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

智力优势：该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特定于被非常大的内含子打断的基因的过程。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准确地从mrna前转录物中去除它们，以避免引入妨碍正确蛋白质产生的错义或无义突变。在发育和细胞调控中起关键作用的基因中经常发现特大内含子。目前的证据表明，递归剪接在促进含有大内含子的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有效和准确地去除内含子，或者它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。剪接在物理上和功能上与mRNA生物发生中的转录和其他过程耦合，新出现的证据表明，剪接本身可以通过影响转录延伸、转录重新启动、聚腺苷酸化和成熟mRNA从细胞核输出来增强基因表达。本项目使用的实验系统是果蝇。递归剪接的特征在这种生物中最为广泛，但也有证据表明它也发生在高等动物中。此外，果蝇为递归剪接转录单位的分析提供了强大的遗传和分子资源。具体目的是：(1)表征递归剪接在基因表达中的作用。等位基因替代技术将用于删除非外显子递归剪接位点的选择，但不同的基因在其原生染色体位置。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。果蝇细胞转染系统中的突变分析将用于解剖和表征RNA上的序列的功能，这些序列指导了Ultrabithorax基因中递归剪接位点RP3的使用。(3)确定介导递归剪接位点活性和功能的反式作用因子。遗传学方法将用于确定Ultrabithorax和zzzzed正确递归剪接所需的因素和/或调节其在基因功能中的作用。生化和分子方法将用于进一步表征已确定因素的机制。更广泛的影响：该项目将有助于更好地了解pre-mRNA剪接机制和策略，以及它们与基因表达和基因结构其他方面的关系。这对于开发遗传控制的综合模型具有重要意义，这些模型可对农业和动物育种、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产生实际影响。本项目所产生的信息将作为一种公共资源纳入现有的递归拼接电子数据库。该项目将为2-4名研究生和6-8名本科生提供为期三年的研究培训。在这两个层次上，这将涉及实验、计算和比较方法的综合训练。三名本科生研究人员已经为该项目发表的研究做出了重要贡献（在过去两年中，四名本科生共同撰写了两篇论文）。他们继续攻读实验生物学和计算生物学的顶级博士学位。更多的本科生将参与其中，包括通过加强多样性的项目招收的学生。首席研究员通过教授相关学科的本科和研究生课程，并作为讲师参加匹兹堡超级计算中心的少数民族生物信息学研究职业暑期学院，将研究与教育和推广结合起来。

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.