Drosophila TAF1 as a model for signal-dependent alternative splicing

研究者的实验室对理解信号转导如何响应发育和应激线索改变前mrna剪接模式感兴趣。在后生动物中，大多数初级转录本都是选择性剪接的，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。虽然在确定反式作用蛋白因子和顺式作用RNA调控元件参与备选剪接机制方面取得了进展，但对激活或抑制这些机制的信号事件知之甚少。本实验室以TAF1 （tbp相关因子1）为模型基因，以果蝇为模型系统，探索信号依赖性选择性剪接的分子机制。TAF1编码TFIID的一个亚基，该亚基指导大多数RNA聚合酶II基因的转录。实验室已经证明，选择性剪接的TAF1 mrna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选择性剪接TAF1 mRNA，该mRNA编码一种结合睾丸特异性启动子DNA的蛋白质异构体，并可能激活男性生殖细胞特异性基因表达程序。因此，对TAF1的研究将对我们理解信号通路如何调节选择性剪接和基因特异性转录产生重大影响。这项研究的目的是描述在DNA损伤反应中调节TAF1选择性剪接的信号通路，这是一个广泛影响细胞生理的事件。实验室将识别信号因子，如启动信号级联反应的受体；效应器，如将起始信号转导到剪接因子的蛋白激酶；剪接因子，如与TAF1前体mrna相互作用的rna结合蛋白；rna调控元件，如调控TAF1替代外显子的内含子剪接沉默子。为了实现这一目标，实验室将在果蝇培养细胞中使用RNA干扰筛选和剪接试验。这项研究意义重大，因为转录因子的信号依赖性替代剪接可能是一种非常常见的调节基因表达的机制，以应对不断变化的细胞环境，然而，文献记载的例子有限，完整的途径尚未描述。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转录调节机制的实验室研究协同，并为实验研究和理解信号通路如何影响果蝇和人类中由选择性剪接调节的众多基因的表达提供一个框架。更广泛的影响。选择性剪接的详细机制以及正常和疾病状态下选择性剪接对基因表达的重要性使得选择性剪接成为一个强大的教育工具，可以通过PI实验室在所有教育水平上进行有效的交流。PI在实验室为研究生、本科生和高中生提供严格的基因、生化和分子训练方面有着良好的记录，其中包括女性和少数族裔。该研究将被纳入由PI指导的真核分子生物学研究生课程。PI还将在针对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，该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.