Genetic Circuitry of Survival Motor Neuron, the Gene Underlying Spinal Muscular Atrophy
Total Page：16
File Type：pdf, Size：1020Kb
Genetic Circuitry of Survival Motor Neuron, the Gene Underlying Spinal Muscular Atrophy The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Sen, Anindya, Douglas N. Dimlich, K. G. Guruharsha, Mark W. Kankel, Kazuya Hori, Takakazu Yokokura, Sophie Brachat, et al. 2013. Genetic Circuitry of Survival Motor Neuron, the Gene Underlying Spinal Muscular Atrophy. Proceedings of the National Academy of Sciences 110, no. 26: E2371–E2380. Published Version doi:10.1073/pnas.1301738110 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12872186 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA 1 69 2 The genetic circuitry of Survival Motor Neuron, the 70 3 71 4 gene underlying Spinal Muscular Atrophy 72 5 73 6 74 Anindya Sen1*, Douglas N. Dimlich1*, K. G. Guruharsha1*, Mark W. Kankel1*, Kazuya Hori1, Takakazu Yokokura1,2, 7 75 Sophie Brachat3,4, Delwood Richardson3, Joseph Loureiro3, Rajeev Sivasankaran3, Daniel Curtis3, Lance S. Davidow5, Lee 8 5 6 1 1 76 9 L. Rubin , Anne C. Hart , David Van Vactor , and Spyros Artavanis-Tsakonas . * Equal contribution 77 10 1.Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. 2.Current affiliation: Okinawa Science and Technology Graduate University, 78 11 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan 3.Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, 250 79 12 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. 4.Current affiliation: Musculoskeletal Diseases, Novartis Institutes for Biomedical Research, 80 13 Novartis Campus, CH-4002 Basel, Switzerland 5.Department of Stem Cell and Regenerative Biology, Harvard Medical School, Boston, MA 02115, USA. 81 6.Department of Neuroscience, Brown University, 185 Meeting Street Box GL-N, Providence, RI 02912, USA. 14 82 15 Submitted to Proceedings of the National Academy of Sciences of the United States of America 83 16 84 17 The clinical severity of the neurodegenerative disorder Spinal though it probed half of the Drosophila genome, identified only a 85 18 Muscular Atrophy (SMA) is dependent on the levels of func- relatively small number of genes that affected the NMJ phenotype 86 19 tional Survival Motor Neuron (SMN) protein. Consequently, cur- associated with Smn loss of function (13). In particular, it did 87 20 rent strategies for developing treatments for SMA generally focus not identify genes involved in snRNP biogenesis, the molecular 88 21 on augmenting SMN levels. To identify additional potential thera- functionality that is most clearly associated with SMN. 89 22 peutic avenues and achieve a greater understanding of SMN, we As the human disease state results from partial loss of SMN 90 23 applied in vivo, in vitro, and in silico approaches to identify genetic function, we reasoned that a screening paradigm using a hypo- 91 24 and biochemical interactors of the Drosophila SMN homolog. We morphic Smn background, (as opposed to a background that com- 92 25 identified more than three hundred candidate genes that alter an pletely eliminates SMN function) would more closely resemble 93 26 Smn-dependent phenotypeSubmission in vivo. Integrating the results from the genetic condition PDF in SMA. Such a screen would therefore 94 27 our genetic screens, large-scale protein interaction studies and enhance our ability to detect novel elements of the Smn genetic 95 28 bioinformatics analysis, we define a unique interactome for SMN network, and, consequently, add significantly to our efforts to 96 29 which provides a knowledge base for a better understanding of both dissect the Smn genetic circuitry as well as identify potential 97 30 SMA. clinically relevant targets with novel mode of action. 98 31 This complementary screen proved to be more sensitive than 99 32 Genetic screen j Interactome j Proteomics j Spinal Muscular Atrophy our previous screen and led to the identification of over 300 100 33 j Survival Motor Neuron genetic interactors. Taking advantage of the recently established 101 34 Drosophila Protein Interaction Map (DPiM) (14), we related 102 35 INTRODUCTION the newly identified genetic interactors to the SMN protein in- 103 36 teractome, producing an integrated Drosophila SMN biological 104 37 Spinal Muscular Atrophy (SMA), the leading genetic cause of network. Finally, the Drosophila SMN network was evaluated 105 38 infant mortality, results from the partial loss of Survival Motor for its relevance to human biology by mapping Drosophila SMN 106 39 Neuron (SMN) gene activity (1). Numerous studies indicate that network genes to their human homologs, and analyzing the hu- 107 40 SMN functions as a central component of a complex which is re- man network using computational biology tools. The projection 108 41 sponsible for the assembly of spliceosomal small nuclear ribonu- of the Drosophila SMN network derived from this study onto 109 42 cleoproteins (snRNPs) [reviewed in (2)]. SMN is also reported the human network derived from prior knowledge provides a 110 43 to play additional roles, including mRNA trafficking in the axon rational basis for novel SMN functional hypotheses and network 111 44 (3). In humans, SMN is encoded by two nearly identical genes, 112 45 SMN1 and SMN2, which are located on chromosome 5 (4). SMN2 113 differs from SMN1 in that only 10% of SMN2 transcripts pro- 46 Significance 114 47 duce functional SMN due to a single nucleotide polymorphism 115 that results in inefficient splicing of exon 7 and translation of a 48 Spinal Muscular Atrophy (SMA), the leading genetic cause of 116 49 truncated, unstable SMN protein (1, 5, 6). The clinical severity of infant mortality, is a devastating neurodegenerative disease 117 50 SMA correlates with SMN2 copy number, which varies between caused by reduced levels of Survival Motor Neuron (SMN) gene 118 individuals (7). As the small amount of functional SMN2 protein activity. Despite well-characterized aspects of the involvement 51 of SMN in snRNP biogenesis, the gene circuitry affecting SMN 119 52 produced by each copy is capable of partially compensating for 120 SMN1 activity remains obscure. Here, we use Drosophila as a model 53 the loss of the gene function, higher copy numbers of system to integrate results from large-scale genetic and pro- 121 54 SMN2 typically result in milder forms of SMA. Therefore, genetic teomic studies, and bioinformatics analyses to define a unique 122 55 modifiers capable of increasing the abundance and/or specific SMN interactome to provide a basis for a better understanding 123 activity of SMN hold promise as therapeutic targets. of SMA. Such efforts not only help dissect the Smn biology but 56 may also point to potential clinically relevant targets. 124 57 The Drosophila genome harbors a single, highly conserved 125 58 ortholog of SMN1/2, the Survival motor neuron (Smn) gene. SMN Reserved for Publication Footnotes 126 59 is essential for cell viability in vertebrates and Drosophila (8, 9). 127 60 In Drosophila, zygotic loss of Smn function results in recessive 128 61 larval lethality (not embryonic as might be expected) due to the 129 62 rescue of early development by maternal contribution of Smn. 130 63 The larval phenotype includes neuromuscular junction (NMJ) 131 64 abnormalities that are reminiscent of those associated with the 132 65 human disease, rendering this invertebrate organism an excellent 133 66 system to model SMN biology (10-12). We previously described 134 67 a genetic screen for modifiers of the lethal phenotype resulting 135 68 from a complete loss of function Smn allele (13). This screen, 136 www.pnas.org --- --- PNAS Issue Date Volume Issue Number 1--?? 137 205 138 206 139 207 140 208 141 209 142 210 143 211 144 212 145 213 146 214 147 215 148 216 149 Fig. 1. Genetic modifiers of Smn using pupal lethal- 217 ity to screen the Exelixis collection of transposon 150 insertions and their functional roles(A) tubulinGAL4 218 151 (tub-GAL4) directed expression of an inducible Smn- 219 152 RNAi construct (UAS-Smn-RNAiFL26B) leads to a fully 220 153 penetrant pupal lethality where approximately 40% 221 154 of the pupae reach a pigmented developmental stage 222 155 (Control). The remaining 60% die at an earlier un- 223 156 pigmented developmental stage. Introduction of an 224 157 Smn deficiency into this background causes the entire 225 158 population of pupae to die at the unpigmented stage 226 (Smn deficiency), while ectopic Smn expression leads 159 to survival to adulthood of the vast majority of pupae 227 160 (Smn rescue). Introduction of previously isolated en- 228 161 hancers (d02492 and d09801) and suppressors (f05549 229 162 Submission PDFand c05057) of Smn (13) lead to quantitative changes 230 163 in the fractions of pigmented vs. unpigmented pupae. 231 164 (B) The screening strategy to identify genetic modi- 232 fiers of the Smn pupal lethality phenotype using the 165 rd 233 166 Exelixis collection (illustrated for insertions on the 3 234 167 chromosome). The lethal phase for all Smn Tb+ TE 235 (16) pupae in individual test crosses are scored and 168 compared to those observed in control crosses (more 236 169 survival = enhancers, more lethality = suppressors). 237 170 (C) Drosophila functional categories over-represented 238 171 in the genetic modifier list. GO biological functions 239 172 with the highest significance relate to known Smn 240 173 functions such as alternative splicing or SMA affected 241 174 processes (neuronal and muscular). Enrichment signif- 242 175 icance is expressed as the –log10 (p-values). 243 176 244 177 245 178 246 179 intervention points that carry potential for so far unexplored pected fashion (Figure 1A).