Drosophila TAF1 as a Model for Signal-dependent Alternative Splicing

研究人员的实验室感兴趣的是了解发育和压力信号如何改变前mRNA剪接模式。在后生动物中，大多数初级转录本是交替剪接的，这使得选择性剪接成为产生蛋白质功能和结构多样性的主要机制。人们对激活或抑制选择性剪接机制的信号事件知之甚少。实验室已经证明，TAF1(TBP相关因子1)的选择性剪接mRNAs编码具有不同DNA结合活性的蛋白质。精子发生过程中的发育信号直接对TAF1基因进行选择性剪接，该基因编码一种蛋白质亚型，与睾丸特异的启动子DNA结合，并可能激活雄性生殖细胞特异的基因表达程序。因此，TAF1的研究将对我们理解信号通路如何调控选择性剪接和基因特异性转录产生重大影响。该项目的目标是了解信号通路如何与剪接机制交互作用，以调节TAF1替代剪接，以应对DNA损伤，这一事件广泛影响细胞生理。该实验室计划使用遗传和生化方法来鉴定TAF1剪接因子的全套，并检查TAF1剪接因子在多大程度上被ATM或ATR信号通路酶翻译后修饰以响应DNA损伤。这项研究意义重大，因为信号依赖的选择性剪接可能是一种非常常见的机制，用于调节基因表达，以应对细胞环境的变化。然而，有记录的例子是有限的，还没有描述完整的途径。因此，阐明控制TAF1表达的信号依赖的选择性剪接途径将与研究者对TAF1转录调控机制的研究协同作用，并为实验研究和理解信号通路如何影响受选择性剪接调控的多种基因的表达提供一个框架。选择性剪接的详细机制以及选择性剪接对正常和疾病状态下的基因表达的重要性使选择性剪接成为一种强大的教育工具，可以由PI的实验室在所有指导水平上有效地交流。PI在为实验室的研究生、本科生和高中生，包括女性和代表性不足的少数民族提供严格的遗传、生化和分子培训方面有着良好的记录。研究成果将被纳入由国际生物研究所指导的真核分子生物学研究生水平课程。PI还将在针对广泛科学受众的论坛上介绍这项工作，例如大学研讨会和同行评议的研究和评论文章。因此，研究将提供整合研究、培训和教学的机会。

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.