An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers.
Numerous disease-associated point mutations exert their effects by disrupting the activity of exonic splicing enhancers (ESEs). We previously derived position weight matrices to predict putative ESEs specific for four human SR proteins. The score matrices are part of ESEfinder, an online resource to identify ESEs in query sequences. We have now carried out a refined functional SELEX screen for motifs that can act as ESEs in response to the human SR protein SF2/ASF. The test BRCA1 exon under selection was internal, rather than the 3'-terminal IGHM exon used in our earlier studies. A naturally occurring heptameric ESE in BRCA1 exon 18 was replaced with two libraries of random sequences, one seven nucleotides in length, the other 14. Following three rounds of selection for in vitro splicing via internal exon inclusion, new consensus motifs and score matrices were derived. Many winner sequences were demonstrated to be functional ESEs in S100-extract-complementation assays with recombinant SF2/ASF. Motif-score threshold values were derived from both experimental and statistical analyses. Motif scores were shown to correlate with levels of exon inclusion, both in vitro and in vivo. Our results confirm and extend our earlier data, as many of the same motifs are recognized as ESEs by both the original and our new score matrix, despite the different context used for selection. Finally, we have derived an increased specificity score matrix that incorporates information from both of our SF2/ASF-specific matrices and that accurately predicts the exon-skipping phenotypes of deleterious point mutations.
Smith
Philip J
PJ
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
Zhang
Chaolin
C
Wang
Jinhua
J
Chew
Shern L
SL
Zhang
Michael Q
MQ
Krainer
Adrian R
AR
D017398:Alternative Splicing / Q000235:genetics*; D020816:Amino Acid Motifs; D001483:Base Sequence; D005544:Forecasting / Q000379:methods*; D019398:Genes, BRCA1; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008962:Models, Theoretical; D009687:Nuclear Proteins / Q000235:genetics*; D016366:Open Reading Frames; D012322:RNA Precursors / Q000378:metabolism; D016601:RNA-Binding Proteins; D050436:Regulatory Elements, Transcriptional; D012107:Research Design; D012689:Sequence Homology, Nucleic Acid; D000068103:Serine-Arginine Splicing Factors
D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors