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Caenorhabditis elegans protein arginine methyltransferase PRMT-5 negatively regulates DNA damage-induced apoptosis.

Yang M, Sun J, Sun X, Shen Q, Gao Z, Yang C - PLoS Genet. (2009)

Bottom Line: PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter.Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms.These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Arginine methylation of histone and non-histone proteins is involved in transcription regulation and many other cellular processes. Nevertheless, whether such protein modification plays a regulatory role during apoptosis remains largely unknown. Here we report that the Caenorhabditis elegans homolog of mammalian type II arginine methyltransferase PRMT5 negatively regulates DNA damage-induced apoptosis. We show that inactivation of C. elegans prmt-5 leads to excessive apoptosis in germline following ionizing irradiation, which is due to a CEP-1/p53-dependent up-regulation of the cell death initiator EGL-1. Moreover, we provide evidence that CBP-1, the worm ortholog of human p300/CBP, functions as a cofactor of CEP-1. PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter. Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms. These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

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prmt-5 mutation causes excessive germ cell apoptosis in response to DNA damage.(A) prmt-5(gk357) is likely a  allele. The top panel shows the schematic representation of prmt-5(gk357) deletion mutation. Solid boxes indicate exons and waved lines indicate introns. The fragment below the gene indicates the deletion region and size. The bottom panel shows the endogenous expression of PRMT-5 in N2 and prmt-5(gk357) animals detected by using anti-PRMT-5 antibody. α-tubulin indicates the loading control of the samples. (B) γ-irradiation induces excessive germ cell apoptosis in prmt-5(gk357) animals. Young adult worms were treated with γ-irradiation of 120 Gy. 36 h later, germ cell corpses were examined under DIC optics and representative images of cell corpses are shown for N2 (a) and prmt-5(gk357) animals (b), respectively. Irradiated worms were also stained with acridine orange (AO) and both DIC and AO-positive germ cell corpses were shown for N2 (c, e) and prmt-5(gk357) mutants (d, f). Germ cell corpses are indicated by arrows (a, c–f) or fragmented circle (b). (C, D) Quantitative analysis of germ cell apoptosis induced by γ-irradiation. Germ cell corpses from one gonad arm of each animal were scored 36 h post irradiation of indicated doses (C), or at indicated time points post irradiation of 120 Gy (D). At least 20 worms were scored at each radiation dose or time point. Error bars represent standard error of the mean (SEM). (E) ENU induces excessive germ cell apoptosis in prmt-5(gk357) mutants. Young adult worms were treated with ENU at indicated concentrations for 4 h and germ cell corpses from one gonad arm of each animal were scored 24 h post ENU treatment. At least 20 worms were scored at each concentration point. (F) Rescuing activity of Ppie-1gfp::prmt-5 in prmt-5(gk357) germline. Worms were irradiated with γ-ray of 120 Gy and germ cell corpses were scored as above 36 h after irradiation.
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pgen-1000514-g001: prmt-5 mutation causes excessive germ cell apoptosis in response to DNA damage.(A) prmt-5(gk357) is likely a allele. The top panel shows the schematic representation of prmt-5(gk357) deletion mutation. Solid boxes indicate exons and waved lines indicate introns. The fragment below the gene indicates the deletion region and size. The bottom panel shows the endogenous expression of PRMT-5 in N2 and prmt-5(gk357) animals detected by using anti-PRMT-5 antibody. α-tubulin indicates the loading control of the samples. (B) γ-irradiation induces excessive germ cell apoptosis in prmt-5(gk357) animals. Young adult worms were treated with γ-irradiation of 120 Gy. 36 h later, germ cell corpses were examined under DIC optics and representative images of cell corpses are shown for N2 (a) and prmt-5(gk357) animals (b), respectively. Irradiated worms were also stained with acridine orange (AO) and both DIC and AO-positive germ cell corpses were shown for N2 (c, e) and prmt-5(gk357) mutants (d, f). Germ cell corpses are indicated by arrows (a, c–f) or fragmented circle (b). (C, D) Quantitative analysis of germ cell apoptosis induced by γ-irradiation. Germ cell corpses from one gonad arm of each animal were scored 36 h post irradiation of indicated doses (C), or at indicated time points post irradiation of 120 Gy (D). At least 20 worms were scored at each radiation dose or time point. Error bars represent standard error of the mean (SEM). (E) ENU induces excessive germ cell apoptosis in prmt-5(gk357) mutants. Young adult worms were treated with ENU at indicated concentrations for 4 h and germ cell corpses from one gonad arm of each animal were scored 24 h post ENU treatment. At least 20 worms were scored at each concentration point. (F) Rescuing activity of Ppie-1gfp::prmt-5 in prmt-5(gk357) germline. Worms were irradiated with γ-ray of 120 Gy and germ cell corpses were scored as above 36 h after irradiation.

Mentions: C. elegans prmt-5 gene is defined by the open reading frame C34E10.5 located on the linkage group III, which encodes a protein of 734 amino acids. The predicted worm PRMT-5 protein shows the highest sequence similarity to human type II protein arginine methyltransferase PRMT5 (34% sequence identity and 48% similarity, respectively). The sequence similarity is particularly strong between the residues 105 to 730 of C. elegans PRMT-5 and residues 58 to 633 of human PRMT5. C. elegans PRMT-5 also shares homology with yeast Skb1 and Drosophila Dart1 (Figure S2). Previously, a genome-wide RNAi screen showed that inactivation of prmt-5/C34E10.5 could cause increased level of spontaneous mutation in C. elegans, suggesting that prmt-5 is important for genome stability [21]. However, it is not known whether prmt-5 also plays a role in DNA damage-induced apoptosis. To further determine this, we analyzed a mutant strain prmt-5(gk357) containing a deletion of 522 bp that removes a small region of the exon 1 and the whole exons 2 and 3 of prmt-5 genomic locus (Figure 1A). Using an antibody generated against recombinant PRMT-5, we detected the expression of PRMT-5 in wild type but not in prmt-5(gk357) mutants, indicating that prmt-5(gk357) is likely a strong loss-of-function allele (Figure 1A). prmt-5(gk357) animals display no obvious developmental defects except that the growth rate is slightly lower than that of wild-type animals. Similar to prmt-5(RNAi) worms, prmt-5(gk357) animals do not show discernible defects in developmental cell deaths (data not shown). However, when exposed to γ-irradiation (IR), prmt-5(gk357) mutants exhibited a strong increase of germ cell apoptosis compared with that in wild-type animals. The IR-induced apoptosis in prmt-5(gk357) animals occurred mostly in the germline meiotic region containing pachytene-stage cells and the apoptotic cells displayed disc-like structures which were morphologically indistinguishable from those in wild-type worms (Figure 1B(a–b)). A further staining of irradiated animals with acridine orange (AO), a fluorescence dye that preferentially stains cell corpses internalized in engulfing cells, also indicated that prmt-5(gk357) worms contained significantly more AO-positive germ cell corpses than wild-type animals (Figure 1B(c–f)). Collectively, these data indicate that prmt-5 loss-of-function mutation leads to excessive germ cell apoptosis following γ-irradiation.


Caenorhabditis elegans protein arginine methyltransferase PRMT-5 negatively regulates DNA damage-induced apoptosis.

Yang M, Sun J, Sun X, Shen Q, Gao Z, Yang C - PLoS Genet. (2009)

prmt-5 mutation causes excessive germ cell apoptosis in response to DNA damage.(A) prmt-5(gk357) is likely a  allele. The top panel shows the schematic representation of prmt-5(gk357) deletion mutation. Solid boxes indicate exons and waved lines indicate introns. The fragment below the gene indicates the deletion region and size. The bottom panel shows the endogenous expression of PRMT-5 in N2 and prmt-5(gk357) animals detected by using anti-PRMT-5 antibody. α-tubulin indicates the loading control of the samples. (B) γ-irradiation induces excessive germ cell apoptosis in prmt-5(gk357) animals. Young adult worms were treated with γ-irradiation of 120 Gy. 36 h later, germ cell corpses were examined under DIC optics and representative images of cell corpses are shown for N2 (a) and prmt-5(gk357) animals (b), respectively. Irradiated worms were also stained with acridine orange (AO) and both DIC and AO-positive germ cell corpses were shown for N2 (c, e) and prmt-5(gk357) mutants (d, f). Germ cell corpses are indicated by arrows (a, c–f) or fragmented circle (b). (C, D) Quantitative analysis of germ cell apoptosis induced by γ-irradiation. Germ cell corpses from one gonad arm of each animal were scored 36 h post irradiation of indicated doses (C), or at indicated time points post irradiation of 120 Gy (D). At least 20 worms were scored at each radiation dose or time point. Error bars represent standard error of the mean (SEM). (E) ENU induces excessive germ cell apoptosis in prmt-5(gk357) mutants. Young adult worms were treated with ENU at indicated concentrations for 4 h and germ cell corpses from one gonad arm of each animal were scored 24 h post ENU treatment. At least 20 worms were scored at each concentration point. (F) Rescuing activity of Ppie-1gfp::prmt-5 in prmt-5(gk357) germline. Worms were irradiated with γ-ray of 120 Gy and germ cell corpses were scored as above 36 h after irradiation.
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Related In: Results  -  Collection

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pgen-1000514-g001: prmt-5 mutation causes excessive germ cell apoptosis in response to DNA damage.(A) prmt-5(gk357) is likely a allele. The top panel shows the schematic representation of prmt-5(gk357) deletion mutation. Solid boxes indicate exons and waved lines indicate introns. The fragment below the gene indicates the deletion region and size. The bottom panel shows the endogenous expression of PRMT-5 in N2 and prmt-5(gk357) animals detected by using anti-PRMT-5 antibody. α-tubulin indicates the loading control of the samples. (B) γ-irradiation induces excessive germ cell apoptosis in prmt-5(gk357) animals. Young adult worms were treated with γ-irradiation of 120 Gy. 36 h later, germ cell corpses were examined under DIC optics and representative images of cell corpses are shown for N2 (a) and prmt-5(gk357) animals (b), respectively. Irradiated worms were also stained with acridine orange (AO) and both DIC and AO-positive germ cell corpses were shown for N2 (c, e) and prmt-5(gk357) mutants (d, f). Germ cell corpses are indicated by arrows (a, c–f) or fragmented circle (b). (C, D) Quantitative analysis of germ cell apoptosis induced by γ-irradiation. Germ cell corpses from one gonad arm of each animal were scored 36 h post irradiation of indicated doses (C), or at indicated time points post irradiation of 120 Gy (D). At least 20 worms were scored at each radiation dose or time point. Error bars represent standard error of the mean (SEM). (E) ENU induces excessive germ cell apoptosis in prmt-5(gk357) mutants. Young adult worms were treated with ENU at indicated concentrations for 4 h and germ cell corpses from one gonad arm of each animal were scored 24 h post ENU treatment. At least 20 worms were scored at each concentration point. (F) Rescuing activity of Ppie-1gfp::prmt-5 in prmt-5(gk357) germline. Worms were irradiated with γ-ray of 120 Gy and germ cell corpses were scored as above 36 h after irradiation.
Mentions: C. elegans prmt-5 gene is defined by the open reading frame C34E10.5 located on the linkage group III, which encodes a protein of 734 amino acids. The predicted worm PRMT-5 protein shows the highest sequence similarity to human type II protein arginine methyltransferase PRMT5 (34% sequence identity and 48% similarity, respectively). The sequence similarity is particularly strong between the residues 105 to 730 of C. elegans PRMT-5 and residues 58 to 633 of human PRMT5. C. elegans PRMT-5 also shares homology with yeast Skb1 and Drosophila Dart1 (Figure S2). Previously, a genome-wide RNAi screen showed that inactivation of prmt-5/C34E10.5 could cause increased level of spontaneous mutation in C. elegans, suggesting that prmt-5 is important for genome stability [21]. However, it is not known whether prmt-5 also plays a role in DNA damage-induced apoptosis. To further determine this, we analyzed a mutant strain prmt-5(gk357) containing a deletion of 522 bp that removes a small region of the exon 1 and the whole exons 2 and 3 of prmt-5 genomic locus (Figure 1A). Using an antibody generated against recombinant PRMT-5, we detected the expression of PRMT-5 in wild type but not in prmt-5(gk357) mutants, indicating that prmt-5(gk357) is likely a strong loss-of-function allele (Figure 1A). prmt-5(gk357) animals display no obvious developmental defects except that the growth rate is slightly lower than that of wild-type animals. Similar to prmt-5(RNAi) worms, prmt-5(gk357) animals do not show discernible defects in developmental cell deaths (data not shown). However, when exposed to γ-irradiation (IR), prmt-5(gk357) mutants exhibited a strong increase of germ cell apoptosis compared with that in wild-type animals. The IR-induced apoptosis in prmt-5(gk357) animals occurred mostly in the germline meiotic region containing pachytene-stage cells and the apoptotic cells displayed disc-like structures which were morphologically indistinguishable from those in wild-type worms (Figure 1B(a–b)). A further staining of irradiated animals with acridine orange (AO), a fluorescence dye that preferentially stains cell corpses internalized in engulfing cells, also indicated that prmt-5(gk357) worms contained significantly more AO-positive germ cell corpses than wild-type animals (Figure 1B(c–f)). Collectively, these data indicate that prmt-5 loss-of-function mutation leads to excessive germ cell apoptosis following γ-irradiation.

Bottom Line: PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter.Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms.These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Arginine methylation of histone and non-histone proteins is involved in transcription regulation and many other cellular processes. Nevertheless, whether such protein modification plays a regulatory role during apoptosis remains largely unknown. Here we report that the Caenorhabditis elegans homolog of mammalian type II arginine methyltransferase PRMT5 negatively regulates DNA damage-induced apoptosis. We show that inactivation of C. elegans prmt-5 leads to excessive apoptosis in germline following ionizing irradiation, which is due to a CEP-1/p53-dependent up-regulation of the cell death initiator EGL-1. Moreover, we provide evidence that CBP-1, the worm ortholog of human p300/CBP, functions as a cofactor of CEP-1. PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter. Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms. These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

Show MeSH