The investigator's laboratory is interested in understanding how developmental and stress signals 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. Little is known about signaling events that activate or repress alternative splicing mechanisms. The laboratory has shown that alternatively spliced mRNAs of TAF1 (TBP-associated factor 1) 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 the project is to understand how signaling pathways interface with the splicing machinery to regulate TAF1 alternative splicing in response to DNA damage, an event that broadly affects cell physiology. The laboratory plans to use genetic and biochemical approaches to identify the full complement of TAF1 splicing factors, and examine the extent to which TAF1 splicing factors are post-translationally modified by ATM or ATR signaling pathway enzymes in response to DNA damage. The research is significant because signal-dependent alternative splicing is likely an exceedingly common mechanism for regulating gene expression in response to changing cellular environments. However, 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 investigator's 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.
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 results 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 broad scientific audiences, such as university seminars and peer-reviewed research and review articles. Thus, the research will provide opportunities to integrate research, training, and teaching.
Intellectual Merit This project was aimed at understanding how developmentally- and environmentally-induced signaling pathways change pre-mRNA alternative splicing patterns. The majority of pre-mRNAs in metazoan organisms are subject to alternative splicing, making this a principal mechanism for increasing the functional and structural diversity of proteins coded in the genome. While there has been significant research progress in identifying the cellular components responsible for alternative splicing, including trans-acting proteins and cis-acting RNA elements, little is known about the signaling events that control the process. The project used TAF1 (TBP-associated factor 1) as a model gene and Drosophila melanogaster as a model organism, to explore the molecular mechanisms of signal-dependent alternative splicing. TAF1 encodes a subunit of the TFIID complex that directs transcription initiation of most RNA polymerase II genes. The PI’s 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 may activate the male germ cell-specific transcription program. Key findings during the grant period include (1) the identification of alternative splicing regulatory proteins that are controlled in a signal-dependent manner, (2) the determination that signaling pathways regulate alternative splicing by controlling the abundance of splicing regulatory proteins, (3) the determination that a given alternative splicing event can be regulated by multiple, independent signaling pathways that control different splicing regulatory proteins, (4) the determination that chromatin structure plays a role in regulating signal-dependent alternative splicing, (5) the identification of RNA sequence elements that control alternative splicing in a signal-dependent manner, (6) the determination that signaling pathways can regulate alternative splicing through splice site recognition-independent mechanisms, and (7) the determination that signal-dependent alternative splicing is likely to regulate testis-specific transcription. These findings are significant because signal-dependent alternative splicing is likely to be an exceedingly common mechanism for regulating gene expression in response to developmental and environmental stimuli, not only in the model system Drosophila but also in humans and other animals. Moreover, a complete pathway has not been described, so elucidation of a signal-dependent alternative splicing pathway that controls TAF1 expression will provide a framework for experimental investigation and understanding of how signaling pathways impact the expression of many other genes that are regulated by alternative splicing. Broader Impacts The research activities have promoted teaching and training, broadened participation by underrepresented students, and enhanced the research endeavor. The research results obtained on alternative splicing have been used as case studies in a graduate school course, Eukaryotic Molecular Biology. Graduate and undergraduate students have received training in molecular, biochemical, and genetic approaches. They have publish their research and present it at regional, national, and international conferences. These experiences have enabled the students to substantially progress in their research careers. The PI has actively participated in the training and mentoring of underrepresented students. He has served as faculty leader of a summer undergraduate research program, as a mentor of undergraduate summer students in his laboratory, and as chair of a graduate program diversity committee. Finally, the PI has facilitated and contributed to the development of a course called Entering Mentoring that is designed to assist graduate students and post-doctoral fellows in becoming effective mentors of both majority and minority students, and he has facilitated a 2-semester course called Entering Research that is designed to guide students through the undergraduate research experience and prepare them for graduate school research.
