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

RNA干扰(RNAi)是真核生物中一种高度保守的、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，包括对病毒的防御和发育过程中基因表达的调节，正被视为治疗包括癌症、神经退行性疾病和病毒感染在内的多种人类疾病的一种有前途的新工具。几年前在分裂酵母中发现了异染色质和RNAi途径之间令人惊讶的联系，最近在各种真核生物中也描述了类似的机制。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途径如何在裂殖酵母中促进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，重要的问题，如RNAi途径的亚细胞定位和时间调控，在很大程度上仍然没有答案。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质将通过将它们表达为来自其内源位点的GFP或mCherry融合蛋白而在活细胞中可视化。这些实验将由RNA-FISH补充，以可视化异染色质RNA和短干扰RNA(SiRNAs)。该项目这一部分的目标是全面了解S.pombe中RNAi的空间组织。此外，还将测试基因组区域的亚核区划是否在异染色质组装中发挥重要作用。最后，将用时间推移显微镜分析RNAi的时间组织，换句话说，将根据细胞周期分析RNAi蛋白和异染色质转录本的分布和丰度。总之，拟议的实验结果将从一个非常不同的角度阐明依赖RNAi的异染色质的形成和维持过程。

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.