Abstract
BackgroundHeat-shock molecular chaperone proteins (Hsps) promote the loading of small interfering RNA (siRNA) onto RNA interference (RNAi) effector complexes. While the RNAi process is coupled with heterochromatin assembly in several model organisms, it remains unclear whether the Hsps contribute to epigenetic gene regulation. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism and investigated the roles of Hsp90 and Mas5 (a nucleocytoplasmic type-I Hsp40 protein) in RNAi-dependent heterochromatin assembly.ResultsUsing a genetic screen and biochemical analyses, we identified Hsp90 and Mas5 as novel silencing factors. Mutations in the genes encoding these factors caused derepression of silencing at the pericentromere, where heterochromatin is assembled in an RNAi-dependent manner, but not at the subtelomere, where RNAi is dispensable. The mutations also caused a substantial reduction in the level of dimethylation of histone H3 at Lys9 at the pericentromere, where association of the Argonaute protein Ago1 was also abrogated. Consistently, siRNA corresponding to the pericentromeric repeats was undetectable in these mutant cells. In addition, levels of Tas3, which is a protein in the RNA-induced transcriptional silencing complex along with Ago1, were reduced in the absence of Mas5.ConclusionsOur results suggest that the Hsps Hsp90 and Mas5 contribute to RNAi-dependent heterochromatin assembly. In particular, Mas5 appears to be required to stabilize Tas3 in vivo. We infer that impairment of Hsp90 and Hsp40 also may affect the integrity of the epigenome in other organisms.
Highlights
Heat-shock molecular chaperone proteins (Hsps) promote the loading of small interfering RNA onto RNA interference (RNAi) effector complexes
Cells harboring a mutation in the genes encoding heatshock protein 90 (Hsp90) or Mas5 formed near-white colonies and exhibited sensitivity to 5-fluoroorotic acid (5-FOA), suggesting that Hsp90 and Mas5 are required for pericentromeric silencing
In this study, we demonstrated that the S. pombe molecular chaperones Hsp90 and Mas5 are required for the silencing, heterochromatin assembly, and chromatin localization of Ago1 in the pericentromere (Figs. 1 and 3)
Summary
Heat-shock molecular chaperone proteins (Hsps) promote the loading of small interfering RNA (siRNA) onto RNA interference (RNAi) effector complexes. In the S. pombe RNAi pathway, formation of the small interfering RNA (siRNA)-containing effector complex is coupled to heterochromatin assembly [1,2,3,4]. The siRNA duplex is loaded onto a non-chromatin-associated complex called Argonaute small interfering RNA chaperone (ARC), which contains the Argonaute family endoribonuclease Ago. The loading of the siRNA duplex onto the Ago subunit requires the two ARC-specific subunits Arb and Arb, which inhibit the release of the passenger strand [12]. This complex changes its subunit composition to form a chromatin-associated effector complex called RNA-induced transcriptional silencing (RITS) [12, 13]. Chp uses a chromodomain to recognize H3K9me [14], whereas Tas bridges Ago and Chp1 [15, 16]
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