A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing.
Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 pre-mRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 5' splice site or weak 3' splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 5' splice site or moderate 3' splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 3' splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.
Marengo
Matthew S
MS
University of Wisconsin School of Medicine and Public Health, Department of Pharmacology, Molecular and Cellular Pharmacology Program, Madison, WI 53706, USA.
Wassarman
David A
DA
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D004249:DNA Damage; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D051548:Histone Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D022821:RNA Splice Sites; D038621:Regulatory Sequences, Ribonucleic Acid; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID / Q000737:chemistry / Q000378:metabolism*
D029721:Drosophila Proteins; D022821:RNA Splice Sites; D038621:Regulatory Sequences, Ribonucleic Acid; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051548:Histone Acetyltransferases; C069001:TATA-binding protein associated factor 250 kDa