Final Report Summary - RNAIEPIMOD (RNA and Epigenetics: RNAi-Driven Chromatin Modifications)
RNA interference (RNAi) is an RNA-dependent process acting in the vast majority of eukaryotes to silence gene expression and the transcriptional and/or post-transcriptional levels. RNAi mediates chromatin modifications in fungi, plants, Drosophila and vertebrates. In the fission yeast Schizosaccharomyces pombe RNA promotes heterochromatin formation, and small interfering RNAs (siRNAs), produced by RNAi, have been proposed to guide the RNA-Induced Transcriptional Silencing (RITS) complex to non-coding and repeated DNA to trigger the formation of heterochromatin.
This project addressed the following questions: What are the physical connections between RITS and other proteins involved in heterochromatin formation and heterochromatin gene silencing? Can RITS localize to other genomic regions, such as euchromatic protein-coding genes or intergenic regions? Does RITS possess other biological functions, beyond the transcriptional silencing of non-coding and repeated DNA? In addition, this project proposed to put together a rather unique combination of approaches consisting of interdisciplinary and innovative methodologies, developed within the time frame of the project, to more classical approaches, such as the yeast molecular genetics and chromatin techniques.
We found new genomic sites to which RITS binds. Surprisingly, the recruitment of RITS depends on an RNA-binding protein named Mmi1 and the exosome, an RNA degradation machinery. Moreover, we found that Mmi1 and the exosome direct the methylation of lysine 9 of histone H3, a mark specifically associated to heterochromatin formation in S. pombe. Other groups have also described similar findings. Additionally, our data favored a role for RNAi in regulating sexual differentiation gene expression program in yeast. In parallel, thanks to the developments we made on a new methodology using the state of the art technology in the field of quantitative proteomics, we found new physical connections between RNAi and chromatin- or RNA-binding proteins. These latter findings bring important insights into the mechanisms of RNAi-mediated heterochromatin formation and gene silencing in general. They also strongly suggest new functions for RNAi.
Altogether, studies conducted in this project tackled fundamental aspects of RNAi-driven chromatin modification and gene silencing. Within the time course of this project, we made technical developments that have now begun to reveal important insights into how RNAi mediates heterochromatin formation as well as possible new functions of RNAi in S. pombe. As these aspects are likely to be conserved through evolution, our studies have the potential to help understand how RNAi-based chromatin modifications and gene silencing take place and are regulated in many other eukaryotes.
This project addressed the following questions: What are the physical connections between RITS and other proteins involved in heterochromatin formation and heterochromatin gene silencing? Can RITS localize to other genomic regions, such as euchromatic protein-coding genes or intergenic regions? Does RITS possess other biological functions, beyond the transcriptional silencing of non-coding and repeated DNA? In addition, this project proposed to put together a rather unique combination of approaches consisting of interdisciplinary and innovative methodologies, developed within the time frame of the project, to more classical approaches, such as the yeast molecular genetics and chromatin techniques.
We found new genomic sites to which RITS binds. Surprisingly, the recruitment of RITS depends on an RNA-binding protein named Mmi1 and the exosome, an RNA degradation machinery. Moreover, we found that Mmi1 and the exosome direct the methylation of lysine 9 of histone H3, a mark specifically associated to heterochromatin formation in S. pombe. Other groups have also described similar findings. Additionally, our data favored a role for RNAi in regulating sexual differentiation gene expression program in yeast. In parallel, thanks to the developments we made on a new methodology using the state of the art technology in the field of quantitative proteomics, we found new physical connections between RNAi and chromatin- or RNA-binding proteins. These latter findings bring important insights into the mechanisms of RNAi-mediated heterochromatin formation and gene silencing in general. They also strongly suggest new functions for RNAi.
Altogether, studies conducted in this project tackled fundamental aspects of RNAi-driven chromatin modification and gene silencing. Within the time course of this project, we made technical developments that have now begun to reveal important insights into how RNAi mediates heterochromatin formation as well as possible new functions of RNAi in S. pombe. As these aspects are likely to be conserved through evolution, our studies have the potential to help understand how RNAi-based chromatin modifications and gene silencing take place and are regulated in many other eukaryotes.