title: A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.
abstract: A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element.
authors: Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla CA 92093-0648, USA.
authors: J M
authors: JM
authors: Yeakley
authors: J P
authors: JP
authors: Morfin
authors: M G
authors: MG
authors: Rosenfeld
authors: X D
authors: XD
authors: Fu
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002116:Calcitonin / Q000096:biosynthesis*; D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis*; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D005091:Exons; D005914:Globins / Q000096:biosynthesis*; D005982:Glutathione Transferase / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis; D016601:RNA-Binding Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic; D014162:Transfection; D018911:Trinucleotide Repeats
chemical_list: D009687:Nuclear Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000068103:Serine-Arginine Splicing Factors; D005914:Globins; D002116:Calcitonin; D005982:Glutathione Transferase; D015740:Calcitonin Gene-Related Peptide