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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.


Ccr4-Not does not regulate turnover or translation of Mmi1 RNA targets. All comparisons presented are based on microarray data. a Venn diagrams comparing genes up-regulated in ccr4 and caf1 mutants relative to wild-type cells. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The p value of the observed overlap is displayed under the diagrams. b As in A, comparing mRNAs associated with Ccr4 and those induced in ccr4 mutants. c As in (a), comparing mRNAs that interact with Ccr4 and those up-regulated in caf1 ccr4 double mutants. d Comparison of expression levels of Ccr4-associated mRNAs between pab2 and ccr4 pab2 mutants (left), and between rrp6 and rrp6 pab2 mutants. Each mRNA is represented by a dot. The lines correspond to a twofold difference between the samples. e As in (a), comparing mRNAs up-regulated in ccr4 mutant and those displaying increased stability in ccr4. f As in (a), comparing mRNAs associated with Ccr4 and those stabilized in ccr4 mutants. g Cell extracts were prepared from the indicated strains, all of which contained TAP-tagged Mei4. Samples were probed with antibodies against TAP (top) or tubulin (bottom). The interaction between TAP-Mmi1 and Red1-myc was used as a positive control. h As in (g), using strains expressing Spo5-TAP.
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Fig3: Ccr4-Not does not regulate turnover or translation of Mmi1 RNA targets. All comparisons presented are based on microarray data. a Venn diagrams comparing genes up-regulated in ccr4 and caf1 mutants relative to wild-type cells. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The p value of the observed overlap is displayed under the diagrams. b As in A, comparing mRNAs associated with Ccr4 and those induced in ccr4 mutants. c As in (a), comparing mRNAs that interact with Ccr4 and those up-regulated in caf1 ccr4 double mutants. d Comparison of expression levels of Ccr4-associated mRNAs between pab2 and ccr4 pab2 mutants (left), and between rrp6 and rrp6 pab2 mutants. Each mRNA is represented by a dot. The lines correspond to a twofold difference between the samples. e As in (a), comparing mRNAs up-regulated in ccr4 mutant and those displaying increased stability in ccr4. f As in (a), comparing mRNAs associated with Ccr4 and those stabilized in ccr4 mutants. g Cell extracts were prepared from the indicated strains, all of which contained TAP-tagged Mei4. Samples were probed with antibodies against TAP (top) or tubulin (bottom). The interaction between TAP-Mmi1 and Red1-myc was used as a positive control. h As in (g), using strains expressing Spo5-TAP.

Mentions: A major function of the Ccr4-Not complex is the shortening of poly(A) tails through its deadenylase activity, which often results in mRNA destabilization. To explore whether Ccr4 regulates the stability of Mmi1 targets, we used DNA microarrays to measure mRNA levels in mutants defective for either deadenylase catalytic subunit (ccr4 and caf1). Both mutants grew slowly compared to wild-type cells, mated with low efficiency, and displayed similar changes in the levels of 78–90 RNAs (Fig. 3a; Additional file 1: Table S2). However, the relative levels of most Ccr4-bound RNAs were not affected in any of the mutants (Fig. 3a, b). The only exception to this was mei4, which showed a small but significant increase of 1.8-fold in caf1 mutants, and a rise of 1.4-fold in ccr4Δ cells (that did not pass the statistical significance threshold, see “Methods”). We examined the possibility that Ccr4 and Caf1 act redundantly by constructing a caf1Δ ccr4Δ double mutant. The double mutant was viable and displayed changes in gene expression similar to those of the single mutants (Additional file 1: Table S2). Notably, none of the Ccr4-bound mRNAs showed significant changes in gene expression (Fig. 3c). Again, the mei4 mRNA was mildly overexpressed (1.6-fold), but the change did not pass the significance threshold. These data indicate that Caf1 and Ccr4 do not regulate the levels of the early meiotic mRNAs with which they associate in a stable manner.Fig. 3


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 does not regulate turnover or translation of Mmi1 RNA targets. All comparisons presented are based on microarray data. a Venn diagrams comparing genes up-regulated in ccr4 and caf1 mutants relative to wild-type cells. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The p value of the observed overlap is displayed under the diagrams. b As in A, comparing mRNAs associated with Ccr4 and those induced in ccr4 mutants. c As in (a), comparing mRNAs that interact with Ccr4 and those up-regulated in caf1 ccr4 double mutants. d Comparison of expression levels of Ccr4-associated mRNAs between pab2 and ccr4 pab2 mutants (left), and between rrp6 and rrp6 pab2 mutants. Each mRNA is represented by a dot. The lines correspond to a twofold difference between the samples. e As in (a), comparing mRNAs up-regulated in ccr4 mutant and those displaying increased stability in ccr4. f As in (a), comparing mRNAs associated with Ccr4 and those stabilized in ccr4 mutants. g Cell extracts were prepared from the indicated strains, all of which contained TAP-tagged Mei4. Samples were probed with antibodies against TAP (top) or tubulin (bottom). The interaction between TAP-Mmi1 and Red1-myc was used as a positive control. h As in (g), using strains expressing Spo5-TAP.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4536793&req=5

Fig3: Ccr4-Not does not regulate turnover or translation of Mmi1 RNA targets. All comparisons presented are based on microarray data. a Venn diagrams comparing genes up-regulated in ccr4 and caf1 mutants relative to wild-type cells. The numbers in parentheses indicate the expected overlap if randomly generated lists of the corresponding sizes were used. The p value of the observed overlap is displayed under the diagrams. b As in A, comparing mRNAs associated with Ccr4 and those induced in ccr4 mutants. c As in (a), comparing mRNAs that interact with Ccr4 and those up-regulated in caf1 ccr4 double mutants. d Comparison of expression levels of Ccr4-associated mRNAs between pab2 and ccr4 pab2 mutants (left), and between rrp6 and rrp6 pab2 mutants. Each mRNA is represented by a dot. The lines correspond to a twofold difference between the samples. e As in (a), comparing mRNAs up-regulated in ccr4 mutant and those displaying increased stability in ccr4. f As in (a), comparing mRNAs associated with Ccr4 and those stabilized in ccr4 mutants. g Cell extracts were prepared from the indicated strains, all of which contained TAP-tagged Mei4. Samples were probed with antibodies against TAP (top) or tubulin (bottom). The interaction between TAP-Mmi1 and Red1-myc was used as a positive control. h As in (g), using strains expressing Spo5-TAP.
Mentions: A major function of the Ccr4-Not complex is the shortening of poly(A) tails through its deadenylase activity, which often results in mRNA destabilization. To explore whether Ccr4 regulates the stability of Mmi1 targets, we used DNA microarrays to measure mRNA levels in mutants defective for either deadenylase catalytic subunit (ccr4 and caf1). Both mutants grew slowly compared to wild-type cells, mated with low efficiency, and displayed similar changes in the levels of 78–90 RNAs (Fig. 3a; Additional file 1: Table S2). However, the relative levels of most Ccr4-bound RNAs were not affected in any of the mutants (Fig. 3a, b). The only exception to this was mei4, which showed a small but significant increase of 1.8-fold in caf1 mutants, and a rise of 1.4-fold in ccr4Δ cells (that did not pass the statistical significance threshold, see “Methods”). We examined the possibility that Ccr4 and Caf1 act redundantly by constructing a caf1Δ ccr4Δ double mutant. The double mutant was viable and displayed changes in gene expression similar to those of the single mutants (Additional file 1: Table S2). Notably, none of the Ccr4-bound mRNAs showed significant changes in gene expression (Fig. 3c). Again, the mei4 mRNA was mildly overexpressed (1.6-fold), but the change did not pass the significance threshold. These data indicate that Caf1 and Ccr4 do not regulate the levels of the early meiotic mRNAs with which they associate in a stable manner.Fig. 3

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.