct phase of oskar localization that occurs during late oogenesis and results in amplification of the germ plasm. Analysis of two newly identified oskar localization factors, Rumpelstiltskin and Lost, that are required specifically for this late phase of oskar localization shows that germ plasm amplification ensures robust abdomen and germ cell formation during embryogenesis. In addition, our results indicate the importance of mechanisms for adapting mRNAs to utilize multiple localization pathways as necessitated by the dramatic changes in ovarian physiology that occur during oogenesis

Germ Plasm Anchoring Is a Dynamic State that Requires Persistent Trafficking

Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP) particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, we show that RNP particle motility is required for long-term germ plasm retention. We propose that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations

Rasputin functions as a positive regulator of orb in Drosophila oogenesis.

The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A) tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1)K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. We have previously shown that Rasputin (Rin), the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP), associates with Orb in a messenger ribonucleoprotein (mRNP) complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. Here we show that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila

Phospho-Rasputin Stabilization by Sec16 Is Required for Stress Granule Formation upon Amino Acid Starvation

Most cellular stresses induce protein translation inhibition and stress granule formation. Here, using Drosophila S2 cells, we investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, we find that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, we identify Sec16, a component of the endoplasmic reticulum exit site, as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein

Phospho-rasputin stabilization by Sec16 is required for stress granule formation upon amino acid starvation

Most cellular stresses induce protein translation inhibition and stress granule formation. Here, using Drosophila S2 cells, we investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, we find that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, we identify Sec16, a component of the endoplasmic reticulum exit site, as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein

Defects in egg chamber packaging and chromosome morphology.

<p>Ovarioles from wild type (A, D) and <i>rin³</i> (B,C,E,F) females were stained with DAPI. A–C) Wild type chambers always contain 15 nurse cells and 1 oocyte, but <i>rin³</i> chambers may have fewer nurse cells and sometimes no oocyte. Typically the number of nurse cells/oocyte in adjacent mis-packaged egg chambers adds up to 15 nurse cells and 1 oocyte. In B the arrow indicates a chamber with a single nurse cell while the arrowhead indicates a chamber with fewer than 15 nurse cells. In C there appears to be three related chambers strung together (arrow, arrowhead and asterisk). Each has fewer than the normal complement of nurse cells (and oocyte). About 15% of the <i>rin</i> mutant ovarioles had at least one egg chamber with partitioning defects. D–F) In wild type (D) the polytenized nurse cell chromosomes disperse at the onset of vitellogenesis. In <i>rin³</i> chambers (E, F) the chromosomes fail to disperse. Instead nuclei with discrete chromosomal blobs (arrows) or chromosomes arranged around the periphery (arrowhead) are observed.</p

<i>orb</i> and <i>rin</i> interact genetically.

<p>The percentage of dorsal-ventral polarity defects in eggs laid by females with different doses of Orb and Rin is shown. Females of the indicated genotypes were crossed to wild-type males at 18°C (black bars), 25°C (gray bars) and 29°C (white bars). Each of the crosses at the indicated temperature was repeated three or more times and a total of between 1,000 to 2000 eggs were scored. Fused dorsal appendage phenotypes range from fusion at the base to fusion along the entire length of the two appendages. <i>Hd19G</i> is a dominant negative transgene carrying sequences of the <i>orb</i> 3′UTR bound by endogenous Orb and sufficient to recapitulate the pattern of localization of the endogenous <i>orb</i> transcript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072864#pone.0072864-Tan1" target="_blank">[13]</a>; <i>orb³⁴³</i>, <i>orb</i> null allele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072864#pone.0072864-Lantz1" target="_blank">[1]</a>; <i>Tub-rin</i>, transgene carrying a wild type copy of <i>rin</i> under control of the <i>tubulin</i> promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072864#pone.0072864-Pazman1" target="_blank">[34]</a>.</p

Molecular characterization of the <i>rin³</i> allele.

<p>(A) The 3.3 kb deletion in the <i>rin³</i> allele was generated by imprecise excision of the P-element P4957 originally isolated from the EMBL lethal collection. The deletion removes DNA encoding the translation start codon, the entire NTF2-like N-terminus, as well as the proline-rich (P-rich) and glutamine-rich (Q-rich) central portions of Rin. The deletion partially affects the RNA Recognition Motif (RRM) at the C-terminus, but leaves the coding region of the arginine/glycine-rich domain (RG-rich) intact. (B) <i>rin³</i> is a null allele. Western blots of protein extracts prepared from dissected ovaries and ovarectomized wild type (wt) and <i>rin³</i> females were probed with antibodies against the RRM domain of Rin. Similar results were obtained with antibodies raised against the N-terminal part of Rin (data not shown). Equal amounts of protein were loaded per lane.</p