The long-term goal of this work is to understand how the RNA interference (RNAi) pathway regulates heterochromatic gene silencing in fission yeast. RNAi is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels and is triggered by double stranded RNA molecules that are processed to small interfering RNAs (called siRNAs). siRNAs guide the inactivation of complementary target nucleic acids by effector complexes. Our laboratory has purified the RITS (RNA-Induced Transcriptional Gene Silencing) complex, containing the Ago1, Chp1, and Tas3 proteins, which physically links the RNAi pathway to heterochromatin. In addition, we have purified the RNAi complexes that synthesize dsRNA, process it into siRNAs, and help load siRNAs onto the RITS complex. We have provided evidence that these RNAi complexes localize to specific chromosome regions via base pairing interactions between siRNAs and nascent non-coding transcripts. This localization leads to recruitment of a histone H3 methyltransferase complex, called the CRC (Clr4-Rik1-Cul4) complex, which methylates histone H3 on lysine-9 and promotes heterochromatin formation. Our recent results suggest that cis-synthesis of dsRNA, at sites of heterochromatin assembly, is required for H3 lysine-9 methylation. The goals of this proposal are to understand how RITS and dsRNA synthesis recruit the CRC histone methyltransferase complex and how the nascent RNA transcript controls the spreading of heterochromatin. In addition, we will perform a biochemical dissection of steps that are involved in the formation of heterochromatic siRNAs.
This project addresses the role of RNA interference (RNAi), a conserved RNA silencing pathway, in gene silencing in the nucleus. Components of this pathway are involved in regulation of cell differentiation and cancer in humans. A basic understanding of the role of RNAi in gene silencing will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention.
|Yu, Ruby; Jih, Gloria; Iglesias, Nahid et al. (2014) Determinants of heterochromatic siRNA biogenesis and function. Mol Cell 53:262-76|
|Egan, Emily D; Braun, Craig R; Gygi, Steven P et al. (2014) Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex. RNA 20:867-81|
|Buker, Shane M; Motamedi, Mohammad R (2011) Purification of native Argonaute complexes from the fission yeast Schizosaccharomyces pombe. Methods Mol Biol 725:1-13|
|Moazed, Danesh (2011) Mechanisms for the inheritance of chromatin states. Cell 146:510-8|
|Halic, Mario; Moazed, Danesh (2010) Dicer-independent primal RNAs trigger RNAi and heterochromatin formation. Cell 140:504-16|
|Gerace, Erica L; Halic, Mario; Moazed, Danesh (2010) The methyltransferase activity of Clr4Suv39h triggers RNAi independently of histone H3K9 methylation. Mol Cell 39:360-72|
|Motamedi, Mohammad R; Hong, Eun-Jin Erica; Li, Xue et al. (2008) HP1 proteins form distinct complexes and mediate heterochromatic gene silencing by nonoverlapping mechanisms. Mol Cell 32:778-90|
|Mekhail, Karim; Seebacher, Jan; Gygi, Steven P et al. (2008) Role for perinuclear chromosome tethering in maintenance of genome stability. Nature 456:667-70|
|Buhler, Marc; Spies, Noah; Bartel, David P et al. (2008) TRAMP-mediated RNA surveillance prevents spurious entry of RNAs into the Schizosaccharomyces pombe siRNA pathway. Nat Struct Mol Biol 15:1015-23|
|Hong, Eun-Jin Erica; Villen, Judit; Gerace, Erica L et al. (2005) A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation. RNA Biol 2:106-11|