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. 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 A Motor Function for Drosophila Gemin3 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 SMN- binding 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 (gemin3W) and P{PZ}Dhh1rL562 (gemin3R). We used PCR to confirm that the transposon insertion site of the gemin3W 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 gemin3W allele were found to have been lost during the insertion. In the gemin3R 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, gemin3R is hypothesised to be an amorph. Figure 1. Conservation of Drosophila Gemin3. (A) Schematic representation of the Drosophila Gemin3 protein sequence showing the helicase motifs, and the SMN interaction domain uncovered in vertebrates. (B) Alignment of the Gemin3 amino acid sequence derived from various species. 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 PLoS Genetics | www.plosgenetics.org 2 November 2008 | Volume 4 | Issue 11 | e1000265