The ER-Bound RING Finger Protein 5 (RNF5/RMA1) Causes Degenerative Myopathy in Transgenic Mice and Is Deregulated in Inclusion Body Myositis Agne` s Delaunay1, Kenneth D. Bromberg2, Yukiko Hayashi3, Massimiliano Mirabella4, Denise Burch1, Brian Kirkwood 1 , Carlo Serra1, May C. Malicdan 3 , Andrew P. Mizisin5, Roberta Morosetti4, Aldobrando Broccolini4, Ling T. Guo5, Stephen N. Jones6, Sergio A. Lira7, Pier Lorenzo Puri1,8, G. Diane Shelton 5 , Ze’ev Ronai1* 1 Signal Transduction, The Burnham Institute for Medical Research, La Jolla, California, United States of America, 2 Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York, United States of America, 3 National Institute of Neuroscience, Tokyo, Japan, 4 Department of Neuroscience, Catholic University, Rome, Italy, 5 Department of Pathology, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 6 Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 7 Immunobiology Center, Mount Sinai School of Medicine, New York, New York, United States of America, 8 Dulbecco Telethon Institute at Fondazione European Brain Research Institute (EBRI)/S.Lucia 00134, Rome, Italy Abstract Growing evidence supports the importance of ubiquitin ligases in the pathogenesis of muscular disorders, although underlying mechanisms remain largely elusive. Here we show that the expression of RNF5 (aka RMA1), an ER-anchored RING finger E3 ligase implicated in muscle organization and in recognition and processing of malfolded proteins, is elevated and mislocalized to cytoplasmic aggregates in biopsies from patients suffering from sporadic-Inclusion Body Myositis (sIBM). Consistent with these findings, an animal model for hereditary IBM (hIBM), but not their control littermates, revealed deregulated expression of RNF5. Further studies for the role of RNF5 in the pathogenesis of s-IBM and more generally in muscle physiology were performed using RNF5 transgenic and KO animals. Transgenic mice carrying inducible expression of RNF5, under control of b-actin or muscle specific promoter, exhibit an early onset of muscle wasting, muscle degeneration and extensive fiber regeneration. Prolonged expression of RNF5 in the muscle also results in the formation of fibers containing congophilic material, blue-rimmed vacuoles and inclusion bodies. These phenotypes were associated with altered expression and activity of ER chaperones, characteristic of myodegenerative diseases such as s-IBM. Conversely, muscle regeneration and induction of ER stress markers were delayed in RNF5 KO mice subjected to cardiotoxin treatment. While supporting a role for RNF5 Tg mice as model for s-IBM, our study also establishes the importance of RNF5 in muscle physiology and its deregulation in ER stress associated muscular disorders. Citation: Delaunay A, Bromberg KD, Hayashi Y, Mirabella M, Burch D, et al (2008) The ER-Bound RING Finger Protein 5 (RNF5/RMA1) Causes Degenerative Myopathy in Transgenic Mice and Is Deregulated in Inclusion Body Myositis. PLoS ONE 3(2): e1609. doi:10.1371/journal.pone.0001609 Editor: Dong-Yan Jin, University of Hong Kong, China Received December 17, 2007; Accepted January 3, 2008; Published February 13, 2008 Copyright: 2008 Delaunay 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: Support by NCI (CA097105 to ZR) is gratefully acknowledged. Competing Interests: The authors have declared that no competing interests exist. *E-mail: ronai@burnham.org Introduction Skeletal muscles are continually subjected to remodeling as a consequence of normal mechanical or metabolic stress, as an adjustment of muscle mass to muscle load during normal activity, or as a response to a muscle disease. While exercise leads to increased protein synthesis and build-up of muscle mass (hypertrophy), disuse is associated with degradation of muscle components (atrophy) [1,2]. Atrophy can also occur during normal aging and as a result of pathological conditions such as primary muscle or peripheral nerve disease, cachexia and cancer [3]. During muscle remodeling, changes in protein turnover due to controlled degradation are balanced by new protein synthesis. The ubiquitin-proteasome system (UPS) is a key player in muscle dynamics both in normal and in pathological conditions. The UPS is able to selectively target for degradation structural and regulatory components following their ubiquitination by different E3 ligases. Increased general UPS components, and in particular specific E3 ligases, have been implicated in muscle remodeling [2,4,5]. The important role of UPS in muscle atrophy is exemplified by MuRF1, a RING finger E3 ligase, and MAFbx, an F-Box protein component of the Skp1-Cullin F-Box protein (SCF) complex [6]. Increased expression of these ligases in mouse models of muscle disuse results in degradation of structural components of the muscle fibers such as the myofibrillar proteins (myosin, titin, troponin, nebulin, myotilin; [7] [8]. Conversely, induced muscle atrophy is prevented in MuRF1 and MAFbx Knockout (KO) mice [6,7]. The E3 ligase MuRF3 has also been shown to elicit a protective effect on myocardial function through its action on two types of filamins involved in muscle mechan- osensing and signaling [9]. Other E3 ubiquitin ligases, such as ZNF216, have been implicated in general muscle physiology and muscle atrophy [10]. Ozz-E3 was identified as an E3 ligase that targets PLoS ONE | www.plosone.org 1 February 2008 | Volume 3 | Issue 2 | e1609