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. 2013 Sep 12;8(9):e72864.
doi: 10.1371/journal.pone.0072864. eCollection 2013.

Rasputin functions as a positive regulator of orb in Drosophila oogenesis

Alexandre Costa  1 , Cecilia Pazman, Kristina S Sinsimer, Li Chin Wong, Ian McLeod, John Yates 3rd, Susan Haynes, Paul Schedl
Affiliations

Rasputin functions as a positive regulator of orb in Drosophila oogenesis

Alexandre Costa et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Orb and Rin are components of an RNase-resistant complex.
Western blot of proteins immunoprecipitated (IP) with the indicated antibodies and probed on Western blots for Rin (A) and Orb (B). Immunoprecipitations were performed using equal amounts of wild-type ovarian extracts and in the presence of RNase A.
Figure 2
Figure 2. Rin protein expression in ovaries.
Confocal analyses of Rin (green) and Orb (red) in the germarium (A), stages 4 and 6 egg chambers (B, B′ and B″), and a stage 10 egg chamber (C, C′, C″ and C‴). Note that Orb is expressed only in the germline and concentrates in the oocyte whereas Rin is expressed both in the germline and surrounding somatic follicle cells. Arrows in A show that Rin is present in stem cells, cystoblasts and 2, 4 and 8 cell cysts while Orb is not. Arrows in B′ and B″ show accumulation of Rin and Orb in the oocyte of stage 4 and 6 cell chambers. Arrows and arrowheads in C′, C″ and C‴ mark overlap between Rin and Orb at the edge of the oocyte cortex in stage 10 egg chambers.
Figure 3
Figure 3. Molecular characterization of the rin3 allele.
(A) The 3.3 kb deletion in the rin3 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) rin3 is a null allele. Western blots of protein extracts prepared from dissected ovaries and ovarectomized wild type (wt) and rin3 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.
Figure 4
Figure 4. Defects in egg chamber packaging and chromosome morphology.
Ovarioles from wild type (A, D) and rin3 (B,C,E,F) females were stained with DAPI. A–C) Wild type chambers always contain 15 nurse cells and 1 oocyte, but rin3 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 rin 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 rin3 chambers (E, F) the chromosomes fail to disperse. Instead nuclei with discrete chromosomal blobs (arrows) or chromosomes arranged around the periphery (arrowhead) are observed.
Figure 5
Figure 5. Defects in ring canals.
A–D) Wild type (A, B) and rin3 (C, D) ovarioles were probed for actin (red) and phosphotyrosine (green) and stained with DAPI (blue). In wild type (A) each nurse cell has a defined number of ring canals (depending on the number of cell divisions) and the ring canals have a regular circular organization of components. In the rin3 chambers the ring canals often fail to maintain a regular circular structure and may disintegrate (D), possibly resulting in nurse cell fusion (C). Ring canal defects, varying in severity, were observed in most vitellogenic stage rin chambers. E, F) Wild type (E) and rin3 chambers probed with phosphotyrosine (PY) antibodies. Note the enlarged (arrow) and fragmented (arrowhead) ring canals in the rin3 chamber.
Figure 6
Figure 6. orb and rin interact genetically.
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. Hd19G is a dominant negative transgene carrying sequences of the orb 3′UTR bound by endogenous Orb and sufficient to recapitulate the pattern of localization of the endogenous orb transcript ; orb343, orb null allele ; Tub-rin, transgene carrying a wild type copy of rin under control of the tubulin promoter .
Figure 7
Figure 7. rin mothers produce ventralized or collapsed eggs.
Egg collections (n = 752) from rin3/rin2 mothers were scored for ventralized eggshells and collapsed eggs. No eggs hatched. The frequency of defective eggs of either type in wild type collections is typically less than a percent.
Figure 8
Figure 8. Rin positively regulates Orb expression.
(A) Orb expression is downregulated in rin3 ovaries. Confocal analysis of wild type (wt) and homozygous rin3 egg chambers at different stages of oogenesis. Samples were processed in parallel and microscopy was carried out under identical settings. (B) Western blot analysis of ovarian protein extracts derived from the indicated genotypes and probed with antibodies against Orb, Rin, and α-tubulin (control). (C) Quantitation of Orb protein levels shown in (B) using the Quantity One software (Bio-Rad).
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