P

P. mutations in the gene have been identified in four distinct human skeletal muscle diseases, reducing body myopathy (RBM) Flecainide acetate (18C20), X-linked dominant scapuloperoneal disease (21), rigid spine syndrome (22), and X-linked myopathy with postural muscle atrophy (XMPMA) (23), which lead to muscle wasting and in severe cases respiratory and cardiac failure. We previously identified SLIMMER as a FHL1 splice isoform of unknown function (15). In this study, we have identified the proapoptotic protein Siva-1 (24) as a SLIMMER-specific binding partner. Significantly we demonstrate that SLIMMER slows the onset of Siva-1-induced apoptosis of myoblasts, identifying SLIMMER as a novel regulator of myoblast cell survival. EXPERIMENTAL PROCEDURES Materials The following cells and materials were used: C2C12 and COS-1 cells (American Type Culture Collection), Opti-MEM (Invitrogen), Lipofectamine 2000, To-Pro-3, SlowFade Gold anti-fade reagent, and TOP10 (Invitrogen), restriction and DNA-modifying enzymes (Promega, MBI Fermentas, or New England Biolabs), DNA oligonucleotides (Geneworks or Micromon), Big Dye version 3.1 sequencing terminators (Applied Systems), death detection kit, TMR red (Roche Applied Science). All other reagents were from Sigma-Aldrich or BDH Chemicals unless indicated. Plasmids and Cloning Oligonucleotides are listed in supplemental Table 1. The plasmids pCGN (25), pCGN–gal (26), pCGN-FHL1, and pCGN-SLIMMER (15) were described previously. The yeast-2-hybrid constructs pGBKT7, pGBKT7-p53, pGADT7-SV40T, and pACT2 human skeletal muscle library were from Clontech. Yeast baits were PCR-amplified and cloned into pGBKT7 (EcoRI site). Murine KyoT2 sequence was from pEFBOS-myc-KyoT2 (16), a gift from Dr. Tasuku Honjo (Kyoto University, Japan). pET-30a(+) (Novagen), pET-30a(+)-(LIM3+ER) was generated by direct cloning from pGBKT7-(LIM3+ER) into the EcoRI site. pACT2-Siva-1 was isolated from the Y2H library. Siva-1 was PCR-amplified from pACT2-Siva-1 and cloned into pGEX-5x-1 (Amersham Biosciences) (EcoRI site) and into pEFBOS-FLAG (27) (MluI site) (from Dr. Tracey Wilson, Walter and Eliza Hall Institute). pTrio12R-HEN was generated by replacing the GST cassette in pTrio12R-HGN (26, 28) with eGFP (henceforth GFP). This vector encodes a tricistronic mRNA using internal ribosome reentry sequences to direct expression of individual HA- and GFP-tagged proteins and G418 resistance. pTrio12R-HEN retains the XbaI site of the parental pCGN plasmid, facilitating direct cloning of -gal and SLIMMER sequences to generate pTrio12R-HA–gal-EN and pTrio12R- HA-SLIMMER-EN. pTrio12R-HA-SLIMMER-GFP-Siva-1-was cloned by adding the Siva-1 sequence from pEFBOS-FLAG-Siva-1 into pTrio12R-HA-SLIMMER-EN (MluI site). Likewise Flecainide acetate pTrio12R-HA–gal-GFP-Siva-1-was cloned in the same manner. Sequence size upstream of an internal ribosome reentry sequence can alter the expression level of downstream cistrons, so the larger Flecainide acetate -gal mRNA sequence was C-terminally truncated to equalize expression of GFP-Siva-1 between -gal and SLIMMER vectors. Truncation was achieved by EcoRV (internal -gal site) and SmaI (3 to -gal) digestion and religation of the major fragment. Antibodies The following antibodies were used: mouse anti-HA.11 clone 16B12 (1:5000 WB, 1:1000 IF, Covance), mouse anti-polyhistidine clone HIS-1 (1:3000 WB, Sigma- Aldrich), goat anti-GST polyclonal (1:1000 WB, Amersham Biosciences), mouse anti-FLAG clone M2 (1:5000 WB, 3 l immunoprecipitation, Sigma-Aldrich), rabbit anti-FLAG (1:500 IF, Sigma Aldrich), mouse anti-myc (3 l of non-immune antibody solution, Invitrogen), goat anti-SLIMMER (1:50 IF-myoblasts/myotubes, 1:100 IF, skeletal muscle sections, 1:1000 WB, Abcam raised to the SLIMMER unique sequence 231KRTVSRVSHPVSKARK246 (15), rabbit Siva (recognizes both Siva-1 and Siva-2, SHC1 1:10C25 IF, 1:500 WB, Santa Cruz Biotechnology M-175), goat Siva (Santa Cruz Biotechnology C-20) mouse anti–tubulin (1:1000 WB, Zymed Laboratories), mouse anti-GFP clones 7.1/13.1 (1:1000 WB, Roche), mouse anti-skeletal muscle -actinin (1:1000 IF, Sigma-Aldrich), mouse anti-Pax-7 (1:20 IF, R&D Systems), and mouse anti-pan actin Ab-5 clone ACTN05 (1:3333 dilution, NeoMarkers). Also used were horseradish peroxidase-conjugated secondary antibodies (1:10,000 WB, Chemicon) and Alexa-Fluor-conjugated 488, 594, and 647 secondary antibodies (1:600 IF, Molecular Probes). Generation of Rabbit Polyclonal Antibodies A unique peptide sequence of SLIMMER 284YRKNRSLAAPRGPG297 (human GenBankTM accession number “type”:”entrez-protein”,”attrs”:”text”:”AAC72886.1″,”term_id”:”3859849″,”term_text”:”AAC72886.1″AAC72886.1) and Siva-1 82ARGQMLIGPDGRL94 (human GenBank accession number “type”:”entrez-protein”,”attrs”:”text”:”NP_006418″,”term_id”:”11277468″,”term_text”:”NP_006418″NP_006418) were synthesized (Mimotopes), and each was conjugated to diphtheria toxoid by a linking N-terminal cysteine. The conjugated peptide was injected subcutaneously into New Zealand White rabbits. SLIMMER and Siva-1 antibodies were purified by affinity chromatography using their respective immunizing peptide-coupled thiopropyl-Sepharose resin (Mimotopes) and eluted in 0.1 m glycine HCl, pH 2.5. Yeast Two-hybrid Analysis The Matchmaker 3 GAL4-based yeast two-hybrid system was used. The yeast strain AH109, was transformed.