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响应发育和压力信号的信号是如何改变前mRNA剪接模式的。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。虽然在识别参与选择性剪接机制的反式作用蛋白因子和顺式作用RNA调控元件方面取得了进展，但对激活或抑制这些机制的信号事件知之甚少。以TAF1(TBP相关因子1)为模型基因，以黑腹果蝇为模型系统，探索信号依赖选择性剪接的分子机制。TAF1编码TFIID的一个亚基，TFIID指导大多数RNA聚合酶II基因的转录。实验室已经证明，选择性剪接的TAF1mRNAs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。这项研究的目标是描绘调控TAF1选择性剪接以响应DNA损伤的信号通路，这一事件广泛地影响细胞生理。该实验室将确定信号因子，如启动信号级联的受体；效应器，如将起始信号转换为剪接因子的蛋白激酶；剪接因子，如与TAF1前-mRNA相互作用的RNA结合蛋白；以及RNA调节元件，如调节TAF1替代外显子包含的内含子剪接沉默因子。为了实现这一目标，实验室将在果蝇培养细胞中使用RNA干扰筛选和剪接分析。这项研究意义重大，因为转录因子的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响应细胞环境的变化，然而，文献中的例子有限，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TAF1转录调控机制的实验室研究协同作用，并为实验研究和理解信号途径如何影响受选择性剪接调控的多种基因在果蝇和人类中的表达提供一个框架。选择性剪接背后的详细机制以及选择性剪接在正常和疾病状态下对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，PI的实验室可以在所有教育水平上有效地进行沟通。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。这项研究将被纳入由国际和平研究所指导的真核分子生物学研究生水平课程。PI还将在面向普通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 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.