RNA Biology in Oxytricha: a Complex Unicellular Model

 描述(由申请人提供)： RNA分子在池塘栖息的纤毛原生动物中显示出一种新的作用模式，在那里，长的和小的非编码RNA都调节着基因组重塑的复杂过程。Oxytricha中母系遗传的piRNA分子标记了225,000个基因组片段，长的模板RNA在大规模基因组重组期间编程这些DNA片段的顺序。本研究的目的是了解这些长的和小的非编码RNA的生物发生和调控，以及模式生物Oxytricha中RNA生产途径中其他步骤的调控。扩大我们研究和操纵这一新兴遗传系统的工具套件也是一项高度优先的任务，其中一个目标是利用我们对这些生物中的piRNA生物学的知识，开发一种强大的基因敲除工具。这反过来将帮助我们更好地了解基因组重排的遗传学和发育生物学的其他方面，以及它对维持和更广泛地调节基因组完整性的影响。拟议的实验将检查参与这些复杂重排事件的分子和它们背后的机制的性质。从拟议的研究中获得的最直接的见解是了解长和小非编码RNA分子可获得的活动范围，特别是Oxytricha中的piRNA途径，并注意该系统与其他真核生物之间的异同。具体目标包括：1.研究棘毛虫中的piRNAs及其生物发生；2.开发piRNA介导的基因敲除工具作为Oxytricha中的第一个基因敲除工具；3.了解长的非编码模板RNA的产生及其相互作用的伙伴；4.从体细胞染色体中鉴定和功能测试调控基因表达的基序。

 DESCRIPTION (provided by applicant): RNA molecules display a novel mode of action in pond-dwelling ciliated protists, where both long and small noncoding RNAs regulate an intricate process of genome remodeling. Maternally-inherited piRNA molecules in Oxytricha mark which 225,000 segments of the genome to retain, and long, template RNAs program the order of those DNA segments during massive genome rearrangements. The proposed goal of this research is to understand the biogenesis and regulation of both these long and small noncoding RNAs, as well as the regulation of other steps in pathways of RNA production in the model organism Oxytricha. Expanding our suite of tools for studying and manipulating this emerging genetic system is also a high priority, and one aim will capitalize on our knowledge of piRNA biology in these organisms to develop a powerful tool for gene knockout. This will, in turn, help us better understand other aspects of the genetics and developmental biology of genome rearrangements, as well as its implications for the maintenance and regulation of genome integrity, more generally. The proposed experiments will examine the properties of both the molecules that participate in these complex rearrangement events and the machinery underlying them. The most immediate insight to be gained from the proposed research is an understanding of the range of activities accessible to both long and small noncoding RNA molecules, and the piRNA pathway in Oxytricha, in particular, with attention to both differences and similarities between this system and other eukaryotic organisms. Specific aims include: 1. Studying piRNAs and their biogenesis in Oxytricha, 2. Develop piRNA-mediated gene knockout as the first gene knockout tool in Oxytricha, 3. Understand long, noncoding template RNA production and their interacting partners, 4. Identify and functionally test motifs that regulate gene expression from somatic chromosomes.