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Self-Oligomerization Regulates Stability of Survival Motor Neuron Protein Isoforms by Sequestering an Scfslmb Degron

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Self-Oligomerization Regulates Stability of Survival Motor Neuron Protein Isoforms by Sequestering an Scfslmb Degron M BoC | ARTICLE Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCFSlmb degron Kelsey M. Graya,b, Kevin A. Kaiferc, David Baillatd, Ying Wenb, Thomas R. Bonaccia,e, Allison D. Ebertf, Amanda C. Raimera,b, Ashlyn M. Springb, Sara ten Haveg, Jacqueline J. Glascockc, Kushol Guptah, Gregory D. Van Duyneh, Michael J. Emanuelea,e, Angus I. Lamondg, Eric J. Wagnerd, Christian L. Lorsonc, and A. Gregory Materaa,b,* aCurriculum in Genetics and Molecular Biology and Lineberger Comprehensive Cancer Center, bIntegrative Program in Biological and Genome Sciences, Department of Biology and Department of Genetics, and eDepartment of Pharmacology, University of North Carolina, Chapel Hill, NC 27599; cMolecular Pathogenesis and Therapeutics Program, Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211; dDepartment of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77550; fDepartment of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226; gCentre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD15EH, UK; hDepartment of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104 ABSTRACT Spinal muscular atrophy (SMA) is caused by homozygous mutations in hu- Monitoring Editor man SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and Yukiko Yamashita University of Michigan production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor Received: Nov 7, 2017 neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and Accepted: Nov 14, 2017 label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant back- ground. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protec- tive effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers. INTRODUCTION Spinal muscular atrophy (SMA) is a common neuromuscular disor- the disease, which is also the most common, become symptomatic der, recognized as the most prevalent genetic cause of early child- in the first 6 mo of life and rarely live past 2 yr (Prior, 2010; Wee hood mortality (Pearn, 1980). Patients with the most severe form of et al., 2010). Because the onset of symptoms and their severity can vary, SMA has historically been classified into three subtypes (Ogino and Wilson, 2004). More recently, clinicians have recognized that This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/ 10.1091/mbc.E17-11-0627) on November 22, 2017. SMA is better characterized as a continuous spectrum disorder, *Address correspondence to: A. Gregory Matera (matera@unc.edu). ranging from acute (prenatal onset) to nearly asymptomatic (Tiziano Abbreviations used: AAV9, adeno-associated virus serotype 9; Ben, Bendless; Cul1, et al., 2013). Clinically, SMA patients experience degeneration of Cullin1; iPSC, induced pluripotent stem cells; NMJ, neuromuscular junction; S2, motor neurons in the anterior horn of the lower spinal cord (Crawford Schneider 2; SkpA, Skp1-related A; Slmb, supernumerary limbs; SMA, spinal mus- cular atrophy; SMN, survival motor neuron; UPS, ubiquitin proteasome system. and Pardo, 1996). This leads to progressive atrophy of proximal © 2018 Gray et al. This article is distributed by The American Society for Cell Biol- muscle groups, ultimately resulting in loss of motor function and ogy under license from the author(s). Two months after publication it is available symmetrical paralysis. The cause of death is often restrictive respira- to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). tory failure (Kolb and Kissell, 2015). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of SMA typically results from homozygous deletion of the survival the Cell®” are registered trademarks of The American Society for Cell Biology. motor neuron 1 (SMN1) gene (Lefebvre et al., 1995). A small fraction 96 | K. M. Gray et al. Molecular Biology of the Cell of SMA patients have lost one copy of SMN1 and the remaining et al., 2012). Although it is highly similar to human SMN1 and SMN2, copy contains a point mutation (Burghes and Beattie, 2009). Humans the entire open reading frame of fruitfly Smn is contained within a have two SMN paralogues, named SMN1 and SMN2, both of which single exon, and so only full-length SMN protein is expressed in contribute to total cellular levels of survival motor neuron (SMN) Drosophila (Rajendra et al., 2007). When modeled in the fly, SMA- protein. SMN2 exon 7 contains a silent base change that alters splic- causing point mutations recapitulate the full range of phenotypic ing to primarily produce a truncated, unstable protein product severity seen in humans (Praveen et al., 2014; Garcia et al., 2016). called SMN∆7 (Lorson et al., 1999; Monani et al., 1999; Lorson and Using this system, we carried out proteomic profiling of Flag-purified Androphy, 2000). The last 16 amino acids of SMN are replaced in embryonic lysates and identified the SCFSlmb E3 ubiquitin ligase SMN∆7 by four amino acids, EMLA, encoded by exon 8. Current complex as a novel SMN interactor. Importantly, this interaction estimates suggest that SMN2 produces 10–15% of the level of full- is conserved from flies to humans. We show that SCFSlmb binding length protein produced by SMN1 (Lorson et al., 2010). Complete requires a phosphodegron motif located within the SMN self-oligo- loss of SMN is lethal in all organisms investigated to date (O’Hearn merization domain, mutation of which stabilizes SMN∆7 and, to a et al., 2016). Although the amount of full-length protein produced lesser extent, full-length SMN. Additional studies in flies, mice, and by SMN2 is not enough to compensate for loss of SMN1, SMN2 is human cells elucidate a disease-relevant mechanism whereby sufficient to rescue embryonic lethality (Monaniet al., 2000). SMA is SMN protein stability is regulated by self-oligomerization. Other E3 therefore a disease that arises due to a hypomorphic reduction in ligases have been reported to target SMN for degradation in SMN levels (Lefebvre et al., 1995). Furthermore, relative levels of cultured human cells (Hsu et al., 2010; Kwon et al., 2013; Han et al., the SMN protein correlate with the phenotypic severity of SMA 2016). Given our findings in fruit-fly embryos, SMN is likely targeted (Coovert et al., 1997; Lefebvre et al., 1997). by multiple E3 ubiquitin ligases. Whereas a causative link between SMN1 and SMA was estab- lished in the early 1990s, the molecular role of SMN in disease etiol- RESULTS ogy remains unclear. SMN is the central component of a multimeric Flag-SMN interacts with ubiquitin proteasome system protein assemblage known as the SMN complex (Li et al., 2014; proteins Matera and Wang, 2014). The best-characterized function of this We previously generated transgenic flies that express Flag-tagged complex, which is found in all tissues of metazoan organisms, is in SMN proteins in an otherwise null Smn background (Praveen et al., the cytoplasmic assembly of small nuclear ribonucleoproteins 2012). To preserve endogenous expression patterns, the constructs (snRNPs), core components of the spliceosome (Fischer et al., 1997; are driven by the native promoter and flanking sequences. As de- Meister et al., 2001; Pellizzoni et al., 2002). scribed under Materials and Methods, we intercrossed hemizygous Although it is ubiquitously expressed, SMN has also been impli- Flag-SmnWT,SmnX7/SmnD animals to establish a stock wherein all of cated in a number of tissue-specific processes related to neurons the SMN protein, including the maternal contribution, is epitope and muscles. These functions include actin dynamics (Oprea et al., tagged. After breeding them for >100 generations, essentially all of 2008; Ackermann et al., 2013), axonal pathfinding (Fan and Simard, the animals are homozygous for the Flag-SmnWT transgene, but 2002; McWhorter et al., 2003; Sharma et al., 2005), axonal transport second-site recessive mutations are minimized due to the use of two of β-actin mRNP (Rossoll et al., 2003), phosphatase and tensin ho- different Smn null alleles. Adults from this stock display no apparent molog-mediated (PTEN-mediated) protein synthesis pathways defects and have an eclosion frequency (∼90%) similar to that of (Ning et al., 2010), translational regulation (Sanchez et al., 2013), wild-type (Oregon-R) animals. neuromuscular junction formation and function (Chan et al., 2003; We collected (0–12 h) embryos from Flag-SmnWT/WT,SmnX7/D Kariya et al., 2008; Kong et al., 2009; Voigt et al., 2010), myoblast (SMN) and Oregon-R (Ctrl) animals and analyzed Flag-purified fusion (Shafey et al., 2005), and maintenance of muscle architecture lysates by “label-free” mass spectrometry. In addition to Flag-SMN, (Rajendra et al., 2007; Walker et al., 2008; Bowerman et al., 2009). we identified SMN complex components Gemin2 and Gemin3, Ubiquitylation pathways have been shown to regulate the stabil- along with all seven of the canonical Sm-core snRNP proteins (Figure ity and degradation of SMN (Chang et al., 2004; Burnett et al., 2009; 1A). We also identified the U7-specific Sm-like heterodimer Hsu et al., 2010) as well as axonal and synaptic stability (Korhonen Lsm10/11 (Pillai et al., 2003) and the Gemin5 orthologue, Rigor and Lindholm, 2004).
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