Drosophila TAF1 as a model for signal-dependent alternative splicing

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

The investigator's laboratory is interested in understanding how signals transduced in response to developmental and stress cues alter pre-mRNA splicing patterns. In metazoan organisms a majority of primary transcripts are alternatively spliced, making alternative splicing a principal mechanism for generating functional and structural diversity in proteins. While progress has been made in identifying trans-acting protein factors and cis-acting RNA regulatory elements involved in alternative splicing mechanisms, little is known about signaling events that activate or repress these mechanisms. Using TAF1 (TBP-associated factor 1) as a model gene and Drosophila melanogaster as a model system, the laboratory is exploring molecular mechanisms of signal-dependent alternative splicing. TAF1 encodes a subunit of TFIID, which directs transcription of most RNA polymerase II genes. The laboratory has shown that alternatively spliced TAF1 mRNAs encode proteins with different DNA-binding activities. Developmental signals during spermatogenesis direct alternative splicing of a TAF1 mRNA encoding a protein isoform that binds testis-specific promoter DNA and may activate the male germ cell-specific gene expression program. Thus, the studies of TAF1 will have a major impact on our understanding of how signaling pathways regulate alternative splicing and gene-specific transcription. The goal of this study is to delineate the signaling pathway that regulates TAF1 alternative splicing in response to DNA damage, an event that broadly affects cell physiology. The laboratory will identify signaling factors, such as receptors that initiate signaling cascades; effectors, such as protein kinases that transduce the initiating signal to splicing factors; splicing factors, such as RNA-binding proteins that interact with the TAF1 pre-mRNA; and RNA-regulatory elements, such as intronic splicing silencers that regulate inclusion of TAF1 alternative exons. To achieve this goal the laboratory will use RNA interference screens and splicing assays in Drosophila cultured cells. The research is significant because signal-dependent alternative splicing of transcription factors is likely an exceedingly common mechanism for regulating gene expression in response to changing cellular environments, yet, documented examples are limited and a complete pathway has not been described. Thus, elucidation of a signal-dependent alternative splicing pathway that controls TAF1 expression will synergize with the laboratories studies of mechanisms of transcriptional regulation by TAF1 and provide a framework for experimental investigation and understanding of how signaling pathways impact expression of the multitude of genes in Drosophila and humans regulated by alternative splicing. Broader impact. The detailed mechanisms that underlie alternative splicing and the importance of alternative splicing for gene expression in both normal and disease states make alternative splicing a powerful educational tool that can be effectively communicated by the PI's laboratory at all instructive levels. The PI has a strong track record of providing rigorous genetic, biochemical, and molecular training for graduate, undergraduate, and high school students in the laboratory, including women and underrepresented minorities. The research will be incorporated into a graduate level course on eukaryotic molecular biology directed by the PI. The PI will also present this work in forums that target general audiences, such as university seminars and peer-reviewed research and review articles. Thus, the research will provide opportunities to integrate research, training, and teaching.