The EJC Binding and Dissociating Activity of PYM Is Regulated in Drosophila

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.


Introduction
In eukaryotes, post-transcriptional regulation of gene expression plays important roles in development and differentiation. These include RNA processing events in the nucleus, such as splicing, which also affects 39 end processing of the RNA, mRNA export, localization, translational enhancement and decay [1][2][3][4][5]. The multi-protein exon junction complex (EJC), which is recruited to RNAs upon splicing, has been linked to most of these steps in RNA maturation. The EJC assembles 20-24 nucleotides (nt) upstream of splice junctions and is organized around a core complex of four proteins: the DEAD box RNA helicase eIF4AIII, which is deposited by the spliceosomal protein CWC22 [6][7][8] and binds the mRNA independently of its sequence, the Y14 (Tsunagi)-MAGOH (Mago nashi, Mago) heterodimer, which stabilizes the complex, and MLN51 (Barentsz, Btz), which associates with the EJC upon RNA export [9].
In Drosophila, asymmetric localization of several key mRNAs during oogenesis is essential for embryonic patterning [10]. While in transport, these mRNAs are translationally repressed, and protein is produced only upon mRNA localization and at a particular developmental stage. The localization of oskar mRNA to the posterior pole of the oocyte requires splicing and the EJC core proteins [4,[11][12][13][14][15], indicating that nuclear events determine mRNA targeting within the cytoplasm. However, in vivo association of an assembled EJC with oskar has not been shown and the basis for the requirement of the complex in RNA transport remains unclear.
Partner of Y14-Mago (PYM) was identified through its association with the Y14-Mago heterodimer in Drosophila S2 cells [16]. The crystal structure of the PYM-Y14-Mago trimeric complex revealed that the PYM N-terminal residues are necessary for its interaction with Y14-Mago [17]; in mammals, this interaction can provoke disassembly of the EJCs from spliced mRNAs [18]. Furthermore, in HeLa cells, the PYM C-terminus, which bears similarity to eIF2A, associates with the 40S ribosomal subunit in the cytoplasm [19]. These observations led to the proposal that cytosolic 'free' PYM binds ribosomes and dislodges EJCs from mRNAs during the pioneer round of translation, thus restricting EJC disassembly to translating mRNAs. However, the function of PYM and its relationship to the EJC has not been characterized in vivo.
In this study, we characterize the function of PYM during Drosophila oogenesis. We show that Drosophila PYM (DmPYM) binds Y14-Mago but that, in contrast to its mammalian ortholog, it does not appear to interact with ribosomes. While DmPYM is not required for viability, it is essential in flies lacking one functional copy of y14 or mago. We demonstrate that overexpression of the N-terminus of DmPYM in the ovary is sufficient to dissociate EJCs from mRNAs in the cytoplasm independently of translation and causes female sterility due to oskar mislocalization in the oocyte. Finally, we show that assembly of the PYM-Y14-Mago ternary complex is modulated by the PYM C-terminal domain, indicating that PYM activity is controlled by a distinct mechanism in Drosophila.

PYM is a non-essential gene in Drosophila
Drosophila pym (wibg, CG30176), situated within intron 1 of the bgcn gene ( Figure 1A) [20], is expressed in the ovary, and the protein maternally deposited in the embryo ( Figure 1C, lane 1 and Figure S1A, lane 1) [21]. Immunostaining of ovaries revealed that DmPYM is present in the germarium, nurse cell and follicle cell cytoplasm, and within the oocyte is uniformly distributed in the cytoplasm ( Figure S1B). A cytoplasmic distribution of PYM has also been reported in Drosophila S2, HeLa and plant cells [17,19,22].
To assess the role of PYM in vivo, we made use of a Drosophila line bearing a P element insertion in the gene (P{lacW}wibg SH1616 ) that constitutes a molecular null allele of pym ( Figure 1B; Figure  S1E). The flies were viable, although the females displayed defective ovarian development, due to impaired bgcn function. Transgenic expression of a bgcn cDNA tagged with GFP (bgcnGFP) restored normal oogenesis, although no PYM protein nor pym RNA was detected ( Figure 1C, lane 2; Figure S1A, lane 2; Figure  S1E). We used such flies, to which we refer as ''pym null'', for our subsequent analyses (Table S1). Severe knockdown of PYM in ovaries and early embryos ( Figure S1A, lanes 3 and 4) by expression of shRNAs targeting pym in the female germline [23] also did not appear to affect oogenesis or embryonic development.
In HeLa cells, PYM (HsPYM) enhances translation of introncontaining reporter mRNAs and is required to stimulate translation of intronless herpesvirus gM transcript by ORF57 protein during the lytic cycle [19,24]. In addition, HsPYM knockdown resulted in increased association of EJC with spliced reporter mRNAs [18], implying a role of PYM in EJC removal. As oskar mRNA transport to the oocyte posterior pole requires the EJC core proteins, and tight control of oskar translation is critical for normal embryonic development [11][12][13][14][15]25], we examined the distribution of oskar mRNA and protein in pym null egg-chambers. As shown in Figure 1D, oskar mRNA was transported into the oocyte during the early stages of oogenesis and accumulated at the posterior pole in of the oocyte at stages 8-9 and Oskar protein was first detected at the posterior pole at stage 9, when the mRNA is localized. Localization of gurken and bicoid mRNAs, as well as expression of Gurken protein, also appeared normal in pym null egg-chambers ( Figure S1C, D).
In Drosophila S2 cells and HeLa cells, PYM interacts with the Y14-Mago heterodimer [17][18][19] which, together with eIF4AIII and Btz, constitute the EJC core [26,27]; in Arabidopsis thaliana, PYM (AtPYM) interacts both with the heterodimer and with Y14 and Mago monomers [22]. Furthermore, in mammalian cells, PYM over-expression has been shown to destabilize EJCs [18]. To probe whether DmPYM might have a role in EJC regulation in vivo, we performed genetic interaction tests between pym and EJC components in the fly (Table S1). y14, mago or eIF4AIII heterozygous mutant flies, like pym null flies, are viable and fertile. Remarkably however, we failed to generate pym null flies harbouring only one functional copy of y14 or mago. In contrast, pym null, eIF4AIII heterozygous flies were viable. The lethality we observed was specific, as transgenic expression of FLAG-tagged Y14 at close to endogenous levels rescued the lethality of the pym null, y14 heterozygous flies. These results show that DmPYM function is essential when Y14 and Mago are present at reduced levels, indicating an important relationship between PYM and these EJC components in vivo.

Drosophila PYM interacts with Y14-Mago but not with ribosomes
To determine the endogenous binding partners of DmPYM during oogenesis, we performed co-immunoprecipitations (coIPs) from cytoplasmic extracts of wild-type ovaries. As shown in Figure 2A, the EJC core proteins Y14 and Mago coprecipitated with PYM when using an anti-PYM antibody, but not an unrelated antibody, demonstrating specificity of the interaction. In contrast, eIF4AIII and Btz did not co-precipitate. Addition of RNase during the coIP did not affect Y14 and Mago recovery, indicating that DmPYM binds to Y14 and Mago by direct protein-protein interaction. This is consistent with a previous study showing that a 35 residue N-terminal domain of Drosophila PYM interacts with Y14 and Mago at their heterodimerization interface [17]. Indeed, the amino acid residues of PYM necessary for this interaction are conserved across metazoa ( Figure S2A).
In HeLa cells, over-expressed PYM interacts with the 48S pre-initiation complex, and components of the eIF4F complex, ribosomal proteins, and translation factors such as CBP80 and

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 overexpression 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 EJCdissociating protein PYM in Drosophila. PABP coIP with HsPYM [19]. Furthermore, sucrose density gradient analysis revealed that the HsPYM C-terminus, which shows a high degree of homology to HseIF2A, is necessary for co-sedimentation with ribosomal fractions [18,19]. Thus it was proposed that PYM physically links the EJC to the translation machinery, enhancing translation of spliced mRNAs [18,19]. To test whether the endogenous Drosophila PYM associates with ribosomal subunits, we performed sucrose cushion centrifugation to pellet the ribosomes from ovarian cytoplasmic extracts and examined the distribution of the endogenous PYM by western blot analysis. As shown in Figure 2B, ribosomal proteins were enriched in the pellet, whereas actin, a predominantly cytoplasmic protein, fractionated in the postribosomal supernatant, validating the assay. Cap-binding proteins and PABP were also detected in the pellet, indicating the presence of translationally competent mRNPs in this fraction. In contrast, the quasi-totality of DmPYM was recovered in the post-ribosomal supernatant ( Figure 2B), suggesting a lack of interaction with ribosomes. In addition, coIP assays using an anti-PYM antibody failed to reveal a significant interaction of DmPYM with ribosomal subunits or components of the translation initiation complex, as compared with Y14 and Mago ( Figure 2A). This suggests that, unlike HsPYM, DmPYM does not associate with the translation initiation machinery. In addition, the absence of a significant association with CBP20 and eIF4E excludes a major role of PYM in cap-dependent translation regulation in Drosophila.

DmPYM over-expression disrupts oskar localization
To analyze the function of the DmPYM during Drosophila oogenesis, we divided the protein into N-terminal (N), middle (M), and C-terminal (C) domains, and generated a set of eGFP-tagged PYM deletion transgenes ( Figure S3A). Upon expression in the female germline, the bulk of the GFP signal in the PYM-GFP eggchambers was distributed uniformly throughout the cytoplasm of the nurse cells ( Figure S3B), similar to endogenous PYM ( Figure  S1B); however, in the case of N-, M-and C-PYM, some GFP signal was also detected in the nurse cell nuclei.
In spite of their similar distribution, the different PYM-GFP proteins had dramatically different effects on embryonic development. Females expressing FL-PYM or DN-PYM in the germline were fertile (Table S1). In contrast, those expressing DCand N-PYM had reduced fertility: most of the progeny embryos failed to hatch due to abdominal patterning defects, and those that did hatch developed into sterile adults. Such a ''grandchildless'' phenotype is suggestive of reduced Oskar protein function. Indeed, immunoblot analysis of ovaries of PYM transgenic females confirmed that Oskar protein levels were substantially reduced in DCand N-PYM-GFP expressing ovaries, compared with FL-or DN-PYM-GFP expressing, or wild-type ovaries ( Figure S4A). This suggested that expression of the PYM N-terminus interferes with Oskar expression.
Expression of the posterior determinant oskar is spatiotemporally controlled such that Oskar protein is produced and accumulates stably only upon localization of the mRNA at the posterior pole of the oocyte during mid-oogenesis [28][29][30]. The low levels of Oskar protein in DCand N-PYM expressing ovaries could therefore be due to a failure in oskar mRNA localization or translation at the posterior pole. To distinguish between these possibilities, we examined the distribution of the oskar mRNP component Staufen and of Oskar protein by immunostaining. As shown in Figure 3C, D, Staufen failed to enrich at the posterior pole of DCand N-PYM oocytes indicating a failure in oskar mRNA localization (see also Table  S1), and Oskar protein was not detected in these oocytes during oogenesis, consistent with the western blot analysis ( Figure S4A). These results show that over-expression of the DmPYM N-terminal domain is sufficient to disrupt posterior localization of oskar and thus explains the absence of Oskar protein and the consequent female sterile phenotype of DCand N-PYM expressing females.
We also noted that oskar localization was somewhat impaired in FL-PYM expressing egg-chambers: although Staufen accumulated at the oocyte posterior pole, the protein was also detected around the cortex (Figure 3, compare panel A with B, E and F), suggesting that, while less potent than DCand N-PYM, FL-PYM also has some capacity to interfere with oskar transport.
To test if the amount of PYM relative to oskar mRNA and EJCs might be important for transport, we expressed FLAG-tagged FL-, DNand DC-PYM transgenes in the germline of osk A87 /+ females, which produce only half the normal dose of oskar mRNA [31] ( Table S1). Both FL-PYM and DC-PYM transgenes caused oskar mislocalization, and no Oskar protein was detected in the osk A87 /+ oocytes ( Figure 3G, H). Western blot analysis revealed a substantial reduction in Oskar protein levels in FL-and DC-PYM expressing ovaries, as compared with DN-PYM ovaries or the wild-type control ( Figure S4B, C). Both FL-and DC-PYM expressing females produced embryos with a strong posterior group phenotype (data not shown); only ,5% of embryos produced by FL-PYM females hatched, and these developed into sterile adults.
In contrast to oskar, gurken and bicoid mRNAs were correctly localized to the antero-dorsal corner and anterior cortex of the PYM over-expressing oocytes ( Figure S4D). The mislocalization of oskar mRNA upon PYM over-expression was independent of the tag and its position in the protein, as expression of PYM transgenes tagged at their C-terminus with eGFP produced a similar effect (data not shown). All subsequent analyses of PYM function in the flies were performed using egg-chambers expressing epitope-tagged PYM constructs in the osk A87 /+ genetic background. The presence of endogenous PYM does not appear to affect oskar localization in wild-type or osk A87 /+ egg-chambers. To assess if the observed effects of PYM over-expression on oskar localization might be due to aberrant interaction of the over-expressed PYM proteins with EJC components, we performed anti-FLAG coIPs from cytoplasmic extracts from ovaries expressing the different FLAG-PYM transgenes. As observed for endogenous PYM (Figure 2A), FL-and DC-PYM proteins co-precipitated Y14 and Mago, but not eIF4AIII, whereas DN-PYM did not interact with any of the EJC core proteins tested ( Figure 3J, K). These data, consistent with previous observation in HeLa cells [18,19], show that the N-terminal portion of DmPYM exclusively mediates its interaction with the Y14-Mago heterodimer.
To test for possible interaction of over-expressed PYM proteins with the ribosomal subunits, we performed coIPs from cytoplasmic extract of the GFP-tagged PYM expressing ovaries. As shown in Figure S5A, similar to the endogenous protein, the over-expressed PYM proteins did not show significant interaction with either of the ribosomal subunits. Consistent with this, sucrose cushion centrifugation assays revealed that the tagged PYM proteins predominantly fractionated in the post-ribosomal supernatant ( Figure S5B). Importantly, the GFP-tagged FL-PYM detected in the ribosomal pellet fraction was not associated with the small ribosomal subunit ( Figure S5B, lane 10), as treatment of the extract with EDTA, which led to redistribution of the small ribosomal subunit to the supernatant fraction, did not affect FL-PYM distribution ( Figure S5C, lane 10). We conclude that epitope-tagging does not affect the interactions of PYM or its distribution in vivo.
Taken together, our data show that increased levels of DmPYM relative to oskar mRNPs disrupt localization of the mRNA. Furthermore, this property of DmPYM is mediated by its Nterminal domain and is down-regulated by the C-terminal domain.

Increasing PYM dosage causes EJC dissociation from oskar mRNA
Although the in vivo association of the EJC with oskar mRNA has never been demonstrated, the EJC core proteins are considered to be essential components of oskar mRNPs [4,11,14]. We therefore hypothesized that oskar mislocalization upon PYM over-expression was due to the loss of EJC association with the mRNA, and assessed the overall integrity of EJCs in the ovary. Over-expression of either FLAG-FL-PYM or FL-PYM-GFP in osk A87 /+ ovaries caused a substantial reduction in coIP of Y14 and Mago with eIF4AIII ( Figure 4A, compare lanes 9 and 10 with lanes 11 and 12), indicating disassembly of the EJC core complex. The interaction of eIF4AIII with Mago was also impaired in eggchambers over-expressing DC-PYM, but not DN-PYM ( Figure 4C, lower panel, lanes 7 and 8). Hence, over-expression of the DmPYM N-terminal domain causes disassembly of the Drosophila EJC core. The fact that in the absence of PYM over-expression intact EJCs are recovered ( Figure 4A, lanes 9 and 10) confirms that endogenous levels of DmPYM are not deleterious to EJC integrity.
To test whether elevated levels of FL-or DC-PYM cause the EJC to dissociate from oskar mRNA, we first performed in vitro splicing assays coupled with RNase H cleavage using osk(E1E2(iftz)) RNA and assessed the degree of protection of the EJC binding site, in the presence or absence of recombinant PYM proteins [32]. Briefly, after the completion of splicing and concomitant EJC deposition, reactions were incubated with GST-tagged FL-, DNor DC-PYM, followed by RNase H cleavage induced by an oligonucleotide (Ol-25) complementary to the EJC deposition site. As shown in Figure 4B, a significant decrease of oskar mRNA correlated with an increase of the corresponding RNase H cleavage product was observed in the presence of GST-FL-and DC-PYM (lanes 6, 7 and 10, 11, respectively; * shows the position of the cleavage product), compared to GST-DN-PYM or the GST control, indicating a loss of EJC binding. This result shows that elevated amounts of DmPYM can cause mature EJCs to dissociate from oskar mRNA in vitro, raising the possibility that oskar-associated EJCs are destabilized by ectopic PYM in vivo.
To test directly whether high PYM levels cause the EJC to dissociate from oskar mRNA in vivo, we co-expressed GFP-Mago and FLAG-tagged PYM constructs in the germline of osk A87 /+ flies and performed RNA-coimmunoprecipitation (RIP) from cytoplasmic extracts of ovaries. The EJC was immunoprecipitated using GFP-Trap beads and the precipitated RNA extracted for semi-quantitative RT-PCR analysis. As shown in Figure 4C, oskar mRNA was enriched in immunoprecipitates of DN-PYM extract, demonstrating that EJCs are assembled on oskar mRNA in vivo and that over-expression of the C-terminal and middle region of DmPYM does not alter its integrity. In contrast, although similar amounts of GFP-Mago were recovered, considerably less oskar mRNA was immunoprecipitated from FL-PYM or DC-PYM ovaries ( Figure 4C). These results show that overexpression of the DmPYM N-terminal domain causes EJC dissociation from oskar mRNA in the ovary. We further investigated whether the association of the EJC with other localized mRNAs was also affected by the expression of PYM transgenes. Interestingly, a profile similar to oskar was observed with bicoid, nanos and gurken mRNAs: these mRNAs were also coprecipitated with a considerably lower efficiency from FL-PYM and DC-PYM, compared with DN-PYM extracts, demonstrating that the N-terminus of PYM is sufficient to dissociate the EJC from the spliced mRNAs in vivo. Since FL-PYM or DC-PYM over-expression leads to oskar mislocalization in the oocyte ( Figure 3G, H), but not that of bicoid or gurken ( Figure S4D), we conclude that although the EJC associates with bicoid and gurken mRNAs in the oocyte, its function is dispensable for their localization. This is consistent with normal localization of bicoid and gurken in y14 mutant oocytes [11].

Ectopic PYM dissociates EJCs independent of the elongating ribosomes
Previous studies have shown that HsPYM interacts with the translation pre-initiation complex and disassembles mature cytoplasmic EJCs from spliced RNAs [18,19]. Having found no evidence of association of DmPYM with ribosomal subunits (Figure 2 and Figure S5), yet shown that DmPYM has the capacity to dismantle EJCs from mRNAs in the egg-chamber (Figure 4), we assessed whether this effect is translationdependent. We monitored the distribution and translational output of oskDi(2,3)-boxB transgenic mRNA [32] in PYM-GFP expressing egg-chambers. Although oskDi(2,3)-boxB mRNA is spliced and localized ( Figure 5A), it is not translated due to the presence of 56 boxB stem-loops between the two oskar start codons ( Figure 5E, lane 2). As shown in Figure 5B-D, upon expression of FL-or DC-PYM, but not DN-PYM, oskDi(2,3)-boxB mRNA was mislocalized in the oocyte, as revealed by anti-Staufen immunostaining. This demonstrates that increased levels of DmPYM cause EJC dissociation from oskar mRNA in the absence of translation. Furthermore, these data provide evidence that the EJC is deposited on the oskDi(2,3)-boxB mRNA in the oocyte and that integrity of the complex is essential for oskar transport.

The C-terminus of DmPYM modulates its interaction with Y14-Mago
To investigate the contribution of the different DmPYM domains to EJC binding and disassembly, we performed coIPs from extracts of Drosophila S2 cells co-expressing HA-tagged eIF4AIII and either GFP (control) or GFP-tagged PYM proteins. To monitor both the PYM-Mago interaction and EJC integrity, PYM-GFP and HA-eIF4AIII were immunoprecipitated separately using GFP-Trap and HA-beads, respectively, and western blots of the bound fractions were probed with anti-Mago antibodies. Neither of the ectopically expressed proteins bound detectably to the agarose beads ( Figure 6A, lanes 8-14, mock), demonstrating specificity of the assay.  Figure 6A, lanes 15-21). In addition, we noted that a greater amount of Mago was recovered in DC-PYM than in FL-PYM immunoprecipitates ( Figure 6A, lanes 16 and 18), suggesting a regulatory function of the C-terminus in DmPYM binding to Mago-Y14. Surprisingly, we observed only low co-precipitation of Mago with N-PYM, compared with DC-PYM, which contains both the Nterminal and middle domain ( Figure 6A, lanes 18 and 19). This implies a role of the PYM middle domain, which itself does not bind Mago, in stabilizing the N-PYM-Y14-Mago interaction.
We next investigated the effect of the different PYM-GFP fusion proteins on EJC integrity, monitoring the ability of HA-eIF4AIII to co-IP Mago ( Figure 6A, lanes 22-28). Remarkably, in spite of their differential ability to co-precipitate Mago, all three fusion proteins, FL-, N-and DC-PYM-GFP, displayed a similar capacity to disassemble the EJC ( Figure 6A, lanes 23, 25 and 26). In contrast, neither DN-, M-, nor C-PYM-GFP, which failed to bind Mago, affected integrity of the EJC ( Figure 6A, lanes 24, 27 and 28). These results show that the ability of the different DmPYM truncations to provoke EJC disassembly correlates with their ability to bind Mago, but not with its co-precipitation efficiency.
Although both N-PYM and DC-PYM affected EJC stability in S2 cells ( Figure 6A, lanes 25 and 26) and were equally potent in causing oskar mislocalization ( Figure 3C, D), the two proteins differed considerably in their ability to co-precipitate Mago ( Figure 6A, lanes 18 and 19). This seemingly low binding of N-PYM to Y14-Mago might reflect a short half-life of the complex. To test this hypothesis, we prepared cytoplasmic extracts from S2 cells expressing the FL-, DC-, N-PYM-GFP or GFP proteins, added the protein cross-linking agent DSP and performed IPs using GFP-Trap beads. The overall immunoprecipitation efficiency was reduced in the presence of DSP. However, substantially greater amounts of Mago and Y14 co-precipitated with N-PYM upon cross-linking, consistent with stabilization of the trimeric complex ( Figure 6B, lane 22).
Quantification of the western blots revealed that, although the binding of both FL-and DC-PYM to Y14-Mago increased upon DSP treatment (1.7 and 3.6 fold, respectively), DC-PYM bound Y14-Mago more effectively than FL-PYM under both native and cross-linking conditions ( Figure 6C). In stark contrast, the efficiency with which N-PYM co-precipitated Mago increased 92-fold (from 0.92%, to 84.88%) upon DSP cross-linking, such that it approximated that of DC-PYM (98.9%, Figure 6C). Hence, while the interaction of N-PYM with Y14-Mago is labile, the binding capacity of the N-terminal domain alone to the EJC is nearly equal to that of DC-PYM, and is far greater than that of FL-PYM. This explains the potent effect of N-PYM over-expression on EJC integrity and thus, on oskar RNA localization.

Discussion
Previous studies carried out in cultured cells have led to the model that PYM -a Y14-Mago binding protein, by virtue of its association with the small ribosomal subunit, dissociates EJCs from spliced mRNAs during the first round of translation (ref. 9 and references therein). To date, however, the physiological role of PYM has remained unclear. Here we have addressed the function of PYM in an animal context.
Although a direct association of the EJC core components with oskar mRNA in vivo has been presumed [4,14,32], our RNA-coimmunoprecipitation experiments on Drosophila ovarian extracts provide the first evidence of a ''physical'' association of EJC core components with oskar mRNA. The effect of PYM on oskar localization can therefore be seen as a direct consequence of EJC dissociation from oskar RNA. This further underscores the importance of EJC association in oskar mRNA localization. PYM over-expression does not affect bicoid or gurken mRNA localization in the oocyte, consistent with previous genetic studies indicating no role of the EJC in this process. However, the fact that upon PYM over-expression EJCs are removed not only from oskar, but also from other templates such as bicoid, gurken and nanos mRNAs, indicates that   1, 7, 13, 19 and lanes 2, 8, 14, 20, the activity of DmPYM in vivo is not restricted to specific mRNPs, but that the protein acts more globally on EJCcontaining mRNP complexes.
Our in vivo analysis shows that the amount of PYM relative to the EJC core proteins, Y14 and Mago, is crucial for Drosophila development: PYM function is essential when the gene dosage of either of its interacting partners Y14 and Mago is reduced. Interestingly, under steady-state conditions, flies that exclusively lack pym function are viable and are easily maintained as a stock in the laboratory. The viability of pym null flies suggests either that in vivo the binding activity of endogenous DmPYM to Y14-Mago has no physiological impact or that it may be negatively regulated. The latter hypothesis is supported by our finding that, in wild-type flies, the EJC-dependent localization of oskar mRNA is only abolished by expression of PYM constructs lacking the C-terminal domain. Over-expression of full-length DmPYM has a mild effect that is increased in ''sensitized'' osk A87 /+ flies, in which oskar RNA dosage is reduced. Such striking differences between the PYM truncations with respect to their capability to mislocalize oskar RNA both suggests that the full-length PYM -in contrast to its truncations -is regulated, and points to the C-terminal domain as a ''key player'' in such a regulatory pathway.
Studies carried out in human cell cultures showed that the Cterminal eIF2A-like domain of HsPYM mediates its association with components of the 48S translation pre-initiation complex [19]. Thus an attractive hypothesis would be that, in Drosophila, PYM is regulated through the interaction of its C-terminal 54 residues with the small ribosomal subunit. However, none of our IP or ribosome pelleting experiments performed on Drosophila ovarian lysates support such an association of endogenous or ectopically expressed DmPYM protein with components of the small ribosomal subunit. This is most likely due to divergence of the amino acid residues in DmeIF2a ( Figure S2B). Furthermore we show that over-expression of DmPYM in oocytes not only impairs localization of endogenous oskar, but also of a non-translatable oskar RNA reporter. Thus a ribosomal interaction with DmPYM in EJC regulation seems unlikely.
Although the interaction of DmPYM with Y14-Mago is essential for EJC disassembly, the stability of the ternary complex is not important for the dissociation. The middle and the C-terminal domains of DmPYM influence its interaction with Y14-Mago, albeit in opposing manners: binding is stabilized by the former and antagonized by the latter (Figure 6). Whether the middle domain somehow stabilizes the PYM N-terminus:Y14-Mago interaction or whether it promotes proper presentation of the N-terminus for Y14-Mago binding remains to be addressed. In support of an antagonistic effect of the PYM C-terminus on Y14-Mago binding, the interaction of FL-PYM with Y14 and Mago does not approximate saturation even in the presence of the protein cross-linker DSP. This clearly indicates that, in vivo, DmPYM must be present in an equilibrium between active and dormant states. The C-terminal domain might be modified posttranslationally or might serve as a binding platform for cofactors that enhance or inhibit the EJC-dismantling activity of DmPYM. Further analysis, including an unbiased proteomics approach to identify novel interacting partners of DmPYM, should provide insights into the regulation of DmPYM and its interaction with the EJC. The fact that PYM function is essential in flies lacking one functional copy of y14 or mago suggests that PYM activity is regulated by a pathway ensuring EJC homeostasis in the fly.

Cloning
The full length pym (FL-PYM) and eIF4AIII coding regions were PCR amplified from ovarian cDNAs using specific primers (Table  S2) and cloned into pENTR/SD/D-TOPO plasmid (Invitrogen) to generate the entry clones. The PYM deletion (DN, DC, N, M and C-PYM) entry clones were generated by PCR using FL-PYM entry clone as template. The entry clones were used for recombination with the destination vector (pPFMW for Nterminal FLAG; pPWG for C-terminal eGFP; pPFMW for Nterminal FLAG-Myc) from the Drosophila Gateway vector collection (gift of Terence Murphy, Carnegie Institution for Science). The eIF4AIII entry plasmid was used for recombination with pAHW vector to generate an N-terminal HA-tagged protein. The primers are listed in Table S2.
For the generation of transgenic flies, pUASp-based destination plasmids containing the PYM fragments were injected together with helper plasmid as described [4].

qRT-PCR analysis
For each biological replicate, 6 adult females (wild-type and pym null) were homogenized in 350 ml TRIzol LS (Invitrogen) and RNA was extracted according to the manufacturer's protocol. The respectively. Bound fractions (20%) were loaded in lanes 4, 10, 16, 22, and the corresponding 56and 206dilutions in lanes 5, 11, 17, 23 and lanes 6, 12, 18, 24 respectively. Lanes 3, 9, 15 and 21 contain 20% of the mock IP precipitates. Antibodies utilized for the western blot analysis are indicated at the right of the panels. (C) CoIP efficiencies of Mago and Y14 with GFP (control) or GFP-tagged FL-, DCand N-PYM proteins under native (left panel) and DSP cross-linked (right panel) conditions. Mago and Y14 coIP efficiency is defined as a percentage of measured GFP enrichment in the corresponding GFP IPs. Plotted bar values represent the mean of two biological and four technical replicates. doi:10.1371/journal.pgen.1004455.g006 RNA samples were treated with TURBO DNase I (Ambion) for 20 min and purified using RNeasy kit (Qiagen). Reverse transcription was performed using Superscript III First-Strand synthesis kit (Invitrogen) following the manufacturer's protocol. The cDNA samples were used for qRT-PCR in One-Step ABI PCR cycler using the primers listed in Table S2.

S2 cell culture
Cells were grown in Express Five SFM Medium (Life technologies) at 25uC in the presence of penicillin-streptomycin (100 U/ml; Life technologies), puromycin (1.8 mg/ml; Sigma) and L-glutamine (2 mM; Life technologies). An S2 cell line stably transfected with pMT-Gal4-puro (gift of S. DeRenzis) was used in this study. For transient transfections, plasmids were transfected into cells in 75 cm 2 cell culture flasks using Effectene reagent (Qiagen) following manufacturer's instructions. After 36 h, CuSO 4 was added to 0.75 mM and the cultures incubated for 6 h for Gal4 induction. Cells were harvested using a cell scraper, washed with PBS and placed on ice for further processing.

Co-immunoprecipitation assay and western blot analysis
All procedures were carried out at 4uC. Cytoplasmic extracts, from fly ovaries or S2 cells, were prepared using the NE-PER kit (Thermo Scientific) following manufacturer's instructions in presence of Halt protease inhibitor cocktail (Thermo Scientific). For protein cross-linking, extracts were supplemented with 1 mM DSP (Dithiobis[succinimidyl propionate], Sigma) and incubated at 4uC for 1 h with mixing. Cross-linking was stopped by addition of 50 mM Tris-HCl pH 7.8 followed by incubation for 15 min. Lysates were pre-cleared with Protein A/G beads (Roche) for 30 min. For immunoprecipitation, extracts were incubated either with rat anti-PYM (1:250), rabbit anti-eIF4AIII (1:150), mouse anti-FLAG (1:100, Sigma #F3165) antibodies or with pre-blocked GFP-Trap (Chromotek) and anti-HA (Sigma) agarose beads for overnight with mixing. The beads were pre-blocked using Western Blocking reagent (Roche). For RNase treatment, 1 ml of RNase cocktail (Ambion) was added to the lysate together with the antibody. When using antibodies, the immuno-complexes were isolated by incubating the lysate with pre-blocked Protein A or G beads for 3 h. The beads were washed four times, 10 min each, with wash buffer (25 mM HEPES-KOH pH 7.5, 300 mM KCl, 4 mM MgCl 2 , 1 mM DTT, 125 mM Sucrose, 0.2% NP-40, 16 Halt protease inhibitor) and once with PBS. The beads were boiled in presence of 26 Laemmli sample buffer and the bound proteins analyzed by SDS-PAGE followed by western blotting.

Quantification of co-immunoprecipitation assays
For quantification of the coIP assays using S2 cell extracts under native and DSP cross-linked conditions, signals obtained from western analysis were subjected to densitometry measurements using ImageJ (http://imagej.nih.gov/ij/) and processed with Excel (Microsoft Inc.). The relative enrichments (RE) for PYM-GFP proteins, Mago and Y14 were defined as relation of measured signals in precipitates and inputs: Since DSP treatment led to lower enrichments for GFP or PYM-GFP fusions ( Figure 6B), all values plotted for Mago and Y14 from the individual immunoprecipitates were defined as the co-IP efficiency (CoIP e ) relative to corresponding enrichments, estimated for GFP and GFP-PYM fusion proteins. For example, the CoIP e of Mago in DC-PYM-GFP IP was defined as:

RNA-coimunoprecipitation and RT-PCR analysis
The ovaries from fly lines co-expressing GFP-Mago and FLAG-PYM constructs were used for preparation of cytoplasmic extract using the NE-PER kit (Thermo Scientific). The extract was precleared using Protein A beads for 1 h at 4uC and incubated with GFP-Trap agarose (ChromoTek) for 3 h. The beads were washed four times, 15 min each, with wash buffer containing 600 mM KCl and once with PBS. 20% of the input and beads were aliquotted for SDS-PAGE analysis and the rest processed for RNA extraction using TRI Reagent (Ambion). The recovered RNA was treated with 2 U TURBO DNase I (Ambion), and used for cDNA synthesis using SuperScript III First-Strand synthesis kit (Invitrogen) according to the manufacturer's protocol. Primers specific for oskar, bicoid, nanos and gurken were used for the PCR amplification (see Table S2). The data from 28 cycles are shown.

Sucrose cushion centrifugation assay
Dissected ovaries were lysed in hypotonic buffer (5 mM Tris-HCl pH 7.5, 1.5 mM KCl, 2.5 mM MgCl 2 , 0.5% TX-100, 0.5% DOC, 16 Halt protease inhibitor), incubated on ice for 15 min with or without 20 mM EDTA and centrifuged at 13,000 g for 10 min. The supernatant was adjusted to the sucrose cushion buffer (10 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM MgCl 2 ), layered on 1 M sucrose (200 ml extract on 700 ml sucrose solution) and centrifuged at 200,000 g for 2 h. The supernatant was concentrated using Amicon Ultra-10K filters and the pellet suspended in sucrose cushion buffer containing 1 mM DTT. The fractions were analysed on SDS-PAGE followed by western blotting.

Recombinant protein purification
The pENTRY/SD/D-TOPO vector containing FL-PYM was used for recombination with the Gateway destination vector pDEST15 (Invitrogen) to generate GST-PYM plasmid. The DNand DC-PYM constructs were generated by PCR using primers pairs O-388/O-390 and O-389/O-391, respectively, and cloned into EcoRI and NotI site of pGEX-4T1 (gift of E. Loeser). All plasmids were fully sequenced.
In vitro splicing reactions were carried out in 25 ml, containing 10 ml of the embryo nuclear extract, 32 P-labeled pre-mRNA substrate in a buffer (26 mM HEPES-KOH pH 7.9, 40 mM KCl, 80 mM KOAc, 4 mM MgOAc, 5 mM Creatine-Phosphate, 4 mM ATP, 2.5% PVA) for 180 min at 20uC. Purified proteins (0.5 or 1 mM of either GST, GST-FL PYM, GST-DN PYM or GST-DC PYM) were added to the 25 ml splicing reactions and further incubated for 30 min at 20uC. RNase H assays were carried out as previously described [32] using oligonucleotide Ol-25.  Figure S5 Over-expressed PYM does not interact with ribosomes. Ovarian cytoplasmic extracts from osk A87 /+ flies or osk A87 /+ flies expressing GFP-tagged FL-, DNand DC-PYM were analysed by immunoprecipitation using GFP-Trap beads (A) and sucrose cushion centrifugation assay (B, C). The proteins bound to the GFP beads (A, lanes 5-8) and the corresponding inputs (1%; A, lanes 1-4) were analyzed by western blotting. For sucrose cushion analysis, the extract was processed either without (B) or with the addition of 20 mM EDTA (C). The input (50%), supernatant and pellet fractions were processed for western blot analysis. The antibodies used for western blot staining are indicated at the right of the panels. The endogenous PYM is shown by the arrow. An asterisk marks a non-specific protein cross-reacting with the anti-Mago antibody. The presence of a fraction of FL-PYM protein in the ribosomal pellet could be an artefact of over-expression or represent an aggregate constituting an insoluble fraction.

(EPS)
Table S1 Summary of genetic interactions observed in pym loss and gain of function analysis. The columns describe the type of fly line (genetic loss or gain of function), their genotype, whether the flies were viable, fertile, and whether oskar mRNA was correctly localized. (PDF)