Marengo Matthew S, Wassarman David A
University of Wisconsin School of Medicine and Public Health, Department of Pharmacology, Molecular and Cellular Pharmacology Program, Madison, WI 53706, USA.
RNA. 2008 Aug;14(8):1681-95. doi: 10.1261/rna.1048808. Epub 2008 Jul 2.
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.
信号依赖的可变剪接对于真核生物基因表达的调控至关重要，但我们对信号如何影响剪接机制的理解仍很有限。解决这一问题的一个模型是果蝇TAF1前体mRNA响应喜树碱（CPT）诱导的DNA损伤信号而发生的可变剪接。用CPT处理果蝇S2细胞会通过ATR信号通路增加TAF1可变盒式外显子12a和13a的包含率。为了评估TAF1前体mRNA序列在可变剪接机制中的作用，我们构建了一个TAF1小基因（miniTAF1）并建立了一种S2细胞剪接检测方法，该方法概括了CPT诱导的内源性TAF1可变剪接的关键方面。对miniTAF1的分析表明，剪接位点强度是控制外显子12a和13a包含的独立且不同机制的基础。将外显子13a的弱5'剪接位点或弱3'剪接位点突变为共有序列足以使外显子13a组成型包含。相比之下，将外显子12a的强5'剪接位点或中度3'剪接位点突变为共有序列仅在存在CPT诱导信号的情况下足以使外显子12a组成型包含。对外显子13的3'剪接位点的类似研究表明，外显子12a的包含涉及组成型和可变剪接位点之间的信号依赖配对。最后，通过进化保守性鉴定的内含子元件对于完全抑制外显子12a的包含或完全激活外显子13a的包含是必需的，并且可能是CPT诱导信号的靶点。总之，这项工作定义了序列元件在响应DNA损伤信号调控TAF1可变剪接中的作用。
