priRNAs are degradation products of abundant transcripts that bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats ( Halic and Moazed, 2010). Recent studies have revealed that Ago1 (fission yeast Argonaute) and the 3′-5′ exonuclease Triman generate Dicer-independent primal small RNAs (priRNAs) and mature siRNAs to initiate de novo assembly of heterochromatin at centromeric repeats ( Marasovic et al., 2013). This step promotes both further dsRNA and siRNA production and a targeted H3K9 methylation and then completes a positive self-reinforcing feedback loop. RITS also recruits the RNA-dependent RNA polymerase complex (RDRC), which contains the conserved proteins Rdp1 (fission yeast RdRP), Hrr1 and Cid12, and is required for synthesis of double-stranded RNAs substrate for Dcr1 (fission yeast Dicer) to generate duplex siRNAs ( Colmenares et al., 2007 Motamedi et al., 2004). The H3K9me mark provides a binding site for the chromodomain-containing proteins Swi6, Chp1 and Chp2 ( Huisinga et al., 2006). The association of RITS with nascent transcripts via Ago1 leads to the recruitment of the Clr4–Rik1–Cul4 (CLRC) methyltransferase/ubiquitin ligase complex to chromatin via the bridging protein Stc1 ( Bayne et al., 2010 Hong et al., 2005), which is followed by additional cycles of histone 3 lysine 9 (H3K9) methylation (H3K9me) ( Zhang et al., 2008). The RITS complex receives short interfering RNAs (siRNAs) passed from the argonaute chaperone (ARC) complex, which consists of Ago1, Arb1 and Arb2 ( Buker et al., 2007). The RITS complex is composed of Argonaute (Ago1), Chp1 and Tas3, in which Tas3 bridges Ago1 and Chp1 to form a linear architecture and uses the single-stranded guide siRNAs to target homologous chromatin containing nascent noncoding RNAs for silencing ( Jain et al., 2016 Schalch et al., 2011 Verdel et al., 2004). It also contributes partly to forming heterochromatin at telomeres, rDNA regions and the mating-type locus ( Martienssen and Moazed, 2015 Motamedi et al., 2004 Noma et al., 2004 Verdel et al., 2004). The RNAi-induced transcriptional silencing (RITS) complex first identified in fission yeast directly regulates heterochromatic gene silencing, and initiates and maintains heterochromatin formation at centromeres ( Verdel et al., 2004). It has been increasingly clear through extensive studies in the past decades that the RNA interference (RNAi) pathway is one of the main pathways mediating heterochromatin formation and post-transcriptional silencing in fission yeast and a variety of multicellular organisms, such as plants like Arabidopsis thaliana ( Martienssen and Moazed, 2015). In the fission yeast Schizosaccharomyces pombe, large blocks of constitutive heterochromatin domains coat centromeres, telomeres, the silent mating-type locus and rDNA regions, and are essential for functional organization of these chromosomal domains ( Cam et al., 2005 Grewal and Jia, 2007 Moazed, 2009). Heterochromatin is a highly condensed form of chromatin in eukaryotes. Thus, this work uncovers a conserved factor critical for promoting RNAi-dependent and -independent heterochromatin assembly and gene silencing through stabilizing multiple effectors and effector complexes. Finally, our genetic analyses demonstrated that increased heterochromatin spreading restores silencing at subtelomeres in the hsp90-G84C mutant. Our ChIP data suggest that Hsp90 regulates the efficient recruitment of the methyltransferase/ubiquitin ligase complex CLRC by shelterin to chromosome ends and targeting of the SHREC and Fft3 to mating type locus and/or rDNA region. We further discovered that Hsp90 is required for stabilization or assembly of the RNA-induced transcriptional silencing (RITS) and Argonaute siRNA chaperone (ARC) RNAi effector complexes, the RNAi-independent factor Fft3, the shelterin complex subunit Poz1 and the Snf2/HDAC-containing repressor complex (SHREC). Also, H3K9me2 enrichment at heterochromatin regions, especially at the mating-type locus and subtelomeres, is compromised, suggesting heterochromatin assembly defects. Here, we show that the silencing of reporter genes at major native heterochromatic loci (centromeres, telomeres, mating-type locus and rDNA regions) and an artificially induced heterochromatin locus is alleviated in a fission yeast hsp90 mutant, hsp90-G84C. In the fission yeast Schizosaccharomyces pombe, both RNAi machinery and RNAi-independent factors mediate transcriptional and posttranscriptional silencing and heterochromatin formation.
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