Muscle-specific Alternative Splicing Research Articles

The RNA-binding protein QKI governs a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes.

In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

The RNA-binding protein QKI governs a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes

The RNA-binding protein QKI governs a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes

Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration

Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.

RNA-binding protein CELF6 is cell cycle regulated and controls cancer cell proliferation by stabilizing p21

CELF6, a member of the CELF family of RNA-binding proteins, regulates muscle-specific alternative splicing and contributes to the pathogenesis of myotonic dystrophy (DM), however the role of CELF6 in cancer cell proliferation is less appreciated. Here, we show that the expression of CELF6 is cell cycle regulated. The cell cycle-dependent expression of CELF6 is mediated through the ubiquitin-proteasome pathway, SCF-β-TrCP recognizes a nonphospho motif in CELF6 and regulates its proteasomal degradation. Overexpression or depletion of CELF6 modulates p21 gene expression. CELF6 binds to the 3′UTR of p21 transcript and increases its mRNA stability. Depletion of CELF6 promotes cell cycle progression, cell proliferation and colony formation whereas overexpression of CELF6 induces G1 phase arrest. The effect of CELF6 on cell proliferation is p53 and/or p21 dependent. Collectively, these data demonstrate that CELF6 might be a potential tumor suppressor, CELF6 regulates cell proliferation and cell cycle progression via modulating p21 stability.

MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells.

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein.

RBM24 Is a Major Regulator of Muscle-Specific Alternative Splicing

Cell-type-specific splicing generates numerous alternatively spliced transcripts playing important roles for organ development and homeostasis, but only a few tissue-specific splicing factors have been identified. We found that RBM24 governs a large number of muscle-specific splicing events that are critically involved in cardiac and skeletal muscle development and disease. Targeted inactivation of RBM24 in mice disrupted cardiac development and impaired sarcomerogenesis in striated muscles. In vitro splicing assays revealed that recombinant RBM24 is sufficient to promote muscle-specific exon inclusion in nuclear extracts of nonmuscle cells. Furthermore, we demonstrate that binding of RBM24 to an intronic splicing enhancer (ISE) is essential and sufficient to overcome repression of exon inclusion by an exonic splicing silencer (ESS) containing PTB and hnRNP A1/A2 binding sites. Introduction of ESS and ISE converted a constitutive exon into an RMB24-dependent alternative exon. We reason that RBM24 is a major regulator of alternative splicing in striated muscles.

Abstract 287: Identification of a Novel PKCα Isoform in Heart Transcriptome via RNA Deep Sequencing

The Protein kinase C (PKC) family of Ca2+ and /or lipid-activated AGC kinases are important regulators of multiple cellular responses including cell growth and programmed cell death. Among these, PKCα is the predominant conventional PKC isoform in heart and has been implicated to have a major impact on cardiac function, hypertrophic response and heart failure. Earlier studies have revealed that the conserved AGC-C-terminal tail containing active site cleft is responsible for regulating AGC kinase activity. The phosphorylation events in AGC-C-terminal tail mediate the activity of kinase by regulating kinase maturation, translocation and substrate interaction. In a genome-wide RNA-Seq analysis in mouse heart, we have identified a novel isoform of PKCα-- due to the insertion of a novel exon in its C-terminal tail. This novel exon has a highly conserved sequence from mouse to human, suggesting a conserved role in PKCα regulation. Tissue analysis showed that this PKCα isoform with Novel Exon (PKCαNE) has enriched expression in heart and skeletal muscle, indicating the involvement of a muscle specific alternative splicing event. The insertion of this novel exon lies between the kinase C-Lobe Tether and Active Site Tether. Biochemistry study in cells showed that both PKCα and PKCαNE were activated and translocated from cytosol to membrane and nucleus in response to PMA. However, upon IGF-1 treatment, PKCαNE showed no activation and could not translocate to either membrane or nucleus in contrast to PKCα. To further explore the mechanism for PKCαNE activation, we have determined the phosphorylation events of this novel isoform. Interestingly, the PKCαNE showed a decreased phosphorylation level at the Turn Motif , which could directly impact its activation and maturation. In summary, we have identified a novel isoform of PKCα that is enriched in heart and skeletal muscle. The PKCαNE isoform has different activation profile and its biological significance in cell signaling in physiology and diseases remains to be further explored.

Tissue-Specific Alternative Splicing of Tak1 Is Conserved in Deuterostomes

Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue-specific isoforms of a target gene that expedites a highly conserved signaling pathway.

The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing.

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.

Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells.

The myotubularin-related 1 (MTMR1) gene belongs to a highly conserved family of eucaryotic phosphatases, with at least 11 members in humans. The founder member of this gene family, MTM1, is mutated in X-linked myotubular myopathy, a severe congenital disorder that affects skeletal muscle, and codes for myotubularin, a specific phosphatidylinositol 3-phosphate [PI(3)P] phosphatase. MTM1 and MTMR1 are adjacent on the X chromosome, and the corresponding proteins share 59% sequence identity. In the present study, we investigated the putative role of MTMR1 in myogenesis by analysing its expression pattern in muscle cells during differentiation and in skeletal muscle throughout development. We have identified three novel coding exons in the MTMR1 intron 2 that are conserved between mouse and human, are alternatively spliced, and give rise to six mRNA isoforms. One of the transcripts is muscle-specific and is induced during myogenesis both in vitro and in vivo, and represents the major isoform in adult skeletal muscle. We show that the two main MTMR1 protein muscular isoforms, like myotubularin, efficiently dephosphorylate PI(3)P in vitro. We have also analysed whether MTMR1 alternative splicing is affected in skeletal muscle cells derived from patients with congenital myotonic dystrophy (cDM1), in which mRNA splicing disturbances of specific genes are thought to constitute an important pathogenic mechanism. We found a striking reduction in the level of the muscle-specific isoform and the appearance of an abnormal MTMR1 transcript in differentiated cDM1 muscle cells in culture and in skeletal muscle from cDM1 patients. Our results suggest that MTMR1 plays a role in muscle formation and represents a novel target for abnormal mRNA splicing in myotonic dystrophy.

Muscle-specific Exonic Splicing Silencer for Exon Exclusion in Human ATP Synthase γ-Subunit Pre-mRNA

Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.

Positive and negative intronic regulatory elements control muscle-specific alternative exon splicing of Drosophila myosin heavy chain transcripts.

Alternative splicing of Drosophila muscle myosin heavy chain (MHC) transcripts is precisely regulated to ensure the expression of specific MHC isoforms required for the distinctive contractile activities of physiologically specialized muscles. We have used transgenic expression analysis in combination with mutagenesis to identify cis-regulatory sequences that are required for muscle-specific splicing of exon 11, which is encoded by five alternative exons that produce alternative "converter" domains in the MHC head. Here, we report the identification of three conserved intronic elements (CIE1, -2, and -3) that control splicing of exon 11e in the indirect flight muscle (IFM). Each of these CIE elements has a distinct function: CIE1 acts as a splice repressor, while CIE2 and CIE3 behave as splice enhancers. These CIE elements function in combination with a nonconsensus splice donor to direct IFM-specific splicing of exon 11e. An additional cis-regulatory element that is essential in coordinating the muscle-specific splicing of other alternative exon 11s is identified. Therefore, multiple interacting intronic and splice donor elements establish the muscle-specific splicing of alternative exon 11s.

TGF-β superfamily members do not promote smooth muscle-specific alternative splicing, a late marker of vascular smooth muscle cell differentiation

Smooth muscle (SM) specific alternate splicing of a number of genes is a late marker of the differentiated vascular smooth muscle cell (VSMC) phenotype and is one of the first differentiation characteristics to be lost during de-differentiation and in disease. An understanding of how this aspect of VSMC phenotype is regulated may provide insights into the earliest events of the atherosclerotic process. TGF-β1 is a potent regulator of VSMC differentiation and can induce expression of SM-specific contractile proteins in both pluripotent stem cells and de-differentiated VSMCs. The purpose of this study was to test the hypothesis that members of the TGFβ-superfamily can also effect SM-specific alternative splicing. Firstly, we established that SM-specific splicing of α-tropomyosin, vinculin and SM-myosin heavy chain (MHC) increases during rat fetal/neonatal development and is decreased in VSMCs following balloon-induced carotid injury in the rat. Treatment of cultured rat VSMCs with TGFβ-superfamily members resulted in a significant reduction in the ratio of SM to non-muscle (NM) α-tropomyosin, but did not effect SM-specific alternative splicing of vinculin or SM-MHC. Treatment of pluripotent C3H10T1/2 cells with TGF-β1, which increased SM differentiation marker expression, did not increase SM-specific α-tropomyosin splicing. Taken together, these results demonstrate differential regulation of SM-specific alternative splicing and indicate that although TGF-β1 promotes VSMC differentiation marker expression, TGF-β1 cannot act as the sole trigger of VSMC differentiation.

Differential Regulation of Exonic Regulatory Elements for Muscle-specific Alternative Splicing during Myogenesis and Cardiogenesis

Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pu-del and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

Fibre type-specific expression of p94, a skeletal muscle-specific calpain.

Members of the calpain proteinase family are present in all mammalian cells, although a novel calpain 94 kDa isoform is found almost exclusively in skeletal muscle. p94 is difficult to purify from muscle and recombinant p94 autolyses rapidly when expressed in COS cells. However, in vivo the enzyme may be stabilised by interaction with titin, which has two well-characterised binding sites for p94 at the N2- and M-lines. Both these titin subdomains are subject to muscle-specific alternative splicing, which could be related to p94 expression level or stability in muscles of different fibre type. In this study, porcine longissimus dorsi (LD), trapezius (TZ) and adductor longus (AL) were characterised as fast, intermediate and slow using commercially available specific anti-human fast- and slow-myosin heavy chain mAbs and also by conventional histochemistry. p94 was quantified both in whole muscle preparations and single fibres by western blotting using an anti-p94 antiserum generated by expressing a recombinant p94 sequence as a GST fusion protein antigen. SDS PAGE and immunoblotting revealed a single band of approximately 94 kDa with identical mobility in all muscle and fibre preparations. The intensity of the 94 kDa band was greater in LD (22 +/- 1.7 densitometric units mean +/- SEM, n = 3) than TZ and AL (10 +/- 2.3 and 6 +/- 0.9 units, respectively). Expressed as a ratio relative to actin immunoreactivity, p94 is present in all types of single fibres isolated from TZ, but at a significantly lower level (P < 0.01) in slow type I (0.08 +/- 0.01, n = 9), compared to fast IIA/IIB fibres (0.22 +/- 0.02, n = 26). No evidence was seen for rapid or variable rate of p94 degradation in either type of fibre. These data suggest a positive correlation between p94 expression level and fast glycolytic characteristics in porcine muscle.

Acidic Stimulation Induces a Negative Regulatory Factor That Affects Alternative Exon Selectionin Vitroin Human ATP Synthase γ-Subunit Pre-mRNA

Tissue-specific alternative RNA splicing in human F1γ pre-mRNA produces muscle- and nonmuscle-type isoforms. Muscle-specific exclusion of exon 9 of the F1γ gene is cell-specifically induced by acidic treatment of human fibrosarcoma HT1080 and rhabdomyosarcoma KYM-1 cells. We constructed an F1γ minigene containing parts of exon 8, intron 8, and exon 9 of the human F1γ gene and then analyzed a negative factor that inhibited inclusion of exon 9 via anin vitrosplicing assay using acid-stimulated HT1080 cell nuclear extract.In vitrosplicing of the F1γ minigene, similarly to the β-globin minigene used as a control, was observed in HeLa cell nuclear extract. Next, we performed supplemental experiments using HeLa and HT1080 cell nuclear extracts. The splicing reaction of the F1γ minigene was specifically inhibited by supplementation with nuclear extract from acid-stimulated HT1080 cells, whereas that of human β-globin was not inhibited. These results indicated that acidic stimulation induced a negative factor that blocked inclusion of alternatively spliced exon in the F1γ minigenein vitro,and a regulatory factor acted in a sequence-specific manner for muscle-specific alternative splicing in F1γ pre-mRNA.

MyoD Is Indispensable for Muscle-specific Alternative Splicing in Mouse Mitochondrial ATP Synthase γ-Subunit Pre-mRNA

Muscle-specific alternative RNA splicing is an essential step during myogenesis. In this paper, we report that a muscle-specific transcription factor, MyoD, plays a central role in the induction of muscle-specific alternative splicing during myogenesis. Recently, we reported that muscle and nonmuscle isoforms of the mitochondrial ATP synthase gamma-subunit (F1gamma) were generated by alternative splicing and that acidic stimulation promoted this muscle-specific alternative splicing (Endo, H., Matsuda, C., and Kagawa, Y. (1994) J. Biol. Chem. 269, 12488-12493). In this report, mouse myoblasts are shown to express the muscle-specific isoform of F1gamma after induction with low-serum medium (differentiation medium) or acidic medium, although myotube formation was not detected after acidic induction. RNA blot analysis revealed that the expression levels of both MEF2 and myogenin were increased by low-serum induction, but not by acidic induction. High expression of MyoD mRNA was observed after both types of induction. Overexpression of exogenous MyoD in fibroblasts showed that MyoD was necessary for muscle-specific alternative splicing in both types of induction. Exogenous Id, a negative regulator of MyoD, blocked muscle-specific alternative splicing of F1gamma pre-mRNA by both types of induction. In addition, MyoD induced several muscle-specific alternative splicings, including structural protein pre-mRNAs such as beta-tropomyosin and neural-cell adhesion molecule and transcriptional protein pre-mRNAs such as MEF2A and MEF2D. Our analysis of the two induction systems shows a common MyoD-dependent mechanism of muscle-specific alternative splicing in several genes, independent of MEF2 and myogenin.

Myogenic conversion of NIH3T3 cells by exogenous MyoD family members: dissociation of terminal differentiation from myotube formation.

Myogenic regulatory factors (MRF) of the MyoD family regulate the skeletal muscle differentiation program. Non-muscle cells transfected with exogenous MRF either are converted to the myogenic lineage or fail to express the muscle phenotype, depending on the cell type analysed. We report here that MRF-induced myogenic conversion of NIH3T3 cells results in an incomplete reprogramming of these cells. Transfected cells withdrew from the cell cycle and underwent biochemical differentiation but, surprisingly, terminally differentiated myocytes absolutely failed to fuse into multinucleated myotubes. Analysis of muscle regulatory and structural gene expression failed to provide an explanation for the fusion defectiveness. However, myogenic derivatives of NIH3T3 cells were shown to be unable to accumulate the transcripts encoding muscle-specific isoforms of the integrin subunit beta1D and the transcription factor MEF2D1b2, that depend on muscle-specific alternative splicing. Our results suggest that the fusion into myotubes is under a distinct genetic control that might depend, at least partially, on differential splicing.

Temporally Distinctive Changes of Alternative Splicing Patterns during Myogenic Differentiation of C2C12 Cells

It is well known that skeletal muscle differentiation is accompanied by the appearance of many muscle-specific components and that some of these components are generated through muscle-specific alternative splicing. It is not clear, however, in what manner, including timing, the system that regulates the muscle-specific splicing reactions is constructed during the process of myogenic differentiation. We simultaneously examined the changes in several splicing patterns for the neural cell adhesion molecule (NCAM), beta-tropomyosin, and M-type pyruvate kinase genes during myogenic differentiation of cultured myoblasts using the reverse transcription-polymerase chain reaction method. The NCAM glycosylphosphatidylinositol anchor form increased in preference to the transmembrane form immediately after the induction of differentiation, while the selection of NCAM MSD1 (muscle-specific domain 1) exons started and abruptly increased at about the time when cell-fusion appeared. M2-type pyruvate kinase was gradually substituted for the M1-type molecule. Skeletal muscle-type beta-tropomyosin was predominantly selected even in myoblasts in the growth medium. As a result, each transcript of these genes independently showed a temporally distinctive pattern of change in isoform selecting during the myogenic differentiation of C2C12 cells. These observations suggest that some independent regulation of alternative splicing reactions should occur during myogenic differentiation.