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Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability.

Kirino Y, Kim N, de Planell-Saguer M, Khandros E, Chiorean S, Klein PS, Rigoutsos I, Jongens TA, Mourelatos Z - Nat. Cell Biol. (2009)

Bottom Line: We found that Piwi proteins are expressed in Xenopus oocytes and we identified numerous Xenopus piRNAs.Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary.Our findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Division of Neuropathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila melanogaster encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). Here we report that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). We found that Piwi proteins are expressed in Xenopus oocytes and we identified numerous Xenopus piRNAs. We report that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, csul/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. Our studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. Our findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification.

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Xenopus laevis Piwi proteins with bound piRNAs are immunoprecipitated by Y12 and contain sDMAs(a) Protein immunoprecipitates from indicated X. laevis tissues; Xili and Xiwi were identified by mass spectrometry (Supplementary Table 3).(b) Immunoprecipitates from X. laevis oocytes were probed on Western blots with indicated antibodies. Band with asterisk is bovine IgG from tissue culture supernatant of anti-Mili hybridoma.(c) RNA-immunoprecipitations from X. laevis.(d) Periodate oxidation and β-elimination of X. laevis piRNAs isolated from Y12 immunoprecipitates.(e) Nucleotide composition of X. laevis piRNAs.(f) Northern blot for XL-piR-3(g) In situ hybridization for XL-piR-3 in X. laevis oocyte; bar = 100μm
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Figure 2: Xenopus laevis Piwi proteins with bound piRNAs are immunoprecipitated by Y12 and contain sDMAs(a) Protein immunoprecipitates from indicated X. laevis tissues; Xili and Xiwi were identified by mass spectrometry (Supplementary Table 3).(b) Immunoprecipitates from X. laevis oocytes were probed on Western blots with indicated antibodies. Band with asterisk is bovine IgG from tissue culture supernatant of anti-Mili hybridoma.(c) RNA-immunoprecipitations from X. laevis.(d) Periodate oxidation and β-elimination of X. laevis piRNAs isolated from Y12 immunoprecipitates.(e) Nucleotide composition of X. laevis piRNAs.(f) Northern blot for XL-piR-3(g) In situ hybridization for XL-piR-3 in X. laevis oocyte; bar = 100μm

Mentions: Next we asked whether the sDMA modification was conserved in Piwi family proteins from other species. A stumbling block in studying the molecular functions of Piwi proteins and piRNAs has been the lack of suitable cell culture systems. We reasoned that Xenopus laevis oocytes might express Piwi proteins and piRNAs and thus prove very useful not only to confirm that sDMAs of Piwi proteins are conserved but also as a model to study the function of Piwi proteins and piRNAs. By searching the Gurdon EST database at Xenbase 23 we identified three Xenopus Piwi proteins which we named Xili, Xiwi and Xiwi2 (Supplementary Figure 4). All three Xenopus Piwi proteins contain putative sDMA motifs (Supplementary Table 2). Immunoprecipitations with Y12 from X. laevis oocytes (defolliculated, mixed Dumont stages I-VI), testis and liver revealed the presence of two proteins at ∼95 kDa and ∼110 kDa specifically in the Y12 immunoprecipitates from oocytes and testis (Figure 2a) that we identified by mass spectrometry as Xiwi and Xili respectively (Supplementary Table 3). As shown in the western blots in Figure 2b, Y12 recognized both Xiwi and Xili, while anti-Mili (17.8) reacted only with Xili. In addition, both Xiwi and Xili were recognized by SYM11, indicating that Xiwi and Xili contain sDMAs.


Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability.

Kirino Y, Kim N, de Planell-Saguer M, Khandros E, Chiorean S, Klein PS, Rigoutsos I, Jongens TA, Mourelatos Z - Nat. Cell Biol. (2009)

Xenopus laevis Piwi proteins with bound piRNAs are immunoprecipitated by Y12 and contain sDMAs(a) Protein immunoprecipitates from indicated X. laevis tissues; Xili and Xiwi were identified by mass spectrometry (Supplementary Table 3).(b) Immunoprecipitates from X. laevis oocytes were probed on Western blots with indicated antibodies. Band with asterisk is bovine IgG from tissue culture supernatant of anti-Mili hybridoma.(c) RNA-immunoprecipitations from X. laevis.(d) Periodate oxidation and β-elimination of X. laevis piRNAs isolated from Y12 immunoprecipitates.(e) Nucleotide composition of X. laevis piRNAs.(f) Northern blot for XL-piR-3(g) In situ hybridization for XL-piR-3 in X. laevis oocyte; bar = 100μm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2746449&req=5

Figure 2: Xenopus laevis Piwi proteins with bound piRNAs are immunoprecipitated by Y12 and contain sDMAs(a) Protein immunoprecipitates from indicated X. laevis tissues; Xili and Xiwi were identified by mass spectrometry (Supplementary Table 3).(b) Immunoprecipitates from X. laevis oocytes were probed on Western blots with indicated antibodies. Band with asterisk is bovine IgG from tissue culture supernatant of anti-Mili hybridoma.(c) RNA-immunoprecipitations from X. laevis.(d) Periodate oxidation and β-elimination of X. laevis piRNAs isolated from Y12 immunoprecipitates.(e) Nucleotide composition of X. laevis piRNAs.(f) Northern blot for XL-piR-3(g) In situ hybridization for XL-piR-3 in X. laevis oocyte; bar = 100μm
Mentions: Next we asked whether the sDMA modification was conserved in Piwi family proteins from other species. A stumbling block in studying the molecular functions of Piwi proteins and piRNAs has been the lack of suitable cell culture systems. We reasoned that Xenopus laevis oocytes might express Piwi proteins and piRNAs and thus prove very useful not only to confirm that sDMAs of Piwi proteins are conserved but also as a model to study the function of Piwi proteins and piRNAs. By searching the Gurdon EST database at Xenbase 23 we identified three Xenopus Piwi proteins which we named Xili, Xiwi and Xiwi2 (Supplementary Figure 4). All three Xenopus Piwi proteins contain putative sDMA motifs (Supplementary Table 2). Immunoprecipitations with Y12 from X. laevis oocytes (defolliculated, mixed Dumont stages I-VI), testis and liver revealed the presence of two proteins at ∼95 kDa and ∼110 kDa specifically in the Y12 immunoprecipitates from oocytes and testis (Figure 2a) that we identified by mass spectrometry as Xiwi and Xili respectively (Supplementary Table 3). As shown in the western blots in Figure 2b, Y12 recognized both Xiwi and Xili, while anti-Mili (17.8) reacted only with Xili. In addition, both Xiwi and Xili were recognized by SYM11, indicating that Xiwi and Xili contain sDMAs.

Bottom Line: We found that Piwi proteins are expressed in Xenopus oocytes and we identified numerous Xenopus piRNAs.Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary.Our findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Division of Neuropathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila melanogaster encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). Here we report that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). We found that Piwi proteins are expressed in Xenopus oocytes and we identified numerous Xenopus piRNAs. We report that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, csul/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. Our studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. Our findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification.

Show MeSH