Dynamic splicing and trans-splicing in the model organism Caenorhabditis elegans

基因组测序项目的一个令人惊讶的发现是，基因组大小或注释基因的数量与相应生物体感知的复杂性之间缺乏令人信服的相关性。另一种基因剪接是生物体复杂性的一种可能机制。然而，许多转录本还没有被注释，包括那些被忽视的低全局表达的转录本，禁止它们用于评估有机体的复杂性。这项研究的目的是以线虫秀丽线虫为模型系统，验证大量全球罕见的转录本在特定发育阶段的特定细胞中具有局部丰富和功能重要性的假设。 拟议的研究包括两个目标。首先，我们将使用与发育阶段和单个细胞相对应的转录组数据来定义这些“全球罕见”但“局部丰富”(GRLA)转录本，这些转录组数据最近才因技术进步而可用。GRLA内含子和外显子是通过搜索识别的内含子和外显子的实例来识别的，这些内含子和外显子分别代表组合的内含子和外显子总数的1%，而在文库中分别对应于特定的发育阶段或细胞类型。我们将研究GRLA转录本的功能意义，并表征它们的转录调控。我们还将研究反式剪接在定义GRLA转录本的瞬时表达中所起的作用。 第二个目的是鉴定线虫GRLA转录本，并比较线虫和线虫中GRLA的进化关系。布里吉萨线虫与线虫关系密切，已成为比较基因组学研究的有力平台。线虫与桥隐杆线虫的进化距离与人与鼠的进化距离相似。尽管存在差异，但它们表现出广泛的染色体组织和共性、发育细胞系以及包括Wnt信号在内的信号通路的保守性。我们通过RNA-Seq注释了桥毛藻中的转录本，并发现了剪接和反式剪接活性的快速进化。尽管有这些发现，但对桥毛线虫转录的研究远远落后于线虫。我们计划构建与发育阶段和细胞类型相对应的RNA-Seq文库，利用它们来鉴定GRLA转录本，并研究它们在桥冠草中的表达和功能，并研究GRLA在线虫中的进化。 这些研究目标的完成将使人们能够研究特定细胞类型在特定发育阶段瞬时基因表达的功能重要性，并探索GRLA转录本在进化中的功能含义。拟议的研究计划还为学生提供了在基因组学、生物信息学和分子生物学方面的极好的跨学科培训机会。

A surprising finding from genome sequencing projects is the lack of convincing correlation between either genome size or number of annotated genes and the perceived complexity of corresponding organisms. Alternative gene splicing is a possible mechanism underlying organismal complexity. However, many transcripts have yet been annotated including the neglected ones with low global expression, prohibiting their use in evaluating organismal complexity. The goal of this research is to test the hypothesis that a large number of globally rare transcripts are “locally abundant” and functionally important in specific cells at specific developmental stages, using the nematode Caenorhabditis elegans as a model system. The proposed research consists of two objectives. First, we will define these “globally rare” but “locally abundant” (GRLA) transcripts using transcriptome data corresponding to developmental stages and to individual cells, which have been made available only recently due to technological advances. GRLA introns and exons are identified by searching for instances of identified introns and exons that represent <1% of the total combined introns and exons, while 1% in libraries that correspond to specific developmental stages or cell types, respectively. We will study the functional significance of GRLA transcripts and characterize their transcriptional regulation. We will also examine the role that trans-splicing plays in defining transient expression of GRLA transcripts. The second objective is to identify GRLA transcripts in the nematode C. briggsae and compare the evolutionary relationship between GRLA in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has been established as a powerful platform for comparative genomics. The evolutionary distance between C. elegans and C. briggsae is similar to that between human and mouse. Despite the divergence, they display extensive conservation of chromosomal organization and synteny, developmental cell linage, and signaling pathways including Wnt signaling. We have annotated transcripts in C. briggsae through RNA-Seq and identified fast evolution of splicing and trans-splicing activities. Despite these findings, research on transcription in C. briggsae lags far behind that in C. elegans. We plan to generate RNA-Seq libraries that correspond to developmental stage and cell types, use them to identify GRLA transcripts and study their expression and function in C. briggsae, and investigate the evolution of GRLA in nematodes. Completion of these research objectives will enable studies of the functional importance of transient gene expression in specific cell types at specific developmental stages, and exploring the functional implications of GRLA transcripts in evolution. The proposed research program also provides excellent interdisciplinary training opportunities for students in genomics, bioinformatics, and molecular biology.