Expression of Large Genes: Functions and Mechanisms of Recursive Splicing in Drosophila

智力价值：这个项目将阐明递归剪接的生物学作用和机制，递归剪接是最近发现的一种过程，专门针对被非常大的内含子打断的基因。这些内含子是许多脊椎动物和无脊椎动物基因组的重要组成部分，在基因表达过程中必须从前mRNA转录本中准确地移除它们，以避免引入错义或无义突变，从而排除正确的蛋白质生产。在发育和细胞调控中起关键作用的基因中经常发现极大的内含子。目前的证据表明，递归剪接在促进大内含子基因的正确表达方面具有广泛的作用，但其潜在的机制仍有待阐明。这一过程可能有助于高效和准确地去除内含子，也可能通过剪接机制介导的相互作用刺激基因表达的其他步骤。剪接在基因发生过程中与转录和其他过程有物理和功能上的耦合，新的证据表明剪接本身可以通过影响转录延长、转录的重新启动、多聚腺苷化以及成熟的mRNA从细胞核输出来增强基因的表达。本项目使用的实验系统是果蝇黑腹果蝇。递归剪接在这种生物体中被最广泛地描述，但也有证据表明它也存在于高等动物中。此外，果蝇还为分析递归剪接转录单位提供了强大的遗传和分子资源。具体目的是：(1)研究递归剪接在基因表达中的作用。等位基因替代技术将被用来删除选定但不同基因在其天然染色体位置上的非外显子递归剪接位点。将确定相应基因对发育表型、转录和RNA加工的影响。(2)描述正确使用非外显子递归剪接位点的辅助元件和机制。在果蝇细胞转基因系统中的突变分析将被用来剖析和表征RNA上的序列的功能，这些序列指导使用超双胸基因中递归剪接位点RP3的使用。(3)确定调节递归剪接位点活性和功能的反式作用因子。遗传学方法将被用来确定正确的超双胸和卷曲递归剪接所需的因子和/或调节其在基因功能中的作用(S)。生物化学和分子方法将被用来进一步表征已确定的因素的机制。广泛的影响：这个项目将导致更好地理解前mRNA剪接的机制和策略，以及它们与基因表达和基因结构的其他方面的关系。这对于开发可在农业和动物育种、病虫害防治以及了解种群内基因组序列突变和变异的后果方面产生实际影响的基因控制的综合模型非常重要。该项目产生的信息将作为公开资源并入现有的递归拼接电子数据库。该项目将在三年内为2-4名研究生和6-8名本科生提供研究培训。在这两个层面上，这将涉及实验、计算和比较方法的综合培训。三名本科生研究人员已经为导致该项目的已发表研究做出了重要贡献(在过去两年中，四名本科生合著了两篇论文)。他们已经成为实验和计算生物学领域的顶级博士项目。更多的本科生将参与其中，包括通过增强多样性的计划招收的学生。首席研究员将研究与教育和推广相结合，教授相关学科的本科生和研究生课程，并作为教师参加匹兹堡超级计算中心的少数族裔访问研究职业暑期生物信息学研究所。

Intellectual Merit: This project will elucidate the biological roles and mechanisms of recursive splicing, a recently discovered process that is specific to genes that are interrupted by very large introns. Such introns are an important component of many vertebrate and invertebrate genomes and they must be removed accurately from the pre-mRNA transcripts during gene expression to avoid introducing mis-sense or nonsense mutations that preclude correct protein production. Extremely large introns are frequently found in genes with key roles in development and cellular regulation. Current evidence indicates that recursive splicing has a widespread role in promoting the proper expression of genes with large introns, but the underlying mechanisms remain to be elucidated. This process may facilitate efficient and accurate removal of the introns, or it may stimulate other steps in gene expression through interactions mediated by the splicing machinery. Splicing is coupled physically and functionally to transcription and other processes in mRNA biogenesis, and emerging evidence indicates that splicing itself can enhance gene expression through effects on transcript elongation, re-initiation of transcription, polyadenylation, and export of mature mRNA from the nucleus. The experimental system used in this project is the fruit fly Drosophila melanogaster. Recursive splicing has been characterized most extensively in this organism but suggestive evidence that it also occurs in higher animals has been presented. In addition, Drosophila provides powerful genetic and molecular resources for analysis of recursively spliced transcription units. The specific aims are: (1) Characterize the roles of recursive splicing in gene expression. Allele substitution techniques will be used to delete non-exonic recursive splice sites in selected but diverse genes at their native chromosomal locations. The effects on developmental phenotypes, transcription and RNA processing from the corresponding genes will be determined. (2) Characterize auxiliary elements and mechanisms for correct use of a non-exonic recursive splice site. Mutational analyses in a Drosophila cell transfection system will be used to dissect and characterize the function of sequences on the RNA that direct the use of recursive splice site RP3 in the Ultrabithorax gene. (3) Identify trans-acting factors that mediate the activity and functions of recursive splice sites. Genetic approaches will be used to identify factors required for correct recursive splicing at Ultrabithorax and frizzled and/or to mediate its role(s) in gene function. Biochemical and molecular approaches will be used to further characterize the mechanisms of identified factors.Broader impacts: This project will lead to a better understanding of pre-mRNA splicing mechanisms and strategies and their relation to other aspects of gene expression and gene structure. This is important for developing integrated models of genetic control that can have practical impact in agriculture and animal breeding, pest control, and understanding the consequences of mutation and variations in genome sequence within populations. Information generated by this project will be incorporated into an existing electronic database on recursive splicing as a publicly available resource. The project will provide research training for 2-4 graduate and 6-8 undergraduate students over a three-year period. At both levels, this will involve integrated training in experimental, computational, and comparative approaches. Three undergraduate researchers have already contributed importantly to published studies leading to this project (4 undergraduate co-authorships on 2 papers during the past two years). They have gone on to top Ph.D. programs in experimental and computational biology. Additional undergraduates will be involved, including students recruited through programs to enhance diversity. The principal investigator integrates research with education and outreach by teaching undergraduate and graduate courses in related subjects and by participating as an instructor in the Pittsburgh Supercomputing Center's Minority Access to Research Careers Summer Institute In Bioinformatics.