A Motor Function for the DEAD-Box RNA Helicase, Gemin3, in Drosophila Ruben J. Cauchi, Kay E. Davies, Ji-Long Liu* Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom Abstract 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. Citation: Cauchi RJ, Davies KE, Liu J-L (2008) A Motor Function for the DEAD-Box RNA Helicase, Gemin3, in Drosophila. PLoS Genet 4(11): e1000265. doi:10.1371/ journal.pgen.1000265 Editor: Gregory A. Cox, The Jackson Laboratory, United States of America Received July 14, 2008; Accepted October 16, 2008; Published November 21, 2008 Copyright: 2008 Cauchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Medical Research Council. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jilong.liu@dpag.ox.ac.uk 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 Sm- like (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]. RNAi- mediated 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 [20–22]. 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. Results 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 PLoS Genetics | www.plosgenetics.org 1 November 2008 | Volume 4 | Issue 11 | e1000265