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Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast.

Cotobal C, Rodríguez-López M, Duncan C, Hasan A, Yamashita A, Yamamoto M, Bähler J, Mata J - Epigenetics Chromatin (2015)

Bottom Line: 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.Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT

Background: 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 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.

Results: Ccr4-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.

Conclusions: Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

No MeSH data available.


Related in: MedlinePlus

Ccr4-Not associates with Mmi1 RNA targets. Venn diagrams comparing RIP-chip and microarray-based gene expression experiments. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The numbers below the diagrams show the p value of the observed overlap (see “Methods”). a mRNAs associated with Ccr4 and early meiotic genes. b mRNAs associated with Ccr4 and with Caf1. c mRNAs associated with Ccr4 and with Rcd1. d mRNAs associated with Mmi1 and genes overexpressed in mmi1Δ mei4Δ mutants. e mRNAs associated with Ccr4 and with Mmi1.
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Fig1: Ccr4-Not associates with Mmi1 RNA targets. Venn diagrams comparing RIP-chip and microarray-based gene expression experiments. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The numbers below the diagrams show the p value of the observed overlap (see “Methods”). a mRNAs associated with Ccr4 and early meiotic genes. b mRNAs associated with Ccr4 and with Caf1. c mRNAs associated with Ccr4 and with Rcd1. d mRNAs associated with Mmi1 and genes overexpressed in mmi1Δ mei4Δ mutants. e mRNAs associated with Ccr4 and with Mmi1.

Mentions: 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. We purified epitope-tagged Ccr4 together with interacting RNAs, and used DNA microarrays to identify the bound RNAs (RNA-binding protein immuno precipitation analysed with DNA chips, or RIP-chip). Ccr4 copurified with ~40 mRNAs and five non-coding RNAs (ncRNAs) 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]. Two other subunits of the complex, Caf1 and Rcd1 (the S. pombe Caf40 homologue), interacted with the same set of RNAs, suggesting that the whole Ccr4-Not complex associates with these mRNAs (Fig. 1b, c; Additional file 1: Table S1). Although the overlap between mRNAs associated with different subunits was highly significant, it was far from complete. This may be due to technical noise (some interactions may be lost during the purification) or reflect the existence of multiple complexes containing different subunits. At present, we cannot distinguish between these two possibilities.Fig. 1


Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast.

Cotobal C, Rodríguez-López M, Duncan C, Hasan A, Yamashita A, Yamamoto M, Bähler J, Mata J - Epigenetics Chromatin (2015)

Ccr4-Not associates with Mmi1 RNA targets. Venn diagrams comparing RIP-chip and microarray-based gene expression experiments. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The numbers below the diagrams show the p value of the observed overlap (see “Methods”). a mRNAs associated with Ccr4 and early meiotic genes. b mRNAs associated with Ccr4 and with Caf1. c mRNAs associated with Ccr4 and with Rcd1. d mRNAs associated with Mmi1 and genes overexpressed in mmi1Δ mei4Δ mutants. e mRNAs associated with Ccr4 and with Mmi1.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536793&req=5

Fig1: Ccr4-Not associates with Mmi1 RNA targets. Venn diagrams comparing RIP-chip and microarray-based gene expression experiments. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The numbers below the diagrams show the p value of the observed overlap (see “Methods”). a mRNAs associated with Ccr4 and early meiotic genes. b mRNAs associated with Ccr4 and with Caf1. c mRNAs associated with Ccr4 and with Rcd1. d mRNAs associated with Mmi1 and genes overexpressed in mmi1Δ mei4Δ mutants. e mRNAs associated with Ccr4 and with Mmi1.
Mentions: 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. We purified epitope-tagged Ccr4 together with interacting RNAs, and used DNA microarrays to identify the bound RNAs (RNA-binding protein immuno precipitation analysed with DNA chips, or RIP-chip). Ccr4 copurified with ~40 mRNAs and five non-coding RNAs (ncRNAs) 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]. Two other subunits of the complex, Caf1 and Rcd1 (the S. pombe Caf40 homologue), interacted with the same set of RNAs, suggesting that the whole Ccr4-Not complex associates with these mRNAs (Fig. 1b, c; Additional file 1: Table S1). Although the overlap between mRNAs associated with different subunits was highly significant, it was far from complete. This may be due to technical noise (some interactions may be lost during the purification) or reflect the existence of multiple complexes containing different subunits. At present, we cannot distinguish between these two possibilities.Fig. 1

Bottom Line: 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.Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT

Background: 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 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.

Results: Ccr4-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.

Conclusions: Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

No MeSH data available.


Related in: MedlinePlus