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. Materials and Methods Fly Stocks and Genetics The y w stock was used as the wild-type control. Transposon insertion alleles gemin3R (P{PZ}Dhh1rL562) and gemin3W (PBac{RB}e03688) were obtained from the Bloomington Drosophila Stock Centre (BDSC) at Indiana University and the Exelixis collection at Harvard Medical School, respectively. Complemen- tation tests, transposon remobilisation and rescue studies were carried out according to standard genetic crossing schemes. The RNAi transgenic constructs UAS-gemin3dwejra (49505) and UAS- gemin3munxar (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- GAL4 (BDSC), elav-GAL4 (BDSC), nrv2-GAL4 (gift from Paul Salvaterra, City of Hope National Medical Center, Duarte, California, USA), D42-GAL4 (BDSC), OK6-GAL4 (gift from Cahir O’Kane, University of Cambridge, Cambridge, UK), C179-GAL4 (BDSC), how-GAL4 (BDSC), mef2-GAL4 (gift from Barry Dickson, Research Institute of Molecular Pathology, Vienna, Austria), G7- GAL4 (gift from Aaron DiAntonio, Washington University, St. Louis, Missouri, USA) and C57-GAL4 (gift from Vivian Budnik, University of Massachusetts, Worcester, Massachusetts, USA); the spatial and temporal expression patterns are described in the Results. All stocks were cultured on standard molasses/maizemeal and agar medium in plastic vials or bottles at 25uC. UAS-CFP::gemin3, UAS-gemin3, and UAS-gemin3DN Transgenic Construct Generation 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-gemin3DN} 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 A Motor Function for Drosophila Gemin3 to microinjection in y w embryos (BestGene Inc., Chino Hills, California, USA). RT-PCR RNA was first extracted using the RNeasy kit (Qiagen Ltd.) and then reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen Ltd.) following manufacturer’s instruc- tions. PCR amplification of mRNA transcripts was performed using primers specific to gemin3 (forward: 59-CACTGGCCAAAATG- GATCTAA-39 and reverse: 59-GGCATTGCCTCAATGAGTTT- 39) and Tbp-1 (forward: 59-CACCGAAAAGATCAAGGTCAA-39 and reverse: 59-CTTTGTTGACTCCGACCAGA-39) mRNAs. RT-PCR products were resolved by electrophoresis on a 1.7% agarose gel containing ethidium bromide and bands were visualized by ultraviolet light. 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 16 PBS followed by grinding into cold 26 protein lysis buffer. Following pre-clearing, lysates were incubated with beads coated with the appropriate target antigen- specific 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 bead- free 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% paraformalde- hyde 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 PLoS Genetics | www.plosgenetics.org 10 November 2008 | Volume 4 | Issue 11 | e1000265