MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1.
The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.
Kino
Yoshihiro
Y
Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan.
Washizu
Chika
C
Oma
Yoko
Y
Onishi
Hayato
H
Nezu
Yuriko
Y
Sasagawa
Noboru
N
Nukina
Nobuyuki
N
Ishiura
Shoichi
S
D017398:Alternative Splicing; D000818:Animals; D000067878:CELF Proteins; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism; D042622:DNA Repeat Expansion; D005091:Exons; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000067878:CELF Proteins; C422243:CELF4 protein, human; C086321:CLC-1 channel; D018118:Chloride Channels; C493663:MBNL1 protein, human; C499444:MBNL2 protein, human; D022821:RNA Splice Sites; D012333:RNA, Messenger; D016601:RNA-Binding Proteins