A Motor Function for the DEAD-Box RNA Helicase, Gemin3, in Drosophila

The survival motor neuron (SMN) protein, the determining factor for spinal muscular atrophy (SMA), is complexed with a group of proteins in human cells. Gemin3 is the only RNA helicase in the SMN complex. Here, we report the identification of Drosophila melanogaster Gemin3 and investigate its function in vivo. Like in vertebrates, Gemin3 physically interacts with SMN in Drosophila. Loss of function of gemin3 results in lethality at larval and/or prepupal stages. Before they die, gemin3 mutant larvae exhibit declined mobility and expanded neuromuscular junctions. Expression of a dominant-negative transgene and knockdown of Gemin3 in mesoderm cause lethality. A less severe Gemin3 disruption in developing muscles leads to flightless adults and flight muscle degeneration. Our findings suggest that Drosophila Gemin3 is required for larval development and motor function.


Introduction
Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterised by degeneration of spinal cord motor neurons, as well as progressive muscular weakness, dysphagia, dyspnoea, and in severe cases, death [1,2]. The majority of SMA patients harbour deletions or mutations in the survival motor neuron (SMN1) gene, which encodes an RNA-binding protein, SMN. In mammalian cells, the SMN protein is stably complexed with a group of proteins including Gemin2 [3], Gemin3 [4,5], Gemin4 [6], Gemin5 [7], Gemin6 [8], Gemin7 [9], and Gemin8 [10]. Biochemical studies in vertebrate systems suggested that the SMN complex plays an essential role in small nuclear ribonucleoprotein (snRNP) assembly. The SMN complex binds directly to small nuclear RNAs (snRNAs) and ensures that a set of seven Sm or Smlike (Lsm) proteins are assembled onto snRNAs [11].
Gemin3, the only RNA helicase in the SMN complex, contains nine conserved motifs including the Asp-Glu-Ala-Asp motif (or DEAD box in one-letter code). The RNA helicase activity of Gemin3 is ATP-dependent with a 59 to 39 direction [12]. RNAimediated knockdown studies indicated a role for Gemin3 in the assembly of snRNP complexes as an integral component of the macromolecular SMN complex [13,14]. Furthermore, a recent study demonstrated that intracellular Gemin3 proteolysis by a poliovirus-encoded proteinase led to reduced Sm core assembly activity in poliovirus-infected cells [14].
In addition to snRNP biogenesis, Gemin3 was also implicated in transcriptional and microRNA regulation. Gemin3 was originally isolated as a cellular factor that associates with the Epstein-Barr virus nuclear proteins EBNA2 and EBNA3C, which play a role in the transcriptional regulation of both latent viral and cellular genes [15]. The non-conserved C-terminal domain of Gemin3 has been shown to interact with and modulate a variety of cellular transcription factors including steroidogenic factor 1 [12,16], early growth response protein 2 [17], forkhead transcription factor FOXL2 [18], and mitogenic Ets repressor METS [19]. Although the majority of Gemin3 and its associated protein, Gemin4, are found in the SMN complex, a less abundant Gemin3-Gemin4 complex has been isolated from HeLa and neuronal cells. The Gemin3-Gemin4 complex contains Argonaute 2 and numerous microRNAs, co-sedimenting with polyribosomes [20][21][22].
Despite the detailed studies in vertebrate systems and a recent study in Drosophila culture cells [23], the function of Gemin3 in Drosophila development remains elusive. Here we identify the orthologue of Gemin3 in Drosophila melanogaster and demonstrate that Drosophila Gemin3, like its vertebrate counterpart, associates with SMN. Loss-of-function gemin3 mutants are lethal as third instar larvae and/or prepupae. Before they perish, gemin3 mutants exhibit dramatic loss of mobility and neuromuscular junction (NMJ) defects. Tissue-specific expression of a dominant-negative transgenic construct and RNAi studies suggest that the function of Gemin3 in mesoderm, particularly in muscles, is essential for animal survival. Furthermore, disruption of Gemin3 in muscles causes flight muscle degeneration and loss of flight. Thus our study demonstrates that Drosophila Gemin3 plays a critical role in development and motor function.

Drosophila Orthologue of the Vertebrate Gemin3
We carried BLAST searches of the Drosophila melanogaster genome using human and mouse Gemin3 sequences, and found that the DEAD/DEAH RNA helicase 1 (Dhh1) or CG6539 is the putative Drosophila Gemin3 orthologue. This gene, renamed for the present studies as gemin3, is located on the third chromosome in region 67E3, and is composed of 2 exons separated by a short intron. The Drosophila melanogaster Gemin3 protein is composed of 1028 amino acids and shows 33% identity and 55% similarity (BLASTP) to the respective human orthologue ( Figure 1A, B). This level of conservation is quite similar to that observed between the Drosophila and human SMN, which have an overall identity and similarity of 31% and 49%, respectively. The N-termini of Gemin3, in which all nine DEAD-box helicase motifs reside, are more conserved than the C-termini. A region in the middle (451-573aa) of Drosophila melanogaster Gemin3 corresponds to the SMNbinding domain identified in higher eukaryotes [5].
Aiming to test whether the physical interaction between SMN and Gemin3 reported in higher eukaryotes [24] is conserved in Drosophila, a co-immunoprecipitation approach was pursued. We have generated a transgenic line expressing CFP::Gemin3. The CFP::Gemin3 gene is functional as it can rescue gemin3 mutants, which we describe later. In extracts derived from CFP::Gemin3 transgenic larvae, anti-SMN antibodies co-immunoprecipitate CFP::Gemin3 ( Figure 2).
gemin3 Is an Essential Gene Two recessive lethal gemin3 alleles were identified: PBac{RB} e03688 (gemin3 W ) and P{PZ}Dhh1 rL562 (gemin3 R ). We used PCR to confirm that the transposon insertion site of the gemin3 W allele is located at 92 nt upstream of the transcription start site ( Figure 3A; Figure S1). Part of the 59 and 39 piggyBac ends in the gemin3 W allele were found to have been lost during the insertion. In the gemin3 R allele, the P element inserted at 108 nt downstream of the transcription start site ( Figure 3A; Figure S1). Since the P{PZ}element insert sequence generates several premature stop codons, gemin3 R is hypothesised to be an amorph.

Author Summary
The childhood disease spinal muscular atrophy (SMA) has a drastic impact on motor neurons and muscles. The cause has been linked to a deficiency in the survival motor neuron (SMN) protein. SMN interacts with various proteins termed Gemins to form the SMN complex, among which Gemin3 is the only one with an RNA unwinding activity. Here, we study the function of D. melanogaster Gemin3 in the context of development. The association of Gemin3 with SMN, which had been reported previously in humans, is conserved in flies. Loss of Gemin3 resulted in death at larval stages. Before they die, gemin3 mutant flies become sluggish and develop large synapses, which are the contacts between motor neurons and muscles. Disruption of Gemin3 in mesodermal tissues, especially muscles, causes development defects, degeneration of flight muscles, and flies that are unable to fly. This study demonstrates that Gemin3 plays a critical role in fruit fly development, especially in motor function, which raises the question of whether disruption of Gemin3 contributes to SMA. High identity is observed at the N-termini where all nine DEAD-box helicase motifs (highlighted in red) are conserved. The C-termini, including the region that binds SMN in higher eukaryotes (highlighted in blue), have lower identity. Conserved residues are shown in light grey (weakly conserved) to black (highly conserved). doi:10.1371/journal.pgen.1000265.g001 Several studies were pursued to demonstrate that the recessive lethality of both transposon insertions is specific to gemin3 disruption, thereby confirming that gemin3 is an essential gene. First, complementation crosses revealed that both gemin3 alleles retain their recessive lethality in trans to each other and to a chromosomal deficiency that completely eliminates the gemin3 gene (Df[3L]ED4457). Second, a re-mobilisation screen of the Pelement in the gemin3 R allele, which is the only transposon that could be excised, recovered homozygous viable precise excision alleles or revertants. Third, both low ubiquitous gemin3 and CFP::gemin3 transgenic expression driven by 1032-GAL4 [25] rescued the lethality of gemin3 R homozygotes and gemin3 R /gemin3 W Figure 2. In vivo association of Drosophila Gemin3 and SMN. Western blot probed for CFP::Gemin3 fusion protein (,143 kDa) using mouse anti-GFP antibody after immunoprecipitation (IP) with rabbit anti-GFP antibody (positive control), rabbit anti-SMN (test) or rabbit non-specific serum (NSS; negative control). Protein extracts were derived from larval muscles ubiquitously expressing CFP::Gemin3. CFP::Gemin3 co-purifies with SMN, but is absent in the negative control lane. Input was one-tenth volume of the whole tissue lysate. doi:10.1371/journal.pgen.1000265.g002 The gemin3 W allele is a transposon insert within the gemin3 gene promoter, specifically at 92 nucleotides upstream of the transcription start site. The transposon insert generating the gemin3 R allele is located 108 and 35 nucleotides downstream of the transcription and translation start site, respectively, thereby generating several premature stop codons. The gemin3 DN transgene lacks 424 amino acid residues from the N-terminal of Gemin3 and is thus devoid of the helicase core. (B) RT-PCR analysis of gemin3 expression in wild-type, gemin3 R /gemin3 W transheterozygotes and gemin3 R heterozygotes. Compared to wild-type, gemin3 mRNA was detected in low amounts in gemin3 R heterozygotic adults. Importantly, gemin3 mRNA expression levels were drastically reduced in gemin3 heteroallelic mutant larvae. The housekeeping Tat-binding protein-1 (Tbp-1) RT-PCR product served as an internal control. Developmental stages at 25uC: F = adult females, M = adult males, L2 = 48 hrs after egg laying (AEL) 2 nd instar larvae, L3a = 72 hrs AEL 3 rd instar larvae, and L3b = 96 hrs AEL 3 rd instar larvae. doi:10.1371/journal.pgen.1000265.g003 transheterozygotes. However, neither of the above gemin3 transgenes can rescue the lethality of homozygous gemin3 W , suggesting that a non-specific mutation may be causing the lethality associated with the gemin3 W allele. Since the lethality observed in gemin3 heteroallelic mutants was specific to the loss of gemin3, further analysis concentrated on this genotype.
Expression of the CFP::gemin3 transgene under the control of tissue-specific drivers such as G7-GAL4 (muscle), elav-GAL4 (neuron), or the combination of both could not rescue the lethality of gemin3 R homozygotes and gemin3 R /gemin3 W transheterozygotes, suggesting that animal survival also depends on the basal level of Gemin3 in tissues not covered by the expression of G7-GAL4 or elav-GAL4 drivers. Homozygous gemin3 R mutants survive to the third instar larval stage, while the transheterozygotic gemin3 R / gemin3 W animals survive to the prepupal stage after both genotypes experience a prolonged wandering third instar larval stage. The expression of gemin3 at different developmental stages was compared by two-step RT-PCR. Essentially gemin3 mRNA was expressed at all developmental stages ( Figure 3B). Supporting the amorphic allele hypothesis, we observed that expression of gemin3 mRNA was dramatically reduced in transheterozygous animals throughout their entire larval life, whereas the housekeeping control Tat-binding protein-1 (Tbp-1) transcripts remained detectable ( Figure 3B). Heterozygous gemin3 R adults have approximately half of the gemin3 mRNA transcript as that in wild-type animals ( Figure 3B).

Motor Defects in gemin3 Mutant Larvae
Although showing no dramatic mobility changes throughout the first and second larval stages, the gemin3 R /gemin3 W transheterozygotes exhibit a significantly decreased contraction rate at the third instar larval stage ( Figure 4A and Video S1). The puparium formed by gemin3 heteroallelic mutants exhibited failed eversion of the spiracles and a large axial ratio ( Figure 4B, C), the latter of which is most probably the result of a failure in body wall muscle contraction. Ubiquitous expression of the CFP::gemin3 transgene heteroallelic mutants (1032-GAL4; gemin3 R /gemin3 W ), and rescued gemin3 mutants (1032-GAL4&UAS-CFP::gemin3; gemin3 R /gemin3 W ) throughout their larval development. Compared to controls, gemin3 transheterozygotes exhibit a significant reduction in body wall contraction at the third instar larval stage. At 25uC, 24 hrs AEL = 1 st instar larvae, 48 hrs AEL = 2 nd instar larvae, and 72-96 hrs AEL = 3 rd instar larvae. Individual bars represent the mean body wall contraction rate 6 1.0 S.E.M. (n = 15-51). (B) Puparia of wild-type, gemin3 heteroallelic mutants and rescued mutants. Mutants form slender and longer puparia compared to wild-type, a defect that was corrected upon expression of gemin3 driven by the constitutive driver, 1032-GAL4. Arrowheads show failure in spiracle eversion. (C) Graph displaying the axial ratios for puparia of the indicated genotype. Compared to wild-type and rescued mutants, axial ratios of mutant puparia were significantly larger. The mean is marked by a horizontal line running through the data points (***p,0.0001; n.31). (D) Wild-type and mutant third instar larval muscle fillets labelled using Alexa Fluor-488-conjugated phalloidin show no apparent disruption in the gross structure of body wall musculature (left panels) and sarcomere organisation (right panels). doi:10.1371/journal.pgen.1000265.g004 within this mutant background rescues the defects in mobility, spiracle eversion and abnormal axial ratio, confirming that the CFP::gemin3 transgene is functional and the above phenotypes exhibited by gemin3 R /gemin3 W transheterozygotes are specifically due to the disruption of Gemin3 function ( Figure 4A-C). Mobility failure is probably not secondary to compromised muscle structure since gemin3 mutant larval fillets have an ordered pattern of muscle fibres without obvious muscle losses. In addition, there are no gross defects in the sarcomeric organisation in the gemin3 mutants ( Figure 4D).
The obvious larval contraction defects of the gemin3 transheterozygotic mutants directed the research focus on the larval neuromuscular junction (NMJ). The present studies focus on the highly characterised type I NMJ innervating ventral longitudinal muscles 6 and 7, and aim at unveiling the presence of any morphological abnormalities in a gemin3 mutant background. To this end, larval muscle fillets were dissected and double-labelled with anti-HRP antibodies, which allow visualisation of the neuronal membrane, and an antibody against Discs-large (Dlg), a primarily postsynaptic scaffold protein localised to the subsynaptic reticulum that surrounds each bouton. Although no obvious motor neuron denervation was detected, gemin3 heteroallelic mutants exhibit an appreciative synaptic overgrowth before pupariation ( Figure 5A) and a significantly increased synaptic area even when normalised to muscle size ( Figure 5B). Expression of a gemin3 transgene in a mutant or wild-type background resulted in an increase in both NMJ and muscle area (data not shown). When normalized to muscle area, the NMJ area and branches in rescued gemin3 mutants restore to the wild-type range, whereas normalized NMJ area and branch numbers within single NMJs are significantly decreased when gemin3 was overexpressed ( Figure 5B, C).

Mesodermal gemin3 Is Critical for Animal Survival
A truncated gemin3 transgene (gemin3 DN ), which lacks 424 amino acid residues from the N-terminus of Drosophila melanogaster Gemin3 and hence lacks the helicase core ( Figure 3A), causes lethality on ubiquitous expression. Whilst highlighting the importance of the helicase domain to the function of Gemin3, the N-terminal truncated Gemin3 isoform is hypothesized to be a dominantnegative mutant. We used various drivers to investigate the effect on animal survival when gemin3 DN is expressed in various temporal and spatial expression patterns (Table 1). No dramatic effect is observed when gemin3 DN is expressed at 25uC under the control of elav-GAL4, nrv2-GAL4, D42-GAL4, OK6-GAL4, mef2-GAL4, or C57-GAL4 drivers ( Figure 6A). However, expression of gemin3 DN at 25uC by Act5C-GAL4, how-GAL4 or C179-GAL4 driver results in lethality, and that by the G7-GAL4 driver leads to a significant decrease in viability ( Figure 6A). When the temperature shifted to 29uC to allow for maximal GAL4 activity, expression of gemin3 DN by Act5C-GAL4, C179-GAL4, how-GAL4, or G7-GAL4 driver causes lethality, while that by mef2-GAL4 and C57-GAL4 drivers results in decreased viability ( Figure 6B). Co-expression of an extra full-length gemin3 transgene but not a control gene such as GFP with the gemin3 DN transgene significantly alleviates the driverassociated lethality ( Figure 6 and data not shown). These experiments indicate that the lethality or low viability associated with the expression of gemin3 DN in the mesoderm and larval muscles is specifically due to the disruption of Gemin3 function.
To confirm the driver-specific lethality pattern induced by the gemin3 DN transgene, several gemin3 RNAi transgenic flies were isolated and tested to establish whether lethality can be induced when gemin3 knockdown occurs ubiquitously throughout the entire organism. Two RNAi transgenes, gemin3 dwejra and gemin3 munxar , fit this criterion. Reducing gemin3 gene activity using elav-GAL4, nrv2-GAL4, or D42-GAL4 has no effect on fly viability (Figure 7). In contrast, Gemin3 knockdown at both 25uC and 29uC via C179-GAL4 resulted in lethality. The how-GAL4 driver gave a similar effect when the gemin3 dwejra and gemin3 munxar RNAi transgene was expressed at both temperatures or at a temperature of 29uC, respectively (Figure 7). The lethality induced by gemin3 munxar could be rescued by co-expressing a functional gemin3 transgene, thus excluding the possibility that lethality is the result of 'off-target' effects ( Figure 7A, B).

Driver-Specific Gemin3 Disruption Results in Pupal Developmental Defects or Loss of Flight
Knockdown of gemin3 in the mesoderm and larval somatic musculature results in lethality at the late pupal stage, that is, pharate adults enclosed in pupae fail to eclose. Animals expressing gemin3 DN under the control of the how-GAL4 driver often lead to Figure 6. Gemin3 disruption in mesoderm and larval muscles has a drastic impact on adult viability. Bar charts showing adult fly viability assayed at 25uC (A) and 29uC (B). Fly viability is unaffected when the gemin3 DN transgene is driven in post-mitotic neuronal tissues via elav-GAL4, nrv2-GAL4, D42-GAL4 and OK6-GAL4. Lethality is however obvious when Gemin3 is disrupted in all tissues via Act5C-GAL4 or in mesoderm and larval muscles through expression by how-GAL4 and C179-GAL4. A significant reduction in viability was also observed when the gemin3 DN was driven in the muscles via G7-GAL4 and at 29uC via C57-GAL4. When highly expressed at 29uC, mef2-GAL4, which expresses in mesoderm and larval muscles, also has a significant effect on viability. Driver-associated lethality was rescued on co-expression of a full-length gemin3 transgene.  Table 1. Documented spatial and temporal expression patterns of GAL4 drivers used in the present study.

Driver name Expression pattern References
Act5C-GAL4 Ubiquitous expression with an early onset [38] elav-GAL4 Expression in all postmitotic neurons with an early onset [39,40] nrv2-GAL4 Nervous system-specific expression from embryo to the adult stage [41] d42-GAL4 Larval and adult stage motor neuron-specific expression [42,43] OK6-GAL4 Expression in all motor neurons, salivary glands, wing discs, and a subset of tracheal branches commencing in the first instar larval stage and persisting until pupation [44] C179-GAL4 Expression in mesoderm and larval muscles [45] how-GAL4 Expression in mesoderm and larval muscles [46,47] mef2-GAL4 Expressed in mesoderm, embryonic stage 12 myoblasts and larval muscles [48,49] C57-GAL4 Expression observed in all larval muscles from mid-first to third instar larval stage, two sensory cell bodies in the body wall and in other mesodermal tissues including the gut [50] G7-GAL4 Expression in all muscles beginning from the second instar larval stage [51][52][53] doi:10.1371/journal.pgen.1000265.t001 pupariation and puparia have increased axial ratios, similar to the defects exhibited by the gemin3 R /gemin3 W transheterozygotes. In addition, how-GAL4&gemin3 DN pupae exhibited several morphological abnormalities, including head eversion defects, short legs, and short wings, although segmentation of the abdomen and mature eye pigments appear normal (Figure 8).
While they can walk and jump normally, eclosed flies with an mef2-GAL4-driven gemin3 DN expression have a reduced ability to fly. In a flight assay, those flies show defective flight ability, similar to wild-type flies with clipped wings, which are flightless ( Figure 9A and Video S2). The indirect flight muscles (IFMs) in mef2-GAL4&gemin3 DN flies are shrunken, resulting in increased spacing, and breakages are obvious between the muscle fibers. Frequently, large tears within the indirect flight muscles are observed in mef2-GAL4&gemin3 DN flies but not in wild-type flies ( Figure 9B).

Discussion
We have shown that CG6539, the Drosophila orthologue of vertebrate Gemin3, plays critical roles in larval and pupal development, especially in motor function.

CG6539 Is the Drosophila Orthologue of Vertebrate gemin3
Gemin3 or DP103 was first identified in mammalian culture cells through biochemical approaches [5,15]. The Gemin3 protein has three critical features. First, the N-terminus of Gemin3 contains multiple helicase motifs including a DEAD-box. Second, Gemin3 interacts with SMN in vitro and in vivo [24]. Third, the Gemin3 and SMN proteins have a similar subcellular localization pattern [5,26].
In Drosophila there are 29 DEAD-box RNA helicases [27]. Using human and mouse Gemin3 to BLAST the Drosophila melanogaster genome, CG6539, previously identified as DEAD/DEAH RNA helicase 1 (Dhh1), is the top hit. In the N-terminus, CG6539 contains 9 conserved RNA helicase motifs including a DEAD-box. A segment in the middle of CG6539, which corresponds to the SMN-binding domain in human Gemin3, is less conserved. Moreover, co-immunoprecipitation experiments using Drosophila larval muscle extracts show that Gemin3 binds to SMN in vivo. We have also carried localization assays, which demonstrate that Gemin3 co-localizes with SMN in the cytoplasm and nucleus [28] (RJC, KED, and JLL, unpublished data). Taken together, we feel confident that we have identified the Drosophila orthologue of vertebrate Gemin3.
Recently, an independent study by Fischer and colleagues also identified CG6539 as Drosophila Gemin3 through bioinformatic and biochemical approaches using Drosophila culture cells [23]. Both their study in Drosophila culture cells and this study in Drosophila tissues have shown that Gemin3 interacts with SMN, suggesting that Gemin3 is a bona fide component of the SMN complex in fruit flies, similar to that in vertebrate systems.

Gemin3 Mutants in Fly and Mouse
In this study, we have multiple lines of evidence demonstrating that Drosophila Gemin3 is essential for animal development and survival. Firstly, homozygous loss of gemin3 through a specific transposon insert (gemin3 R ) or a transheterozygous combination of two transposon inserts which do not complement each other (gemin3 R /gemin3 W ) results in lethality at the larval and/or prepupal stage. Secondly, a functional gemin3 transgene specifically rescues the lethality and developmental defects caused by loss of gemin3. Thirdly, expression of a dominant-negative allele of gemin3 (gemin3 DN ) or Gemin3 knockdown by RNAi ubiquitously or even in a tissue-specific pattern results in lethality or reduced viability.
Gemin3-null mutants have recently been described in the mouse [29]. Heterozygous gemin3 mutant mice are healthy and fertile, with minor defects in the female reproductive system, whereas homozygous gemin3 knockout in mice leads to death at the 2-cell embryonic stage [29]. Thus, the lethality caused by loss of Gemin3 in Drosophila is consistent with the findings in Gemin3-null mice. However, while Gemin3-null mice died at an early embryonic stage, gemin3 mutant flies exhibit delayed lethality, which probably results from maternal contribution of the gemin3 transcript. In a separate study in female ovaries, we observed severe defects in nurse cells and oocytes when gemin3 is disrupted in germline cells (RJC, KED, and JLL, unpublished data).

Motor Function of Gemin3: Pre-or Post-Synaptic?
The earliest clues pointing towards a motor function were a progressive loss of mobility and consequent long and thin puparia when Gemin3 function is lost. Similar phenotypes have previously been observed in mutants with disrupted Mlp84B, a muscle sarcomeric protein [30], or Tiggrin, an extracellular matrix ligand for the position-specific 2 integrins [31]. We also observe that gemin3 mutants have an overgrown NMJ though these could be a secondary response to the progressive loss of muscle power. The size ratio of NMJs to muscles is reduced when gemin3 is overexpressed raising the possibility that Gemin3 might also play a role in synaptic growth.
The requirement of Gemin3 in mesoderm and larval muscles for adult viability suggests a function of Gemin3 at the postsynaptic side. Based on the tissue-specific phenotypes uncovered, such a function is critical for pupal metamorphic changes and flight muscles. However, another possible explanation is that an earlier and wider disruption of Gemin3 by mesodermal-related drivers is responsible for the lethality, while late and local disruption of Gemin3 by neuroectodermal-related drivers causes milder phenotypes. More studies on the expression details of Gemin3 in pre-and post-synaptic tissues would help to distinguish those views.

Relationship between Gemin3 and SMN
Studies in vertebrate systems, in vitro and in vivo, have shown that Gemin3 directly binds to SMN [24]. A recent study in Drosophila culture cells [23] and this study in fly tissues confirm that the interaction between Gemin3 and SMN is conserved from fly to human. showing that in how-GAL4&gemin3 DN flies the abdomen remains elongated as in the larval stage, and red-pigmented eye discs are often clearly visible within the thorax, denoted by an arrow. The abdomen shows normal segmentation as discerned by the complete bristle pattern on the abdominal tergites. (G-I) Dorsal view showing that compared to controls, in how-GAL4&gemin3 DN flies, leg discs have everted but the appendages do not appear fully elongated compared to those of controls (arrowhead). In (C-I), the puparium was removed whilst the pupal cuticle was left intact. Pupae were photographed at the same magnification at approximately 3 days following puparium formation. doi:10.1371/journal.pgen.1000265.g008 This study raises the possibility of a functional interaction between Gemin3 and SMN. Loss of gemin3 phenocopies the larval mobility phenotypes observed in smn mutants [32]. Strong Gemin3 disruption in mesoderm and muscles led to striking developmental defects during metamorphosis, similar to those reported on disruption of SMN in a similar expression pattern [33]. A less severe gemin3 disruption in the developing musculature results in viable but flightless adult flies, which have flight muscle degeneration, similar to the phenotype in a hypomorphic smn mutant [34]. We observed that gemin3 mutants exhibit an overgrown NMJ before puparation and overexpression of gemin3 leads to a significant decrease in NMJ area and branches relative to muscle size. Interestingly, two studies describe a range of NMJ phenotypes for smn mutants [32,35]. It is still not clear whether smn and gemin3 mutants have similar morphologic defects at the NMJ as the parameters and the segments used for NMJ analysis vary in different studies. Comparison of smn and gemin3 mutant NMJs with the same standard, as well as analysing the NMJ phenotype in smn and gemin3 double mutants would help to address this question.
The motor defects unravelled on disruption of Gemin3 function in Drosophila are very intriguing in view of its association with SMN, and the possible link to SMA. More studies are necessary to clarify the roles of SMN-Gemin3 interaction in development, which may help us to understand the molecular mechanisms of the devastating neurodegenerative disorder SMA.

Fly Stocks and Genetics
The y w stock was used as the wild-type control. Transposon insertion alleles gemin3 R (P{PZ}Dhh1 rL562 ) and gemin3 W (PBac{RB}e03688) were obtained from the Bloomington Drosophila Stock Centre (BDSC) at Indiana University and the Exelixis collection at Harvard Medical School, respectively. Complementation tests, transposon remobilisation and rescue studies were carried out according to standard genetic crossing schemes. The RNAi transgenic constructs UAS-gemin3 dwejra (49505) and UAS-gemin3 munxar (49506) were obtained from the Vienna Drosophila RNAi Center and their generation was described in Dietzl et al. [36]. GAL4 lines used in this study included 1032-GAL4, Act5C- For the generation of the P{CFP::gemin3} transgenic construct, the PCR-amplified full-length coding sequence of gemin3 was ligated into the KpnI and XbaI restriction sites of the pUAST vector. The NotI and KpnI restriction sites of the resulting recombinant vector were then used to insert the cyan fluorescent protein (CFP) coding portion of the pECFP-C1 vector (BD Biosciences Clontech, Palo Alto, California, USA) upstream of the gemin3 sequence. The P{UAS-gemin3} construct was produced by ligating the gemin3 cDNA (Drosophila Genomics Resource Centre, Indiana University) in the pUAST vector using the KpnI and NotI restriction sites. The generation of the P{UAS-gemin3 DN } involved PCR-amplification of the C-terminus of gemin3 followed by ligation into the KpnI and XbaI restriction sites of the pUAST vector. In both cases, the ligation products were used to transform E. coli competent cells using standard protocols. Correct transformants were further propagated and their harbouring plasmids were purified (Qiagen HiSpeed Plasmid Midi Kit, Qiagen Ltd., West Sussex, UK) prior to microinjection in y w embryos (BestGene Inc., Chino Hills, California, USA).

Behavioural Assays
Measurement of larval mobility involved placing age-matched larvae individually at the centre of a 0.7% agar plate and measuring the forward body wall contractions exhibited by each larva for 1 minute. Puparial axial ratios were calculated by dividing the length by the width of the puparia, both of which were measured from still images.
Adult viability assays were conducted by crossing GAL4 driver stocks to lines harbouring knockdown or truncated gemin3 transgenes. A week following eclosion, adult flies were screened and counted. Adult viability was calculated as the percentage of the number of adult flies with the appropriate genotype divided by the expected number for the cross.
The flight assay was done according to a modified protocol originally designed by Benzer [37]. In brief, a 1000 ml-graduated cylinder divided into 5 sectors was coated internally with mineral oil. Flies were introduced into the top of the cylinder through a funnel and the flies stuck in each sector were counted. The height flies stick in the cylinder is indicative of their flight capabilities.

Co-Immunoprecipitation and Western Blotting
Protein A beads washed and suspended in protein lysis buffer (26 protein lysis buffer [50 mM Tris pH8, 150 mM NaCl, 1 mM EDTA, and 1% v/v NP-40]+216 protease inhibitor cocktail [complete, Mini; Roche Diagnostics Ltd.]) were incubated with preimmune serum or an antigen-specific antibody, including rabbit anti-GFP (Abcam plc., Cambridge, UK) and rabbit anti-SMN (gift from Marcel van den Heuvel, University of Oxford). Sample lysates were prepared by dissecting body wall larval muscle fillets (,30/IP) into cold 16PBS followed by grinding into cold 26 protein lysis buffer. Following pre-clearing, lysates were incubated with beads coated with the appropriate target antigenspecific antibody. The beads were then washed in lysis buffer, and mixed with 46 NuPAGE LDS Sample Buffer (Invitrogen Ltd., Paisley, UK), 106 NuPAGE Reducing Agent (Invitrogen Ltd.) and deionised water. The mixture was then heated at 70uC in order to dissociate the immunoprecipitated antigen and any other macromolecules bound to it, followed by a brief spin. The beadfree supernatant was loaded onto a 4-12% NuPAGE Novex Bis-Tris pre-cast gel (Invitrogen Ltd.), resolved and probed for GFP according to standard Western blotting procedures.

Immunostaining and Analysis of NMJs
Larvae were dissected in 16 PBS, fixed in 4% paraformaldehyde in PBS and then washed in 16 PBS+0.1% (v/v) Triton X-100 (PBT). The tissues were next subjected to overnight staining at 4uC by mouse anti-Discs large antibodies (1:100; Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA). The next day, tissues were washed in PBT and stained for ,2 hours at room temperature with anti-rabbit Alexa Fluor 488-conjugated secondary goat antibodies (1:50), and anti-HRP goat antibodies conjugated to TRITC (1:50; Jackson ImmunoResearch Laboratories Inc, West Grove, Pennsylvania, USA). Samples were then counterstained with nuclear-staining Hoechst 33342 (1:500) and Cy5-conjugated actin-binding phallodin (1:200) and mounted in Vectashield medium (Vector Laboratories Ltd., Peterborough, UK) prior to viewing with a Zeiss LSM 510 META confocal microscope.
ImageJ software (NIH) was used to quantify branch number, NMJ area, and muscle area from z-projections of confocal image stacks capturing ventral longitudinal muscles 6 and 7 (Segment A1). NMJ area constituted the presynaptic region stained by the anti-HRP antibody whereas branch number calculates the number of arborisations containing at least two boutons within a single NMJ. Both NMJ area and branch numbers were normalised through dividing each by the total muscle area of ventral longitudinal muscles 6 and 7.

Histology of Adult Flight Muscles
Adult flies were fixed overnight in 4% (v/v) paraformalde-hyde+2.5% (v/v) glutaraldehyde+0.1 M phosphate buffer pH7.2. The flies were then washed in 0.1 M phosphate buffer pH7.2 and post-fixed with 2% (w/v) osmium tetroxide for 2 hours at room temperature. Following a wash in water, the samples were subjected to a series of progressive dehydration steps in ethanol : water mixtures prior to embedding in Spurr's resin. Ultrathin sections were then made with a diamond knife, stained with Toluidine Blue and viewed under a light microscope. Figure S1 The gemin3 alleles. Schematic showing location and characteristics of the gemin3 alleles. Sequence upstream of the gemin3 transcription start site (black) flanks the PBac{RB} insert (red) of the gemin3 W allele, whilst the sequence encoding the gemin3 exon 1 fringes the P{PZ} insert (red) of the gmn3 R allele. Transcribed but untranslated sequences are coloured in purple and the predicted translation of the gemin3 R allele is also shown, with asterisks representing premature stop codons. On transposition, P-elements integrate into an 8 bp target site (underlined) that becomes duplicated at either end of the insertion. A schematic of the structure of each transposon construct is also shown [adopted from 54,55]. The P{PZ} construct of the gemin3 R allele has a plasmid backbone with an E. coli origin of replication (ori) and an antibiotic resistance gene (kan, kanamycin). The PBac{RB} construct of the gemin3 W allele lacks some of the piggyBac 59 and 39 end sequences (denoted by square brackets). Both insert constructs are inserted in the reverse orientation. Video S1 Loss of gemin3 disrupts normal locomotive behaviour in third instar larvae. Compared to wild-type, gemin3 heteroallelic mutants are sluggish in their movement.