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doi: 10.1242/10.1242/jcs.00108

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Two mammalian UNC-45 isoforms are related to distinct cytoskeletal and muscle-specific functions

Maureen G. Price¹, Megan L. Landsverk², Jose M. Barral^2,* and Henry F. Epstein^1,2,

¹ Department of Neurology, Baylor College of Medicine, Houston, TX 77030, USA
² The Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA
^* Present address: Max-Planck Institute for Biochemistry, D82152 Martinsried, Germany

View larger version (94K): [in a new window]   Fig. 1. Alignment of general cell (GC) and striated muscle (SM) isoforms of human (hUNC-45) and mouse (mUNC-45) UNC-45. The TPR domain (red), central region (green) and UCS domain (purple) are indicated. Identity is indicated by black boxes, similarity by grey boxes. The GC and SM isoforms are 55-56% identical, while respective human and mouse isoforms are 94-95% identical. Intron/exon boundaries, indicated by arrowheads (blue for GC, red for SM), are identical for the same mouse and human isoform, and furthermore, are identical for homologous coding regions in GC and SM genes. Red asterisks indicate conserved residues corresponding to C. elegans temperature-sensitive UNC-45 mutations (Barral et al., 1998), and blue asterisks indicate S. pombe Rng3p mutations (Wong et al., 2000). Blue and red arrows indicate the chymotryptic and tryptic cleavage sites, respectively, present in C. elegans UNC-45 (J.M.B. and H.F.E., unpublished).

View larger version (120K): [in a new window]   Fig. 2. Alignment of UCS domains from fungal homologues and animal UNC-45 proteins. Only one UCS protein is present in fungi and invertebrates. Two UNC-45 isoforms, SM (striated muscle) and GC (general cell) are present in the bony fish Fugu rubripies, suggesting that the gene divergence is ancient. Note that the fungal proteins have extra residues in five places, compared with the animal proteins. Identity is indicated by black boxes, similarity by grey boxes. There is a clear distinction between invertebrate and vertebrate proteins. Blue arrowheads indicate completely conserved residues, and red arrowheads indicate 90-95% identity. Asterisks indicate conserved residues corresponding to C. elegans temperature-sensitive UNC-45 (Barral et al., 1998) and S. pombe Rng3p mutations (Wong et al., 2000). Ano. gam., Anopheles gambiae (coding sequence compiled from acc. no. AAAB01008844.1); Dros. melan., Drosophila melanogaster (acc. no. AAK93568); C. elegans, Caenorhabditis elegans [(Venolia et al., 1999) acc. no. AAD01976]; C. briggsae, Caenorhabditis briggsae [(Venolia et al., 1999) acc. no. AAD01960]; Daniorerio, Danio rerio (acc. no. AAL57031); Fugurub., Fugu rubripies (SM, coding sequence compiled from JGI Fugu genome project scaffold 6404; GC, coding sequence compiled from JGI Fugu genome project scaffold 465); Human, Homo sapiens (SM, coding sequence compiled from AC022916; GC, acc. nos. BAB20273, AAH06214); N.c., Neuropsora crassa (direct submission acc. no. T49461); Mouse, Mus musculus (SM, coding sequence compiled from acc. no. AL603745; GC, acc. no. AAH04717); P.a., Podospora anserina [(Berteaux-Lecellier et al., 1998) acc. no. CAA76144]; S.c., Saccharomyces cerevisiae [(Jansen et al., 1996) acc. no. CAA63795]; S.p., Schizosaccharomyces pombe [(Wong et al., 2000) acc. no. O74994], Xen. laev., Xenopus laevis (coding sequence compiled from acc. nos. AW765658, BJ074261 and BJ091725), Xen. trop., Xenopus tropicalis (coding sequence compiled from acc. nos. AL650279, AL656414, AL661438 and AL646713).

View larger version (59K): [in a new window]   Fig. 3. The two murine UNC-45 mRNAs are differentially expressed. Duplicate northern blots of total RNA from various adult mouse organs and from 12 day embryos were labeled with isoform-specific probes (SM, skeletal muscle; He, heart; Ut, uterus; In, large intestine; Ki, kidney; Sp, spleen; Lu, lung; Br, brain; Li, liver; Ov, ovary; Em, embryo). The GC UNC-45 isoform was detected in all adult organs examined, and the SM UNC-45 isoform was detected in organs consisting mainly of striated muscle. A small amount of GC UNC-45 mRNA was also present in striated muscle tissues. The minor SM UNC-45 band in the lung sample is of unknown origin. Labeling of 18 S RNA indicated comparable loading.

View larger version (53K): [in a new window]   Fig. 4. The SM unc-45 gene is strongly expressed in the contractile heart during embryogenesis. In situ hybridization was done on whole mouse embryos using gene-specific anti-sense and sense control probes. (A) SM UNC-45 mRNA was detected in the functional heart, as shown here at 8.75 days. (B) Comparison with an 8.5 day embryo labeled with the sense control demonstrates that the SM UNC-45 mRNA was not expressed in other tissues above background level.

View larger version (60K): [in a new window]   Fig. 5. SM UNC-45 mRNA expression begins when skeletal myogenic cells fuse to form myotubes while expression of GC UNC-45 mRNA decreases in differentiating cultures. (A) Phase-contrast images of C2C12 cultures at different stages of myogenesis; P shows proliferating myoblasts, F shows fusing myocytes, Y mt shows young myotubes, and O mt shows older myotubes. Bar, 20 µm. Total RNA was prepared from these cultures. (B) Differential expression of GC and SM UNC-45 mRNA at the proliferative stage and during the stages of myotube formation, detected in duplicate northern blots. GAPDH mRNA levels show that approximately equal amounts of RNA were loaded per lane. (C) Quantification of the expression levels of GC (gray bars) and SM (black bars) UNC-45 mRNAs at different stages of in vitro muscle differentiation, shown as pixels per band normalized to the levels of GAPDH mRNA per sample (mt, myotube cultures).

View larger version (65K): [in a new window]   Fig. 6. Cell proliferation was retarded when levels of GC UNC-45 mRNA were reduced. (A) Treatment of C2C12 cultures with 2.5 µM GC UNC-45 antisense oligonucleotides suppressed GC UNC-45 mRNA expression by 50%. Northern blot of GC UNC-45 mRNA in 10 µg of total RNA from proliferating cells treated 3 days with none (0), a negative control (C), GC UNC-45 antisense (G) or SM UNC-45 (S) antisense oligonucleotide. Note that SM UNC-45 mRNA is not expressed in proliferating cells (see Fig. 5). GAPDH labeling demonstrated equal RNA loads per lane. (B) Phase-contrast images of proliferating C2C12 cells treated with a negative control or GC UNC-45 antisense oligonucleotide, demonstrating reduced proliferation in GC UNC-45 antisense treated cultures. Cells treated with no or SM UNC-45 antisense oligonucleotide resembled control cultures. Bar, 50 µm. (C) The relative reduction in cell proliferation caused by 3 days of GC or SM UNC-45 antisense treatment, determined from pair-wise comparisons with cultures treated with the control oligonucleotide. Total DNA content was used to gauge cell number. The mean of five experiments is given ±s.d.

View larger version (83K): [in a new window]   Fig. 7. GC and SM UNC-45 have different functions in muscle differentiation. C2C12 skeletal myocytes were treated for 8 days with none (0), negative control (C), GC UNC-45 antisense (G) or SM UNC-45 (S) antisense oligonucleotides. (A) SM UNC-45 antisense treatment specifically reduced SM UNC-45 mRNA expression by 50%. Northern blot of 10 µg total RNA from myotube-containing cultures treated for 8 days, labeled with a SM UNC-45 and a GAPDH probe. SM UNC-45 mRNA was expressed at control levels in the GC UNC-45 antisense-treated cultures, reflecting the presence of short myotubes. (B) Muscle differentiation was assessed by immunostaining with EA53 antibody to sarcomeric -actinin. Nuclei were stained blue with DAPI. Unstained cells appear black with blue nuclei. Robust, multinucleated myotubes were observed in cultures treated with the negative control oligonucleotide. Cultures not treated were indistinguishable from these. SM UNC-45 antisense-treated cultures had fewer myotubes than controls. The majority of differentiated cells in GC UNC-45 antisense-treated cultures were short myotubes with one to four nuclei, suggesting that GC UNC-45 is necessary for normal levels of myoblast fusion. Bar, 100 µm. (C) Subnormal levels of SM UNC-45 reduced the extent of striated myofibrils, as shown by this high magnification view of sarcomeric -actinin staining in myotubes from negative control and SM UNC-45 antisense-treated cultures. Bar, 10 µm. (D) Graphical representation of the effects of GC and SM UNC-45 antisense treatment on myotube differentiation in vitro. Bar colors are: stippled for no treatment; white for control oligonucleotide; black for SM antisense; and grey for GC antisense. Six hundred cells were counted for each sample; those not represented here were mononuclear. GC UNC-45 appears to function in cell fusion while SM UNC-45 has a role in fusion and formation of striated myofibrils within myotubes.

View larger version (106K): [in a new window]   Fig. 8. Reduction of SM UNC-45 mRNA did not alter skeletal myosin expression in myotube cultures. A Commassie blue-stained 7.5% polyacrylamide gel shows equal amounts of total protein from myotube cultures treated for 8 days with none (0), a negative control (C), GC UNC-45 antisense (G) or SM UNC-45 (S) antisense oligonucleotide. Myosin heavy chain (MHC) (arrow) and molecular weight markers are indicated. The lower panel shows the results of immunoblotting with MF 20 antibody to specifically detect skeletal MHC in the samples above. Equal amounts of skeletal MHC were in all but the GC UNC-45 antisense-treated cultures, which contained more myoblasts and few large myotubes.

View larger version (13K): [in a new window]   Fig. 9. Schematic summary of the proposed roles of GC and SM UNC-45, based on the processes inhibited by antisense reduction of specific mRNAs. GC UNC-45 functions in cytoskeletal processes in proliferating, non-differentiating cells. Both GC and SM UNC-45 function in myotube formation through cell fusion. Myofibril formation requires both GC and SM UNC-45, consistent with the fact that the cytoskeleton is necessary for the development and maintenance of organized myofibrils.