High-throughput binding analysis determines the binding specificity of ASF/SF2 on alternatively spliced human pre-mRNAs.
High-throughput immunoprecipitation studies of transcription factors and splicing factors have revolutionized the fields of transcription and splicing. Recent location studies on Nova1/2 and Fox2 have identified a set of cellular targets of these splicing factors. One problem with identifying binding sites for splicing factors arises from the transient role of RNA in gene expression. The primary role of most splicing factors is to bind pre-mRNA co-transcriptionally and participate in the extremely rapid process of splice site selection and catalysis. Pre-mRNA is a labile species with a steady state level that is three orders of magnitude less abundant than mRNA. As many splicing factors also bind mRNA to some degree, these substrates tend to dominate the output of location studies. Here we present an in-vitro method for screening RNA protein interactions that circumvents these problems. We screen approximately 4000 alternatively spliced exons and the entire Hepatitis C genome for binding of ASF/SF2, the only splicing factor demonstrated to function as an oncogene. From the pre-mRNA sequences returned in this screen we discovered physiologically relevant ASF recognition element motifs. ASF binds two motifs: a C-rich and a purine rich motif. Comparisons with similar data derived from the hnRNP protein PTB reveals little overlap between strong PTB and ASF/SF2 sites. We illustrate how this method could be employed to screen disease alleles with the set of small molecules that have been shown to alter splicing in search for therapies for splicing diseases.
Chang
Brian
B
Department of Molecular and Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Levin
J
J
Thompson
William A
WA
Fairbrother
William G
WG
D001665:Binding Sites; D005091:Exons; D016678:Genome; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors
D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors