Fission yeast Dicer harbours a unique dsRBD that participates in the spatial organization of the RNAi pathway

Abstract

The term RNAi describes a set of conserved pathways found in most eukaryotes. RNAi is involved in various cellular processes, ranging from the control of gene expression to the establishment of heterochromatic structures. Common to all RNAi pathways is the association of small RNAs with members of the Argonaute family of proteins, forming the core component of a diverse set of protein-RNA complexes. The small RNAs guide these complexes via base-pairing interactions to homologous sequences, which usually results in reduced activity of these targets. The vast majority of small RNA molecules are generated by Dicer enzymes by endonucleolytically processing double-stranded RNAs. The Schizosaccharomyces pombe RNAi pathway is required for the formation of centromeric heterochromatin, and this process has been biochemically characterized in great detail. However, our knowledge about the spatial organization of RNAi in Schizosaccharomyces pombe is very limited. The few experiments performed so far, which have mainly addressed the cellular localization of Dicer and Argonaute, have resulted in data conflicting with the biochemical observations. In my PhD thesis, I have employed yeast genetics, biochemical and proteomics approaches to untangle these conflicting data with a major focus on the investigation of Dicer localization. I was able to demonstrate for the first time that Dicer is primarily localized to the nucleus where it associates with the nuclear periphery. Furthermore, I showed that nuclear retention of Dicer is essential for the formation of centromeric heterochromatin. These findings are consistent with the existing biochemical data and further support our model proposed for the formation of centromeric heterochromatin by the RNAi pathway. My early work demonstrated that nuclear localization of Dicer depends on its C-terminus. In a subsequent collaborative effort, we have solved the solution structure of this C-terminus, which showed that it encodes for a unique type of dsRBD and revealed novel insights into the mechanisms of nuclear retention of Dicer. Importantly, I have found that binding of this domain to RNA is dispensable for RNAi. Rather, the dsRBD represents a novel regulatory module for RNAi, which can mediate nucleo-cytoplasmic shuttling of Dicer. This feature seems to be conserved in higher eukaryotes. My work does also suggest a new function for RNAi in fission yeast, which is different from the well-established RNAi-mediated formation of heterochromatin at the centromeres and is likely to function in controlling environmentally regulated genes. Future studies in our laboratory will focus on the dissection of the mechanistic details of this novel mode of gene regulation.