Drosophila TAF1 as a Model for Signal-dependent Alternative Splicing

该项目研究了发育和环境诱导的信号通路如何改变前mrna选择性剪接模式。在后生动物中，大多数前mrna都受到选择性剪接的影响，这是增加基因组编码蛋白质功能和结构多样性的主要机制。虽然在识别负责选择性剪接的细胞成分（包括反式作用蛋白和顺式作用RNA元件）方面已经取得了重大的研究进展，但对控制这一过程的信号事件知之甚少。本项目将以TAF1 （tbp相关因子1）为模式基因，以黑腹果蝇为模式生物，探索信号依赖性选择性剪接的分子机制。TAF1基因编码TFIID复合物的一个亚基，该亚基指导大多数RNA聚合酶II基因的转录起始。PI的实验室已经证明，可选择剪接的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，导致一种可能激活雄性生殖细胞特异性转录程序的蛋白质异构体的产生。因此，对TAF1的研究有望对我们理解信号通路如何调节选择性剪接和细胞类型特异性转录产生重大影响。研究的具体目标是：1)确定信号通路如何控制剪接调节蛋白；2)确定信号通路如何协调控制多个剪接调节蛋白；3)确定染色质结构与选择性剪接之间的因果关系。为了实现这些目标，将使用分子、生化和基因组学方法。实现这些目标将为信号依赖的选择性剪接建立一个范例，并将允许研究更复杂的问题，例如发育信号如何调节选择性剪接。这项研究意义重大，因为不仅在果蝇模型系统中，而且在人类和其他动物中，信号依赖的选择性剪接可能是调节基因表达以响应发育和环境刺激的一种极其常见的机制。此外，完整的途径尚未被描述，因此阐明控制TAF1表达的信号依赖性替代剪接途径将为实验研究和理解信号通路如何影响许多其他由替代剪接调节的基因的表达提供一个框架。更广泛的影响研究活动将促进教学和培训，扩大弱势学生对科学的参与，并加强研究工作。关于选择性剪接的研究结果将在研究生院的真核分子生物学课程中作为案例研究。研究生和本科生将接受分子、生化和遗传方法方面的培训。他们将在地区、国家和国际会议上展示他们的研究成果，并在同行评议的国际期刊上发表论文。这些经历将使学生在他们的研究生涯中取得实质性的进步。PI将积极参与培训和指导代表性不足的学生。他将担任一个夏季本科生研究项目的教师领导，在他的实验室担任本科生暑期学生的导师，并担任一个委员会的主席，以增加研究生项目的多样性。此外，PI是NSF本科生研究经验（REU）项目的共同PI，该项目旨在使全国范围内准备入学和成功参加生物科学研究生培训计划的学生多样化。最后，PI将促进和促进一门名为“进入指导”的课程的发展，该课程旨在帮助研究生和博士后成为来自多数和少数民族背景的学生的有效导师，他将促进一门为期两个学期的名为“进入研究”的课程，该课程旨在指导学生完成本科研究经验，并为研究生院的研究做好准备。

Intellectual MeritThis project addresses 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 major mechanism for increasing the functional and structural diversity of proteins encoded by 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 will use 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. The TAF1 gene 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 TAF1 mRNA, leading to production of a protein isoform that may activate the male germ cell-specific transcription program. Thus, research on TAF1 is expected to have a major impact on our understanding of how signaling pathways regulate alternative splicing and cell type-specific transcription. The specific objectives of the research are: 1) to determine how signaling pathways control splicing regulatory proteins, 2) to determine how signaling pathways coordinately control multiple splicing regulatory proteins, and 3) to determine the cause-and-effect relationship between chromatin structure and alternative splicing. To achieve these objectives, molecular, biochemical, and genomics approaches will be used. Achieving these objectives will establish a paradigm for signal-dependent alternative splicing and will permit investigation of more complex issues, such as how developmental signals regulate alternative splicing. The research is 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, the 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 expression of many other genes that are regulated by alternative splicing. Broader ImpactsThe research activities will promote teaching and training, broaden participation in science by underrepresented students, and enhance the research endeavor. The research results obtained on alternative splicing will be used as case studies in a graduate school course, Eukaryotic Molecular Biology. Graduate and undergraduate students will receive training in molecular, biochemical, and genetic approaches. They will present their research at regional, national and international conferences and publish in peer-reviewed international journals. These experiences will enable the students to substantially progress in their research careers. The PI will actively participate in the training and mentoring of underrepresented students. He will serve as faculty leader of a summer undergraduate research program, as a mentor of undergraduate summer students in his laboratory, and as chair of a committee to increase graduate program diversity. In addition, the PI is co-PI of an NSF Research Experiences for Undergraduates (REU) project aimed at diversifying the pool of students prepared for admission and success to graduate training programs in biological sciences across the nation. Finally, the PI will facilitate and contribute to the development of a course called "Entering Mentoring" that is designed to assist graduate students and post-doctoral fellows in becoming effective mentors for students from majority as well as minority backgrounds, and he will facilitate 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.