Drosophila Syncrip Modulates the Expression of Mrnas Encoding Key Synaptic Proteins Required for Morphology at the Neuromuscular Junction

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Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

SUZANNE M. MCDERMOTT,1,2,3 LU YANG,1,2 JAMES M. HALSTEAD,1,2,4 RUSSELL S. HAMILTON,1,5 CARINE MEIGNIN,1,6 and ILAN DAVIS1 1Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

ABSTRACT Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function. Keywords: Drosophila; mRNA localization; localized translation; neuromuscular junction

INTRODUCTION et al. 2002; Ashraf et al. 2006; Lyles et al. 2006; Sanchez- Carbente Mdel and Desgroseillers 2008; Wang et al. 2009; Localized translation is a widespread and evolutionarily an- Buxbaum et al. 2014). The Drosophila neuromuscular junc- cient strategy used to temporally and spatially restrict specific tion (NMJ) is an excellent model system for studying the proteins to their site of function and has been extensively general molecular principles of the regulation of synaptic de- studied during early development and in polarized cells in velopment and plasticity. Genetic or activity-based manipu- a variety of model systems (Holt and Bullock 2009; Martin lations of synaptic translation at the NMJ has previously and Ephrussi 2009; Meignin and Davis 2010). It is thought been shown to affect the morphological and electrophysio- to be of particular importance in the regulation of neuronal logical plasticity of NMJ synapses (Sigrist et al. 2000, 2003; development and in the plastic changes at neuronal synapses Zhang et al. 2001; Menon et al. 2004, 2009; Pepper et al. that underlie memory and learning, allowing rapid local 2009). However, neither the mRNA targets nor the molecular changes in gene expression to occur independently of new mechanism by which such translational regulation occurs are transcriptional programs (Guzowski et al. 2000; Miller fully understood. We previously identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, 2These authors contributed equally to this work. 3 which we named Syncrip (Syp). Mammalian SYNCRIP/ Present address: Seattle Biomedical Research Institute, Seattle, WA 98109, USA hnRNPQ is a component of neuronal RNA transport 4Present address: Friedrich Miescher Institute for Biomedical Research, granules that contain CamKIIα, Arc, and IP3R1 mRNAs 4058 Basel, Switzerland 5 (Bannai et al. 2004; Kanai et al. 2004; Elvira et al. 2006) Present address: CEGX, Babraham Research Campus, Cambridge CB22 3AT, UK and is thought to regulate translation via an interaction 6Present address: CNRS-UPR9022, IBMC–University of Strasbourg, Strasbourg 67084, France Corresponding author: [email protected] Article published online ahead of print. Article and publication date are at © 2014 McDermott et al. This article, published in RNA, is available under http://www.rnajournal.org/cgi/doi/10.1261/rna.045849.114. Freely available a Creative Commons License (Attribution 4.0 International), as described at online through the RNA Open Access option. http://creativecommons.org/licenses/by/4.0/.

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McDermott et al. with the noncoding RNA BC200/BC1, itself atranslational repressor (Duning et al. 2008). Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro (Svitkin et al. 2013) and regulates dendritic morphology (Chen et al. 2012) via association with, and localization of, mRNAs encoding components of the Cdc-42/N- WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. We previously showed that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation. Further- more, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott et al. 2012). Here, we show that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. We show that Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 FIGURE 1. Syp is present throughout the muscle in third instar larvae and is enriched both in (nrx-1), futsch, highwire (hiw), discs large muscle nuclei and postsynaptically at NMJs. (A) Wild-type Oregon-R (OrR) third instar larval 1 (dlg1), and α-spectrin (α-spec). The fillet preparations were stained with an anti-Syp antibody (red). anti-HRP antibody was used to label neurons (green) and DAPI used to label muscle nuclei (blue). (A′) Syp is present protein levels of a number of these throughout the muscle cytoplasm and is enriched in muscle nuclei (small arrowheads). Syp mRNA targets, including msp-300 and is also enriched postsynaptically at NMJs (large arrow). (B–B′′) The NMJ (white box) high- dlg1, are significantly affected in syp lighted in A shown at a higher magnification. (C–C′′) The same staining in a syp null mutant e00286 null mutants. Furthermore, in addition (syp /Df124) third instar larva. This shows no detectable Syp protein and demonstrates the specificity of the Syp antibody. Images are mean intensity 5-μm projections. Scale bar: (A)40 to regulating MSP-300 protein levels, µm, (B,C) 5 µm. Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphol- RESULTS ogy that resemble those observed in msp-300 mutants. We Syp is required for synaptic morphology propose that Syp coordinates the protein levels from a num- at the Drosophila NMJ ber of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Droso- The in vivo function of both the mammalian SYNCRIP and phila NMJ. Drosophila Syp is not well understood in the nervous system.

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To address this, we first analyzed the Syp protein expression pattern in third instar larvae. We detected Syp in the nervous system of third instar larvae, previously in the brain (McDermott et al. 2012), and postsynaptically at the NMJ in a wild-type third instar larval fillet prepa- ration (Fig. 1A,B). Syp is also present throughout the larval body wall muscles, both in the cytoplasm and particularly enriched in muscle nuclei (Fig. 1A). These signals are not detected in syp null mutant larvae, confirming that the protein detected in wild-type larvae is specific to Syp (Fig. 1C). Many RNA binding proteins are critical regulators of synapse structure (Sigrist et al. 2000; Zhang et al. 2001; Menon et al. 2004, 2009). To test whether Syp regulates synapse morphology, we characterized the morphology of the NMJs of syp mutant larvae. We focused on the highly characterized type 1 NMJs in- nervating muscles 6 and 7 of the larvae. Wild-type control (OrR) (Fig. 2A) and syp null mutant (sype00286/sype00286 or sype00286/Df(3R)BSC124) (Fig. 2B,C; Supplemental Fig. 1A–C) NMJs were stained with anti-HRP to visualize neuronal membrane and with anti-DLG to visualize the postsynaptic scaffold surrounding each type 1b bouton. syp mutants show a synaptic overgrowth phenotype, with significantly increased numbers of synaptic branches and boutons, particularly type 1b boutons (Fig. 2G–J), while bouton shape and size were similar in both. While these significant changes in branch (1.2-fold increase) and bouton number (1.5-fold increase in 1b bouton number; 1.3-fold increase in total bouton number) are small, they resemble, and are comparable to, those seen in mutants of other regulators of subsynaptic translation at the NMJ (Menon et al. 2004, 2009). A genomic syp rescue construct containing most syp isoforms (Fig. 2D) that restores ∼33% wild-type Syp protein expression (data not shown) was able to reverse these phenotypes and restore branch and bouton numbers to those observed in wild-type larvae (Fig. 2G–J). To test whether excess Syp protein can FIGURE 2. (Legend on next page) also lead to a disruption of synaptic

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McDermott et al. morphology, we overexpressed Syp in muscles or neurons et al. 2012) and is present in RNA transport granules in the using the muscle-specific driver MHC-GAL4 or the pan-neu- dendrites of mammalian neurons (Bannai et al. 2004; ronal driver Elav-GAL4 (Supplemental Fig. 1A–C). Overex- Kanai et al. 2004; Elvira et al. 2006; Chen et al. 2012), we pression in the muscle results in underelaboration of the sought to determine the downstream mRNA targets of Syp synapse and generates opposite branching and bouton num- in third instar larvae. Regulation of a number of different ber phenotypes to those in the syp loss-of-function mutant RNA targets in different cell types could also account for (Fig. 2E,G–J). Moreover, overexpression in the muscle leads the complex phenotypes that we observe at the NMJ. We per- to an alteration in the morphology of 1b boutons so that they formed RNA-immunoprecipitation (RIP) of native RNP appear abnormally large and fused (Fig. 2E). In contrast, pan- complexes from wild-type (OrR) and syp null mutant larval neuronal overexpression in the CNS using Elav-GAL4 gener- lysate, followed by deep sequencing (RIP-Seq). For a given ates a synaptic overgrowth phenotype with significantly in- transcript with a substantial number of fragments per kb of creased numbers of synaptic branches and boutons (Fig. exon model per million mapped reads (FPKM), we calculat- 2F–J). We conclude that the levels of Syp protein are impor- ed the ratio of the number of reads before and after immuno- tant for regulating NMJ synaptic morphology. precipitation. We then focused on those 274 transcripts with In order to clarify the relative requirement for Syp protein a substantial number of reads where there was an enrichment post- and presynaptically, we selectively knocked down syp due to immunoprecipitation with anti-Syp antibody from expression by driving two syp RNAi lines (independent wild-type larval lysate and not in syp null mutant lysate insertions on the second and third chromosomes) with the (Fig. 4C; Table 1; Supplemental Table 1; Supplemental Fig. muscle-specific driver MHC-GAL4 (Supplemental Fig. 2) 4). Analysis of our RIP-Seq results revealed that Syp associ- or the motoneuron-specific driver OK6-GAL4. We found ates with a large number of mRNAs encoding proteins with that presynaptic expression of RNAi in motoneurons did key roles in synaptic growth and function. This was illustrated not result in a significant decrease in branch number or bou- in the results of a Gene Ontology (GO) functional annotation ton number (Fig. 3D–G). We conclude that presynaptic syp clustering analysis (Huang et al. 2009a,b) of the 274 RNAs knockdown in motoneurons does not affect synaptic mor- with a log2 enrichment >1 (Supplemental Tables 1,2). In phology. In contrast, expression of either RNAi insertion this analysis, the top GO annotation cluster corresponds to in muscle leads to an increase in bouton number (Fig. 3B– neuron/cell projection morphogenesis or development (Fig. G), with a particular increase in type 1s boutons (arrows, 5; Supplemental Table 2). msp-300 (Elhanany-Tamir et al. Fig. 3B,C). These results, combined with the observed con- 2012; Volk 2013; Morel et al. 2014), syd-1 (Owald et al. sequences of overexpression of Syp and the fact that Syp is 2010, 2012), nrx-1 (Li et al. 2007; Zeng et al. 2007; Owald only detectable postsynaptically indicate that Syp is required et al. 2012), and futsch (Roos et al. 2000; Zhang et al. 2001) postsynaptically in muscle to suppress NMJ overgrowth. were among the top hits, while hiw (Wan et al. 2000; Interestingly, pan-neuronal Syp overexpression and syp McCabe et al. 2004; Wu et al. 2005; Collins et al. 2006), RNAi (Supplemental Fig. 3) driven by Elav-GAL4 also result dlg1 (Lahey et al. 1994; Budnik et al. 1996; Guan et al. in overgrowth defects in NMJ morphology as assessed by 1996; Tejedor et al. 1997; Thomas et al. 1997), and α-spec branch and bouton numbers. Therefore, Syp in neuronal (Featherstone et al. 2001; Pielage et al. 2005, 2006) were cell types other than the motoneurons may also regulate syn- also found to associate with Syp (Fig. 4A; Table 1; Supple- aptic morphology. mental Table 1; Supplemental Fig. 4). In contrast, abundant transcripts with no known synaptic function in larvae were not associated with Syp. These include rp49, lsp2, lk6, Syp protein associates specifically with key synaptic and ubi-p63E. Collectively, these results suggest that the transcripts RIP-Seq experiment was successful in identifying specific As Syp is an RNA-binding protein which regulates the local- mRNA that interact with Syp. The RIP-Seq results were fur- ization and translation of mRNAs in the oocyte (McDermott ther confirmed by qRT-PCR (Fig. 4B).

FIGURE 2. Syp is required for proper growth and morphology of NMJs. (A–F) Muscle 6/7 NMJs Syp regulates the protein levels of of A3 hemi-segments labeled with anti-DLG (magenta) and anti-HRP (green), showing the de- associated transcripts gree of branching and number of synaptic boutons. (A) Wild-type (OrR); (B) sype00286/sype00286; (C) sype00286/Df124; (D) mutant rescued with a genomic fragment containing all syp isoforms ex- To further investigate the regulation of Rescue e00286 e00286 cept A and H (syp is Rescue/Rescue; syp /syp ); (E) overexpression of Syp in muscle Syp target mRNAs identified by RIP- (MHC-Gal4>UAS-Syp-GFP), and (F) overexpression of Syp in neurons (Elav-Gal4>UAS-Syp- GFP). Scale bar, 20 µm. (G–J) Quantification of synaptic structural phenotypes. Error bars indi- Seq and confirmed by qRT-PCR, we ex- cate the mean ± SEM. Statistical significance was calculated using Student’s t-test. (∗) P < 0.05, amined the levels of MSP-300, Syd-1, (∗∗) P < 0.01, (∗∗∗) P < 0.001. Numbers of scored NMJs were as follows: OrR, n = 31; sype00286/ e00286 e00286 Rescue Hiw, Nrx-1, and DLG protein in wild- syp , n = 27; syp /Df, n = 31; syp , n = 30; Elav-Gal4>UAS-Syp-GFP, n = 30; type (OrR) and syp null mutant larval MHCGal4>UAS-Syp-GFP, n = 30. In all four graphs, muscle 6/7 NMJs of A3 hemi-segments were analyzed. Muscle sizes for all of the indicated genotypes did not differ significantly from body wall lysates. Our results showed wild type. that there are significant changes in the

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Syncrip regulates synaptic morphology

protein levels of these mRNA targets in the body wall of syp null mutants; MSP- 300 and Nrx-1 are reduced (Fig. 6), while Syd-1, Hiw (Fig. 6), and DLG (both 116- kDa and 97-kDa isoforms) (Supplemen- tal Fig. 5A,B; Mendoza-Topaz et al. 2008) are increased. Conversely, in lysate from larvae overexpressing Syp in muscle, the levels of both DLG isoforms are reduced compared to wild-type controls (Supple- mental Fig. 5A,B). Furthermore, at least in the case of DLG, changes in protein levels are observed without changes in dlg1 mRNA level (Supplemental Fig. 5C). We conclude that Syp is required for the correct levels of proteins that are encoded by the mRNAs with which it associates in RIP, and that loss of Syp can result in differential effects on the levels of target transcripts. In the case of some proteins, Syp functions to keep the protein levels low and in others to keep the protein levels high. msp-300 was the most highly enriched transcript following anti-Syp immunoprecipitation observed in RIP- Seq (∼5.68-log2 enrichment following immunoprecipitation), and MSP-300 protein levels are markedly reduced in syp null mutant larval body wall lysates as compared to wild type. To further as- sess the effect of loss of Syp on MSP-300 function, we stained wild-type (OrR) and syp null mutant third instar larval fillet preparations with an anti-MSP-300 antibody. MSP-300 is highly expressed in striated muscles of wild-type third instar larvae (Fig. 7A), and previous studies have indicated that its distribution in the myoplasm reflects an association with the Z-discs (Elhanany-Tamir et al. 2012; Morel et al. 2014). MSP-300 is also detected in a ring-like structure that surrounds each myonucleus (Fig. 7A). Consistent with the reduction in MSP-300 levels that we observed in Fig- ure 6, syp mutant larvae exhibit a decrease in myoplasmic Z-disc MSP- 300 staining, an elongation of the nuclear ring beyond the circumference of each myonucleus (Fig. 7B), and ectopic expression of MSP-300 in discrete circular structures (Fig. 7C). syp mutant larvae also display myonuclear clustering and FIGURE 3. (Legend on next page) aberrant nuclear shape and size without

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McDermott et al. change in number (Fig. 7B,E,F). These defects in nuclear dis- plasm but not MSP-300 function in controlling GluRIIA tribution and morphology resemble those seen in msp-300 density at the NMJ. Indeed, it has previously been shown mutants (Elhanany-Tamir et al. 2012; Morel et al. 2014) that these roles of MSP-300 are distinct and can be discon- and potentially reflect the requirement for Syp in maintain- nected (Morel et al. 2014). It is possible that this is a reflection ing the correct level and localization of MSP-300 protein. of distinct functions for multiple MSP-300 isoforms and of In particular, the MSP-300 nuclear ring staining in syp mu- the regulation of specific msp-300 isoforms by Syp as suggest- tant larvae is reminiscent of that observed in an msp-300 mu- ed by the pattern of MSP-300 immunostaining in syp mutant in which exons encoding the C-terminal KASH domain tants (Fig. 7). The second possible reason could be that are deleted (Elhanany-Tamir et al. 2012). The msp-300 gene changes in the expression of one or a number of Syp target produces KASH as well as non-KASH isoforms, and in the transcripts in the syp mutant, in turn, affect GluRIIA expres- ▵ msp-300 KASH mutant, nuclear ring staining was attributed sion. This may mask any differences that we may have seen in to non-KASH sequences (the antibody recognizes the spec- GluRIIA levels due to MSP-300 alone. Given that Syp associ- trin repeat domain) that had dissociated from the nuclear en- ates with so many key regulators of NMJ function, the NMJ velope. It is possible that the staining and phenotype we phenotype in syp mutants likely represents a complex func- observe are due to the regulation of specific msp-300 isoforms tion of multiple regulators. by Syp. A genomic syp rescue construct was able to restore MSP-300 distribution to wild type. However, MSP-300 levels still appeared slightly reduced (Fig. 7D), and the nuclear clus- DISCUSSION tering phenotype was not rescued (Fig. 7E). This is possibly Drosophila Syp is present throughout the larval body wall due to the caveat that the genomic syp rescue fragment muscles, both in myonuclei and in the cytoplasm, and is en- does not fully rescue Syp protein expression (∼33% of riched postsynaptically at the NMJ. Our results demonstrate wild-type levels) (data not shown) and does not contain all that Syp is required for the appropriate branching of the syp isoforms. A small number of annotated isoforms are motoneurons and the number of synapses they make at not present in the rescue construct, suggesting that missing the muscle. These observations are potentially explained isoforms are required for full MSP-300 expression and func- by our finding that Syp is also required for the correct level tion. This interpretation is consistent with the fact that mul- of expression of msp-300, dlg1 and other mRNA targets. tiple mammalian Syp isoforms are also required for the Given that we have previously shown that Syp regulates correct expression of single target proteins. For example, mRNA localization and localized translation in the three mammalian hnRNPQ isoforms can have differential ef- Drosophila oocyte (McDermott et al. 2012), and studies by fects on the splicing of smn mRNA (Chen et al. 2008). others have shown that mammalian SYNCRIP/hnRNPQ ▵ msp-300 KASH mutants display a locomotion defect and a inhibits translation initiation by competitively binding significant decrease in GluRIIA fluorescence density com- poly(A) sequences (Svitkin et al. 2013), we interpret these pared to the wild-type control at the NMJ (Morel et al. functions of Syp as occurring at the level of translational 2014). To address whether syp mutants also have decreased regulation of the mRNAs to which Syp binds. Our data are GluRIIA density at the NMJ, we stained wild-type (OrR) also consistent with other work in mammals showing that and syp null mutant third instar larval fillet preparations SYNCRIP/hnRNPQ is a component of neuronal RNA trans- with an anti-GluRIIA antibody. We detected no difference port granules (Bannai et al. 2004; Kanai et al. 2004; Elvira in distribution or levels of GluRIIA in the syp mutant (Sup- et al. 2006) that can regulate dendritic morphology via the lo- plemental Fig. 6). GluR complexes are also appropriately ap- calized expression of mRNAs encoding components of the posed to active zones in syp mutants (Halstead et al. 2014). Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting com- That we do not detect a reduction in GluRIIA could be due plex (Chen et al. 2012). to a number of reasons; first, that Syp regulates MSP-300 Translation at the Drosophila NMJ is thought to provide a function in nuclear positioning and anchoring in the myo- mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in FIGURE 3. Syp is required in the muscle to suppress NMJ overgrowth. (A–C) Muscle 6/7 NMJs the surface area of body wall muscles or of A3 hemi-segments labeled with anti-HRP (green) and anti-DLG (red) antibodies in (A) MHC- in response to changes in larval locomo- Gal4 third instar larvae, and (B,C) larvae expressing two independent RNAi insertions in muscle tion (Sigrist et al. 2000, 2003; Zhang et al. using the MHC-Gal4 driver (MHC-Gal4>sypRNAi 1 and 2). Expression of either RNAi construct 2001; Pepper et al. 2009). The pheno- in muscle leads to overgrowth of the NMJ, with a particular increase in type 1s boutons (arrows). Scale bar, 30 μm. (D–G) Quantification of synaptic structural phenotypes. Error bars indicate the types we observed here resemble, and mean ± SEM. Statistical significance was calculated using Student’s t-test. (n.s.) P > 0.5, (∗) P < are comparable to, those seen when ∗∗ ∗∗∗ 0.05, ( ) P < 0.005, ( ) P < 0.001. Numbers of scored NMJs were as follows: MHC-Gal4, n subsynaptic translation is altered geneti- = 27; MHC-Gal4>sypRNAi 1, n = 24; MHC-Gal4>sypRNAi 2, n = 24; OK6-Gal4, n = 25; OK6- cally or by increased locomotor activity Gal4>sypRNAi 1, n = 28; OK6-Gal4> sypRNAi 2, n = 28. In all four graphs, muscle 6/7 NMJs of A3 hemi-segments were analyzed. Muscle sizes for all of the indicated genotypes did not differ (Sigrist et al. 2003; Menon et al. 2004, significantly from wild type. 2009). In syp null mutants, NMJ synaptic

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FIGURE 4. Syp associates with key synaptic transcripts. (A) RNA immunoprecipitation (RIP) from larval lysates using an anti-Syp antibody was followed by RNA-Seq to identify RNAs that can associate with Syp. RNA-Seq read coverage plots aligned against gene models for msp-300, futsch, hiw, nrx-1, dlg1, and α-spec for anti-Syp RIP from wild-type larvae (WT-antiSyp). Reads are plotted for each of three RIP replicates in red, green, and blue. The color density of the exons represents the relative occurrence in the known FlyBase transcripts. (B) Validation of RNA-Seq results using qRT-PCR following WT-antiSyp RIP. Percent inputs for negative control transcripts that were not enriched (rp49, lsp2, lk6, ubi-p63E), and transcripts that were enriched by anti-Syp immunoprecipitation (msp300, futsch, hiw, nrx-1, dlg1, and α-spec) were determined. Data shown represent mean ± SEM from three independent experiments. (C) RNA-Seq enrichment plot showing log2 FPKM values for WT-antiSyp RIP against total WT larvae RNA-Seq. Points have been colored by enrichment (green, ≥1 and red, <1). terminals are overgrown, containing more branches and syn- syp null mutant may be attributed to Syp function in neuronal aptic boutons. Similarly, bouton numbers are increased cell types other than the motoneurons, such as glial cells, by knocking down Syp in the muscle using RNAi. In con- which are known to influence NMJ morphology (Berger trast, overexpression of Syp in the muscle has the opposite et al. 2007; Fuentes-Medel et al. 2012). Finally, while Syp is phenotype, resulting in an inhibition of synaptic growth not required in the motoneuron to regulate synapse growth and branching. Furthermore, expressing RNAi against syp and is not detected in the motoneuron (Halstead et al. in motoneurons alone does not result in a change in NMJ 2014), we cannot exclude the possibility that Syp is present morphology, indicating that Syp acts postsynaptically in at low levels in the presynapse and regulates processes inde- muscle, but not presynaptically at the NMJ to regulate mor- pendent of synapse morphology. A further detailed character- phology. Interestingly, pan-neuronal syp knockdown or over- ization of the cell types and developmental stages in which Syp expression using Elav-GAL4 also results in NMJ growth is expressed and functions is required to better understand the defects, revealing that some of the defects observed in the complex phenotypes we observe.

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McDermott et al.

presynaptic motoneurons does not result in any defects in TABLE 1. Ranked table of selected transcripts identified by RNA- Seq to have a substantial number of reads where there was an NMJ morphology. The RIP-Seq experiments were carried enrichment due to anti-Syp immunoprecipitation from wild-type out using whole larvae and will, therefore, identify Syp targets syp larval lysate, and not null larval lysate in a range of different tissues and cells, the regulation of Average FPKM which may or may not contribute to the phenotype we observe in syp mutants. It is, therefore, possible that Syp as- Total log2 WT- WT SypNull- (Expt1/ sociates with these presynaptic targets in other neuronal Rank Gene Transcript antiSyp larvae antiSyp Expt2) cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are ex- 0 msp-300 FBtr0303383 15 0 0 6 pressed and required postsynaptically in the muscle, but 2 syd-1 FBtr0302179 3 0 0 5 8 nrx-1 FBtr0334611 1 0 0 4 this has not been definitively determined (Wu et al. 2005; 11 futsch FBtr0307597 5 0 1 4 Chen et al. 2010). syp alleles may provide useful tools to ex- 56 hiw FBtr0073970 8 1 0 3 amine where key synaptic genes are expressed and how they 69 α-spec FBtr0332878 54 11 0 2 are regulated. 74 dlg1 FBtr0073484 3 1 0 2 The identity of localized mRNAs and the mechanism of lo- See Supplemental Table 1 for all enriched transcripts. The enrich- calized translation at the NMJ are major outstanding ques- ment is calculated by comparing the log2 FPKM for WT-antiSyp to tions in the field. To date, studies have shown that GluRIIA total WT larvae RNA-Seq. mRNA aggregates are distributed throughout the muscle (Currie et al. 1995; Karr et al. 2009; Ganesan et al. 2011). The Syp targets we have identified here, such as msp-300, RNA binding proteins have emerged as critical regulators hiw, nrx-1, α-spec, and dlg1, are now excellent candidates of both neuronal morphology and synaptic transmision (Sig- for localized expression at the NMJ. Ultimately, conclusive rist et al. 2000; Zhang et al. 2001; Menon et al. 2004, 2009), demonstration of localized translation will involve the visual- suggesting that protein production modulates synapse effica- ization of new protein synthesis of targets during activity-de- cy. Consistent with this, we have shown in a parallel study pendent synaptic plasticity. Biochemical experiments will that Syp is required for proper synaptic transmission and also be required to establish the precise mode of binding of vesicle release and regulates the presynapse through expres- Syp to its downstream mRNA targets, the basis for interac- sion of retrograde Bone Morphogenesis Protein (BMP) sig- tion specificity, and the molecular mechanism by which nals in the postsynapse (Halstead et al. 2014). In this role, Syp differentially regulates the protein levels of its mRNA tar- Syp may coordinate postsynaptic translation with presynaptic gets at the Drosophila NMJ. Despite the fact that mammalian neurotransmitter release. These observations provide a good SYNCRIP is known to associate with poly(A) tails, our study explanation for how Syp influences the presynapse despite and other published work have revealed that Syp can associ- being only detectable in the postsynapse. Here, we have ate with specific transcripts (Chen et al. 2012; McDermott shown that Syp associates with a large number of mRNAs et al. 2012). How Syp associates with specific mRNAs is un- within third instar larvae, many of which encode proteins known, and future studies are needed to uncover whether the with key roles in synaptic growth and function. Syp mRNA interaction of Syp with specific transcripts is dictated by di- targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and rect binding of the three Syp RRM RNA binding domains α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein or by binding to other specific mRNA binding proteins. It levels in the larval body wall but positively regulates MSP-300 is also possible that specific mRNA stem–loops, similar to and Nrx-1 protein levels. Dysregulation of these multiple the gurken localization signal, are required for Syp to bind mRNA targets likely accounts for the phenotypes we observe. to its mRNA targets. Postsynaptically expressed targets with key synaptic roles that Our study shows that msp-300 is the most significant could explain the synaptic phenotypes we observe in syp al- mRNA target of Syp. MSP-300 is the Drosophila ortho- leles include MSP-300 (Elhanany-Tamir et al. 2012; Morel log of human Nesprin proteins. These proteins have been et al. 2014), α-Spec (Pielage et al. 2006), and DLG (Lahey genetically implicated in various human myopathies. For et al. 1994; Budnik et al. 1996; Guan et al. 1996; Tejedor example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated et al. 1997; Thomas et al. 1997; Zito et al. 1997; Chen and with Emery-Dreifuss muscular dystrophy (EDMD) (Puckel- Featherstone 2005). For example, mutants in dlg1 and mu- wartz et al. 2009) as well as severe cardiomyopathies (Puck- tants where postsynaptic DLG is destabilized or delocalized elwartz et al. 2010). Moreover, Syp itself is increasingly have NMJ morphology phenotypes similar to those observed linked with factors and targets that can cause human neuro- upon overexpression of Syp in the muscle (Koh et al. 1999; degenerative disorders. Recent work has revealed that SYN- Zhang et al. 2007; Bachmann et al. 2010). Presynaptically CRIP/hnRNPQ and Fragile X mental retardation protein expressed targets include syd-1, nrx-1, and hiw (Wu et al. (FMRP) are present in the same mRNP granule (Chen 2005; Owald et al. 2010, 2012; Tian et al. 2011; Xiong et al. et al. 2012), and loss of expression of FMRP or the ability 2012). However, we have shown that syp knockdown in of FMRP to interact with mRNA and polysomes can cause

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FIGURE 5. Functional annotation clustering of the Syp RIP-Seq data set. The top ten significant clusters from a DAVID GO Analysis for RNAs with a log2 enrichment >1 (see Materials and Methods). Number given is GO enrichment score (number of genes in group).

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Antibodies All antibodies used in this study are described in Supplemental Material.

Immunoprecipitation Guinea pig anti-Syp, rabbit anti-GFP (Invitrogen), and control pre- immune sera were cross-linked to Protein A Sepharose using standard protocols and the beads equilibrated in IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, 10% glycerol, and Complete EDTA-free protease inhibitor). Twenty microliters of a 50% slurry were added to 500 μg precleared lysate and the mixture incubated overnight at 4°C with mixing. The beads were then rinsed once with cold IP buffer and washed four times for 5 min each at 4°C. After the final wash, the beads were resuspended in 25 μL pro- FIGURE 6. Syp regulates protein levels of associated transcripts. MSP- tein sample buffer (Invitrogen), boiled for 5 min, and the superna- 300, Syd-1, Hiw, and Nrx protein levels in body wall muscle extracts of tant loaded onto a gel for SDS-PAGE and Western blotting. For wild-type Oregon-R (OrR) and syp null mutant (sype00286/Df124) third RNase treatment, the incubation and wash steps were carried out instar larvae were assessed by Western blot analysis and quantified with μ μ the Odyssey imaging system, using actin for normalization. in the presence of 200 g/mL RNase A or 0.12 units/ L RNasin Ribonuclease Inhibitor (Promega). For RNA immunoprecipitation, all steps were carried out in the cases of Fragile X syndrome. Separate studies have also shown presence of RNasin, and immunocomplexes were eluted from that SYNCRIP interacts with wild-type survival of motor the beads by the addition of 100 μL extraction buffer (50 mM neuron (SMN) protein but not the truncated or mutant Tris-HCl, pH 8.0, 10 mM EDTA, and 1.3% SDS) plus 100 units forms found to cause spinal muscular atrophy (Rossoll RNasin and incubated for 30 min at 65°C. Following centrifu- et al. 2002), and Syp genetically interacts with Smn mutations gation, the elution step was repeated and the two supernatants combined. RNA was extracted from input samples and immuno- in vivo (Sen et al. 2013). Understanding Syp function in the precipitates using Trizol LS reagent (Invitrogen). Following DNase regulation of such diverse and complex targets may, there- (Ambion) treatment, immunoprecipitated RNA and 10% total fore, provide new avenues for understanding the molecular RNA were then used as a template for cDNA synthesis in combina- basis of complex disease phenotypes and potentially lead to tion with SuperScript III (Invitrogen) RT and random hexamer future therapeutic approaches. primers (Invitrogen). cDNA was then processed for sequencing (RNA-Seq) using the Illumina Genome Analyzer IIx platform (Ge- nomics Services Group, Wellcome Trust Centre for Human Ge- MATERIALS AND METHODS netics, University of Oxford) or used directly as a template for real-time PCR. Genetics

Stocks were raised on standard cornmeal-agar medium at 25°C. RNA-Seq data analysis The wild-type (WT) control strain was Oregon R (OrR). The allele sype00286 is the PBac{RB}CG17838e00286 insertion line (Exelixis Col- Alignment of the RNA-Seq reads to the Drosophila genome (Release lection at Harvard). syp mutant third instar larvae were sype00286/Df 3) was performed by the sequencing facility using the Stampy align- (3R)BSC124 (Df(3R)BSC124; Bloomington Deletion Project, ment tool (version 1.0.22) (Lunter and Goodson 2011). BAM for- Bloomington Stock Centre). mat files were created by the Oxford University Genomics Services The syp genomic rescue construct was generated using the clone Group. The analysis of the RNA-Seq data was performed with a cus- FlyFos024580 from a genomic fosmid library in the pFlyFos vector tom-written Perl script (freely available under the GPL license from (Ejsmont et al. 2009). This clone covers all syp isoforms except A and http://www.darogan.co.uk/RNA-Seq). Calls to SAMtools (version H and was integrated using ΦC31-mediated site-specific recombi- 0.1.18) (http://samtools.sourceforge.net) for reading the BAM se- nation into the attP40 site on chromosome II (stock 25709; Bloom- quencing files are made via the Bio-SamTools Perl Module (version ington Stock Center) and were generated using the services of 1.33) (http://search.cpan.org/~lds/Bio-SamTools/). Input files for Genetic Services Inc. the script are the RNA-Seq BAM files (each experiment in tripli- GAL4 drivers were ElavC155-GAL4 (Bloomington Stock Center), cate), chromosome sequences (D. mel release 3), and GFF format MHC82B-GAL4 (Schuster et al. 1996), and OK6-GAL4 (Aberle Flybase annotation file (D. mel release 5.49). For each BAM file, et al. 2002; Sanyal 2009). C-terminally GFP-tagged syp cDNA was the reads are extracted for each annotated gene region. The reads cloned into pUASt for somatic expression in larvae. Several inde- are then assigned to the exons of annotated transcripts for the pendent transgenic lines were obtained following transformation gene. Fragments per kilobase of exon model per million mapped with this plasmid, and UASt-SypGFP (chromosome III) was used reads are calculated for paired-end read experiments as defined in in this study. Mortazavi et al. (2008). The output from the program is a ranked syp RNAi lines were stocks 33011 (line 1) and 33012 (line 2) table of all transcripts above a defined cut-off for RNA enrichment (Vienna Drosophila RNAi Center). (Table 1; Supplemental Table 1). The enrichment is calculated by

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FIGURE 7. syp null mutants have nuclear distribution defects. (A) Wild-type Oregon-R (OrR) third instar larval fillet preparations were stained with an anti-MSP-300 antibody (green). DAPI was used to label muscle nuclei (blue). A wild-type distribution of MSP-300 is typified by a striated pattern throughout the myoplasm and a nuclear ring that tightly fits the muscle nuclei circumference (yellow circles). (B) The same staining in a syp null mutant (sype00286/Df124) third instar larva. syp mutants exhibit a decrease in striated MSP-300 staining and an elongation of the nuclear ring beyond the nuclei circumference. Muscle nuclei are misplaced in syp mutants. (C) syp null mutant (sype00286/Df124) third instar larvae also exhibit ectopic expression of MSP-300 in discrete circular structures. (D) Expression of a genomic fragment containing most all syp isoforms (sypRescue) in the syp mutant restores the MSP-300 distribution to wild type, though MSP-300 levels still appear slightly reduced. Scale bar, 40 μm. (E,F) Quantification of nuclear clustering phenotypes. Error bars indicate the mean ± SEM. Statistical significance was calculated using Student’s t-test. (∗) P < 0.05. Nuclear clustering is increased in syp mutants relative to wild-type controls, though the number of nuclei per muscle remains unchanged.

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comparing WT anti-Syp to WT larvae using log2 FPKM values were acquired with Olympus 20x/0.75, 40x/0.95, 60x/0.9 or 100x/ (Zhao et al. 2010). Coverage statistics were calculated using SAM- 1.4 objective lenses and then de-convolved (Parton and Davis 2006). tools and Picard-tools (http://picard.sourceforge.net/version 1.89) and are shown in Supplemental Table 3. The Perl script was also SUPPLEMENTAL MATERIAL used to generate custom images for selected gene models, showing the WT-anti-Syp IP reads aligned to the gene models. The images Supplemental material is available for this article. are generated as scalable vector graphics (SVG). The Gene Ontology analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/version 6.7) (Fig. 5; Supplemental ACKNOWLEDGMENTS Table 2; Huang et al. 2009a,b). A functional annotation analysis We thank Stephan Sigrist (Freie Universität, Berlin); Talila Volk was performed on the 274 RNAs with a log enrichment >1 and 2 (Weizmann Institute of Science, Rehovot, Israel); Developmental compared to a background of the Drosophila genome. Studies Hybridoma Bank, who provided the indicated antibodies (Supplemental Materials and Methods); Flybase, Bloomington Real-time quantitative PCR Drosophila Stock Center, and Vienna Drosophila RNAi Center resources, who made a critical contribution to this work. We also Real-time PCR was carried out using iQ SYBR Green Supermix thank Hugo Bellen, David Finnegan, Neil Brockdorff, Kim Na- (Bio-Rad Laboratories) with a Qiagen Rotor-Gene Q according to smyth, and Dave Sherratt for discussions and comments on the the manufacturer’s instructions. Ten percent of the RT product manuscript. This work was supported by a Wellcome Trust Senior was used as a template for real-time PCR analysis. Cycle threshold Basic Biomedical Research Fellowship (096144) to I.D., a Wellcome (C[T]) values were determined by the second differential maximum Trust Strategic Award (091911) supporting advanced microscopy at method as calculated by the Rotor-Gene software. Calculation of Micron Oxford (http://micronoxford.com), Wellcome Trust PhD relative mRNA levels was done by using the 2 [-ΔΔC(T)] method studentships to S.M.M. and J.H., and a Marie Curie postdoctoral fel- (Livak and Schmittgen 2001), where the C(T) values of the mRNA lowship to C.M. level were normalized to the C(T) values of rp49 mRNA in the same sample and mRNA levels represented as relative fold change Received April 20, 2014; accepted June 9, 2014. over control. C(T) values used were the means of triplicate repeats. Experiments were repeated three times. 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Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

Suzanne M. McDermott, Lu Yang, James M. Halstead, et al.

RNA 2014 20: 1593-1606 originally published online August 29, 2014 Access the most recent version at doi:10.1261/rna.045849.114

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