hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc.
The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.
Box 3053 (424 CARL), Duke University Medical Center, Research Drive, Durham, NC 27710, USA.
David M
DM
Mauger
Carolina
C
Lin
Mariano A
MA
Garcia-Blanco
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics*; D020868:Gene Silencing; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D000068103:Serine-Arginine Splicing Factors; D013329:Structure-Activity Relationship
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D009687:Nuclear Proteins; C454924:RBFOX2 protein, human; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D000068103:Serine-Arginine Splicing Factors; D051497:Receptor, Fibroblast Growth Factor, Type 2