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

Abstract

Muscle-specific isoform of the mitochondrial ATP synthase ␥ subunit (F 1 ␥) 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 ␥ gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F 1 ␥ 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 ␥ Pudel and F 1 ␥ Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum-or acid-stimulated splicing induction system in mouse myoblasts, Pudel 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 ␥ 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 ␥ Pu-rich minigene mice revealed that the F 1 ␥ 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 ␥ pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.Alternative pre-mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of gene expression at the posttranscriptional level. Recently, both cis-acting elements and transacting factors have been reported to varying degrees (1-3). Significant progress has been made in identifying the alternative splicing mechanism involved in the Drosophila sex determination pathway (4 -7).Exonic and intronic cis-acting regulatory elements for RNA splicing have been reported in a number of mammalian genes located near weak 5Ј and 3Ј splice sites, and these elements appear to be involved in the control of stage-or tissue-specific splicing events (5, 8 -13). For example, a majority of exonic splicing enhancer (ESE) 1 elements have been reported to be abundant in purine nucleotides (14,15), ...

Alternative Splicing of the Adenylyl Cyclase Stimulatory G-protein Gαs Is Regulated by SF2/ASF and Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNPA1) and Involves the Use of an Unusual TG 3′-Splice Site

“…Several studies have also indicated that cis-acting elements within exons can function as splicing enhancers and contribute to the accuracy and efficiency of alternative splicing when bound by specific trans-acting factors (30 -32). Purine-rich splicing enhancer sequences (ESEs) have been identified in exons from a number of different tissue-specific or developmentally regulated precursor mRNA species (33,34). These and other degenerate short RNA sequences, which are recognized by one or more SR proteins, are capable of influencing 5Ј-and 3Ј-splice site selection by affecting the activity of weak splice sites in adjacent introns (32,35,36).…”

Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred by α-Tropomyosin Regulatory Elements in Transgenic Mice

“…Moreover, because the transgene was more widely expressed than the Ig gene, it could be seen that neither of the splicing patterns in resting or lipopolysaccharide-stimulated B cells represented a default or regulated state. Analysis of transgene tissue-specific splicing has been carried out for exon 9 of the F1␥ gene (38). However, only two mutant minigenes were tested in transgenic animals so that a direct comparison with a wild type construct could not be made.…”

An erythroid differentiation–specific splicing switch in protein 4.1R mediated by the interaction of SF2/ASF with an exonic splicing enhancer

“…11 These elements become particularly important in the presence of weak splice sites or in alternative splicing. 12,13 Exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs), which are ubiquitous in both constitutively and alternatively spliced exons, account for some of these signals. 14,15 ESEs act as binding sites for serine/ arginine-rich (SR) proteins, a family of highly conserved and structurally similar splicing factors that act at multiple steps of the pre-mRNA splicing pathway.…”