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

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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.