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

Morisada Hayakawa,Eiji Sakashita,Eriko Ueno,Shin-Ichi Tominaga,Toshiro Hamamoto,Yasuo Kagawa,Hitoshi Endo

doi:10.1074/jbc.m110138200

Abstract

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.

Highlights

□S The on-line version of this article contains a description of methods of plasmid construction and one table
Exon 9 Is Not Excluded by Acidic Stimulation in HeLa Cells but Is Excluded in HT1080 Cells—The pattern of alternative splicing in F1␥ pre-mRNA represented in Fig. 1A shows that exon 9 is excluded in heart and skeletal muscle tissues
We previously reported that musclespecific exclusion of exon 9 was reversibly induced by cultivation under acidic medium in human fibrosarcoma HT1080 cells and mouse myoblast C2C12 cells (29, 31)

Summary

Introduction

□S The on-line version of this article (available at http://www.jbc.org) contains a description of methods of plasmid construction and one table. Mutation analyses on the F1␥Ex8-9 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. This in vitro system demonstrates that nuclear extracts from acid-stimulated HT1080 cells contain a trans-acting regulatory factor for muscle-specific exclusion of exon 9.

Results

Conclusion