Molecular Psychiatry (2014) 19, 486–494 & 2014 Macmillan Publishers Limited All rights reserved 1359-4184/14 www.nature.com/mp ORIGINAL ARTICLE The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing G Barry1, JA Briggs2, DP Vanichkina1, EM Poth3, NJ Beveridge4,5, VS Ratnu6, SP Nayler2, K Nones7,JHu8, TW Bredy6, S Nakagawa9, F Rigo10, RJ Taft1, MJ Cairns4,5, S Blackshaw3, EJ Wolvetang2 and JS Mattick1,11,12 Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders. Molecular Psychiatry (2014) 19, 486–494; doi:10.1038/mp.2013.45; published online 30 April 2013 Keywords: alternative splicing; Gomafu; neuronal activation; quaking homolog; schizophrenia INTRODUCTION LncRNAs are arbitrarily defined as transcripts longer than 200 bp 10 Schizophrenia (SZ) disorder, a debilitating mental disorder that possess no protein-coding capacity. Conservative affecting about 1% of the population,1 is believed to be the annotations currently estimate 49500 independent lncRNA result of combinations of genetic and environmental factors genes producing 415 500 transcripts from regions antisense, resulting in brain dysfunction.2 Despite a high level of heritability overlapping or intronic to protein-coding genes, or from observed in SZ,3 exome sequencing has uncovered only a small bidirectional promoters or completely intergenic.11–13 Recent percentage of disease-causing genetic alterations associated with studies have indicated that lncRNAs may act as modular the etiology of the disorder.4,5 Therefore, an understanding of scaffolds, combining multiple nucleic acid and protein binding other regulatory mechanisms involved in SZ may explain the domains to direct the localization and/or regulate the activity of ‘missing heritability’ of the disorder. multiple effector proteins simultaneously.14,15 They are involved in It is well accepted that alternative splicing has a role in SZ multiple processes critical to normal cellular function, including pathology.6 Many of the archetypal genes associated with SZ are the regulation of gene expression,15 cell proliferation and aberrantly spliced, including disrupted in schizophrenia 1 (DISC1)7 differentiation,16 and disease.17 The lncRNA Gomafu is localized and v-erb-a erythroblastic leukemia viral oncogene homolog 4 to a novel nuclear compartment, which is enriched in pre-mRNA (ERBB4).8 However, the molecular mechanism underpinning this splicing factors,18 and its dysfunction is associated with risk of aberrant alternative splicing is unknown, which precludes the myocardial infarction.19 Additional studies have demonstrated development of rationally designed therapies. Here, we present roles for Gomafu in retinal cell development,20–22 brain data demonstrating that the long non-coding RNA (lncRNA) development23 and post-mitotic neuronal function,24,25 although Gomafu (also known as MIAT and RNCR2), which has recently a mechanistic explanation of these functions has been lacking. been shown to be associated with alternative splicing through its Gomafu localizes to specific subset of neurons in adult mice,25 interaction with splicing factor 1 (SF1),9 binds several additional including the CA1 region of the hippocampus and large cortical nuclear splicing factor proteins, and is involved in alternative neurons suggesting a role in neuronal excitatory transmission. splicing of the SZ pathology-related genes DISC1 and ERBB4. Interestingly, Gomafu has also been shown to be upregulated in 1Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia; 2Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia; 3Department of Neuroscience, Neurology and Ophthalmology, Center for High-Throughput Biology and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Schizophrenia Research Institute, Sydney, NSW, Australia; 5The University of Newcastle, Callaghan, NSW, Australia; 6Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia; 7Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia; 8Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 9RNA Biology Laboratory, RIKEN Advanced Research Institute, Wako, Saitama, Japan; 10Isis Pharmaceuticals, Carlsbad, CA, USA; 11Garvan Institute of Medical Research, Sydney, NSW, Australia and 12St Vincent’s Clinical School and School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia. Correspondence: Professor JS Mattick, RNA Biology and Plasticity, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia. E-mail: [email protected] Received 14 September 2012; revised 8 February 2013; accepted 18 March 2013; published online 30 April 2013 Gomafu in neuronal activity and schizophrenia G Barry et al 487 the nucleus accumbens of cocaine and heroin users,26,27 a region resulting normalized signal intensity called the z-score. Arrays contained involved in behavior and addiction,28 suggesting that dysregu- two identical probes corresponding to each protein and were repeated lation of Gomafu can influence behavior. Notably, Gomafu is also three times. To be considered as a positive interaction, both probes found in a chromosomal region linked to SZ (22q12.1).29 Here, we corresponding to a protein were required to have a z-score of X3 demonstrate that Gomafu is acutely regulated by neuronal (Po0.0015) in at least 2 of the three array experiments conducted, which resulted in 88 positive hits. Median z-score for the six conducted replicates activity, and is dysregulated in post-mortem cortex of SZ is presented in Figure 3a as a kernel density plot. Gomafu probes were subjects. Combined with its role in SZ-related alternative synthesized as depicted in Supplementary Figure 1. Briefly, two probes splicing, these results implicate Gomafu in SZ pathogenesis. were synthesized from mouse genomic DNA. One probe contained the repeat sequence ‘ACUAACC’ (Probe A) and the other lacked the sequence (Probe B). These two probes were assayed on the human protein MATERIALS AND METHODS microarray to determine the protein hits specific to the repeat region. Mouse primary neurons Additionally, a human probe was synthesized that uses the endogenous Cortices from embryonic day 14 C57BL/6J mice were isolated and sequence with three repeat sequences within the probe. A graphical mechanically dissociated (as previously described in Martinowich et al.30 representation of median z-scores (Supplementary file 2) for significant where B90% of the cells were postmitotic neurons) and plated onto poly- protein hits using the human probe is shown in Figure 3a. L-ornithine hydrochloride- and fibronectin-coated plates at a density of 105 cells per cm2. Cultures were maintained in serum-free neurobasal Electromobility shift assay medium, supplemented with B27 Supplement, L-glutamine and penicillin/ Gomafu plasmid was linearized and in vitro transcribed using the Promega streptomycin (Life Technologies, Brisbane, QLD, Australia). Riboprobe in vitro transcription kit (Promega, Madison, WI, USA). Reactions were spiked with 5 mlof50mCi of a-32P CTP and incubated at 37 1C for 4 h. Depolarization and quantitative RT-PCR Cy5-labeled riboprobes were purified using 50:50 phenol:chloroform and ethanol precipitated using 5 M NaCl. Electromobility shift assay analysis Depolarization (50 mM KCl) was performed at 7 days in vitro for mouse was conducted as previously reported.41 RNA probes were incubated with primary neuronal cultures and at week 8 for human-induced pluripotent 5, 10 or 20 mg of QKI protein. stem cells (hiPSCs). Total RNA was isolated using Trizol as per manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), quantified by NanoDrop (Thermo Scientific, Delaware, ME, USA), DNase treated (Turbo RNA immunoprecipitation DNase, Life Technologies) and reverse transcribed (Superscript III; SH-SY5Y cells (10 Â 106 cells) were lyzed in 1 ml of polysome lysis buffer Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using the ViiA (10 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT), 7 Real-Time PCR System (Life Technologies) and SYBR Green master mix RNAse OUT (Invitrogen) protease inhibitor cocktail