Spatiotemporal analysis of RNAi-mediated heterochromatin assembly and maintenance in the fission yeast Schizosaccharomyces pombe

裂殖酵母裂殖酵母中 RNAi 介导的异染色质组装和维持的时空分析

• 批准号: 146863529
• 负责人: Dr. Heiko Schober
• 金额: --
• 依托单位: Friedrich Miescher Institut für Biomedizinische Forschung
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/146863529?language=en
• 关键词: Spatiotemporal; analysis; RNAi; mediated; heterochromatin

RNA interference (RNAi) is a highly conserved, sequence-specific gene regulatory mechanism among eukaryotes, critical for a variety of important biological functions, including defense against viruses and regulation of gene expression during development, and is being pursued as a promising new tool for the treatment of a variety of human diseases including cancer, neurodegenerativ diseases, and viral infections. A surprising link between heterochromatin and the RNAi pathway was discovered a few years ago in fission yeast, and similar mechanisms have more recently been described in various eukaryotes. Thus, RNAi-mediated chromatin modification leading to heterochromatic gene silencing seems to be a widespread phenomenon in eukaryotes. Biochemical and genetic analyses over recent years have greatly improved our understanding of how the RNAi pathway contributes to heterochro-matin assembly in the fission yeast Schizosaccharomyces pombe. However, little work has been done on a cell biological level and important questions such as subcellular localization and temporal regulation of the RNAi pathway remain largely unanswered. Therefore I propose to carry out experiments that tackle these questions. Proteins involved in RNAi-mediated heterochromatin assembly will be visualized in living cells by expressing them as GFP or mCherry fusion proteins from their endogenous loci. These experiments will be complemented by RNA-FISH to visualize heterochromatic RNAs and short interfering RNAs (siRNAs). The goal of this part of the project is to get a comprehensive picture of the spatial organization of RNAi in S. pombe. In addition, it will be tested whether subnuclear compartmentalization of genomic regions plays an important role in heterochromatin assembly. Finally, the temporal organization of RNAi will be analyzed by time-lapse microscopy, or in other words, the distribution and abundance of RNAi proteins and heterochromatic transcripts will be analyzed with respect to the cell cycle. In summary, the results of the proposed experiments will shed light on the process of RNAi-dependent heterochromatin formation and maintenance from a very different angle.

RNA干扰（RNAi）是真核生物中高度保守的、序列特异性的基因调控机制，对多种重要的生物学功能至关重要，包括在发育过程中防御病毒和调节基因表达，并且作为一种有前途的新工具被用于治疗各种人类疾病，包括癌症、神经退行性疾病和病毒感染。几年前在裂变酵母中发现了异染色质和RNAi途径之间的惊人联系，最近在各种真核生物中也发现了类似的机制。因此，rnai介导的染色质修饰导致异染色质基因沉默似乎是真核生物中普遍存在的现象。近年来的生化和遗传分析极大地提高了我们对RNAi途径如何促进裂变酵母裂糖酵母（Schizosaccharomyces pombe）中异染色质组装的理解。然而，在细胞生物学水平上所做的工作很少，并且诸如亚细胞定位和RNAi途径的时间调节等重要问题在很大程度上仍未得到解答。因此，我建议进行一些实验来解决这些问题。参与rnai介导的异染色质组装的蛋白将在活细胞中通过内源性基因座将其表达为GFP或mCherry融合蛋白来可视化。这些实验将辅以RNA-FISH来可视化异染色质rna和短干扰rna （sirna）。该项目的这部分目标是获得S. pombe中RNAi空间组织的全面图像。此外，还将测试基因组区域的亚核区隔化是否在异染色质组装中起重要作用。最后，将通过延时显微镜分析RNAi的时间组织，或者换句话说，RNAi蛋白和异色转录本的分布和丰度将根据细胞周期进行分析。综上所述，这些实验结果将从一个非常不同的角度揭示rnai依赖性异染色质形成和维持的过程。