Abstract
BackgroundHeterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway.ResultsCcr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.ConclusionsTaken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0018-4) contains supplementary material, which is available to authorized users.
Highlights
Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity
The Mmi1 protein recruits the Ccr4‐Not complex to its target mRNAs As part of a project to understand the regulation of RNA decay in fission yeast, we sought to identify mRNAs associated with the Ccr4-Not complex in vegetatively growing cells
Ccr4 copurified with ~40 mRNAs and five non-coding RNAs that were highly enriched in early meiotic genes (Fig. 1a; Additional file 1: Table S1); these genes are weakly expressed in vegetative cells but are induced during pre-meiotic S phase and meiotic prophase [25]
Summary
Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The control of cytoplasmic mRNA turnover by Ccr4-Not is the best understood function of the complex This role is mediated by Ccr4-Not deadenylase activity, which reduces the length of the poly(A) tail that protects mRNAs from degradation and destabilizes them [1]. Ccr4-Not is involved in nuclear RNA degradation, and associates with the nuclear exosome and the non-canonical polyadenylation complex TRAMP (Trf4/ Air2/Mtr Polyadenylation), which recognizes and tags
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