Figure 7. Gemin3 knockdown in mesoderm and larval muscles affects adult viability. (A, B) Knockdown of Gemin3 through expression of the gemin3munxar RNAi construct in all tissues via Act5C- GAL4 or in mesoderm and larval muscles via C179-GAL4 and how-GAL4 at 29uC (B) leads to lethality. No effect on adult viability was observed when Gemin3 was knockdown in post-mitotic neuronal tissues via elav- GAL4, nrv2-GAL4, D42-GAL4 and OK6-GAL4. Driver-associated lethality was rescued on co-expression of a full-length gemin3 transgene. (C) Similarly, knockdown of Gemin3 via an independent gemin3 RNAi construct (gemin3dwejra) induces lethality when expression is driven ubiquitously (ActC-GAL4) or in a strong mesodermal and larval muscle pattern (C179-GAL4 and how-GAL4). Both gemin3 RNAi transgenes targeted the 59 end sequence of the gemin3 mRNA transcript. Individual bars represent the mean viability 6 1.0 S.E.M. of 4 independent replicates. The ¤ and e symbols indicate lethality. doi:10.1371/journal.pgen.1000265.g007 pupariation and puparia have increased axial ratios, similar to the defects exhibited by the gemin3R/gemin3W transheterozygotes. In addition, how-GAL4&gemin3DN pupae exhibited several morpho- logical abnormalities, including head eversion defects, short legs, and short wings, although segmentation of the abdomen and mature eye pigments appear normal (Figure 8). A Motor Function for Drosophila Gemin3 While they can walk and jump normally, eclosed flies with an mef2-GAL4-driven gemin3DN 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&gemin3DN 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&gemin3DN 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 (gemin3R) or a transheterozygous combination of two transposon inserts which do not complement each other (gemin3R/gemin3W) 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 (gemin3DN) 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 PLoS Genetics | www.plosgenetics.org 7 November 2008 | Volume 4 | Issue 11 | e1000265