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Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster.

Kotelnikov RN, Klenov MS, Rozovsky YM, Olenina LV, Kibanov MV, Gvozdev VA - Nucleic Acids Res. (2009)

Bottom Line: We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction.Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of beta-gal activity.In cell culture, exogenous Su(Ste) dsRNA dramatically decreases beta-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics of Cell, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.

ABSTRACT
Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA-Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of beta-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases beta-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation.

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Degradation of Stellate mRNA by the piRNA machinery occurs both in the nucleus and the cytoplasm. (A) Deletion of Su(Ste) repeats leads to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts quantity both in the nuclear (n) and cytoplasmic (c) fractions. (B) Estimation of the purity of nuclear and cytoplasmic fractions. Upper rows: northern analysis with probes complementary to mitochondrial methionine tRNA (mt tRNA M) and cytoplasmic lysine tRNA (tRNA K) in nuclear (n) and cytoplasmic (c) fractions. Lower rows: western analysis with antibodies against nuclear lamin, cytoplasmic γ-tubulin and membrane marker calnexin proteins. (C) Su(Ste) piRNAs and the Aub protein are found both in nuclear and cytoplasmic fractions. Upper row: northern analysis with a riboprobe complementary to a pool of Su(Ste) piRNAs. Middle row: northern analysis with a DNA oligonucleotide complementary to a unique Su(Ste)-4 piRNA. Lower row: Western analysis with antibodies against Aub. (D) Amount (%) of mitochondrial methionine tRNA (from B) and Su(Ste) piRNAs (from C) in the nuclear fraction as compared to the cytoplasm. (E) Northern analysis confirms the proportionality of the hybridization signal to the amount of loaded RNA (methionine tRNA probe). (F) Localization of Aub in Drosophila testis. Testes were immunostained with anti-Aub (shown in red) and anti-lamin (shown in green). Specificity of anti-Aub was verified by immunostaining of testes of aubHN/aubQC42 (–/–) trans-heterozygous mutant flies. Scale bars: 10 µm. (G) Aub co-localizes with lamina in the nuclei. Nuclear fraction was immunostained with anti-Aub and anti-lamin. A dot-like signal of Aub co-localizes with lamina.
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Figure 2: Degradation of Stellate mRNA by the piRNA machinery occurs both in the nucleus and the cytoplasm. (A) Deletion of Su(Ste) repeats leads to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts quantity both in the nuclear (n) and cytoplasmic (c) fractions. (B) Estimation of the purity of nuclear and cytoplasmic fractions. Upper rows: northern analysis with probes complementary to mitochondrial methionine tRNA (mt tRNA M) and cytoplasmic lysine tRNA (tRNA K) in nuclear (n) and cytoplasmic (c) fractions. Lower rows: western analysis with antibodies against nuclear lamin, cytoplasmic γ-tubulin and membrane marker calnexin proteins. (C) Su(Ste) piRNAs and the Aub protein are found both in nuclear and cytoplasmic fractions. Upper row: northern analysis with a riboprobe complementary to a pool of Su(Ste) piRNAs. Middle row: northern analysis with a DNA oligonucleotide complementary to a unique Su(Ste)-4 piRNA. Lower row: Western analysis with antibodies against Aub. (D) Amount (%) of mitochondrial methionine tRNA (from B) and Su(Ste) piRNAs (from C) in the nuclear fraction as compared to the cytoplasm. (E) Northern analysis confirms the proportionality of the hybridization signal to the amount of loaded RNA (methionine tRNA probe). (F) Localization of Aub in Drosophila testis. Testes were immunostained with anti-Aub (shown in red) and anti-lamin (shown in green). Specificity of anti-Aub was verified by immunostaining of testes of aubHN/aubQC42 (–/–) trans-heterozygous mutant flies. Scale bars: 10 µm. (G) Aub co-localizes with lamina in the nuclei. Nuclear fraction was immunostained with anti-Aub and anti-lamin. A dot-like signal of Aub co-localizes with lamina.

Mentions: It was shown that siRNA-mediated degradation of transcripts may occur in the cytoplasm (25,26) and in the nucleus (27,28), but it is poorly understood where piRNA-mediated degradation takes place. We examined the effect of Su(Ste) deletion on Stellate transcript abundance in the nuclei and the cytoplasm separately. Nuclear and cytoplasmic fractions were obtained from lysates of Su(Ste)-deficient and wild-type testes. Quantitative RT-PCR revealed that Su(Ste) deletion leads to 38- and 13-fold increase of spliced heterochromatic Stellate mRNA amount in the cytoplasmic and nuclear fraction, respectively (Figure 2A). A similar result was obtained for euchromatic Stellate mRNA. The purity of the fractions was evaluated by western analysis using antibodies against lamin and γ-tubulin, which are nuclear and cytoplasmic proteins, respectively (Figure 2B). According to the procedure of fractionation nuclear fraction should be free of nuclear membranes (21). In order to control this, western analysis with an antibody against the endoplasmic reticulum membrane marker calnexin protein was done (Figure 2B). Contamination of the nuclear fraction by the cytoplasm was also estimated by northern analysis with a probe complementary to cytoplasmic lysine tRNA and mitochondrial methionine tRNA (Figure 2B). According to both western and northern analyses, the extent of nuclear fraction contamination by the cytoplasm does not exceed 10% (Figure 2D). Thus, the observed significant accumulation of Stellate transcripts in the nuclear fraction can not be explained by cytoplasmic contamination. We conclude that Stellate mRNA degradation occurs both in the nucleus and the cytoplasm. This result suggests that the complex of the Aub protein and Su(Ste) piRNAs performing silencing of Stellate genes (11,14) may be found both in the nuclear and cytoplasmic fractions. Using northern hybridization, we detected Su(Ste) piRNAs in both fractions (Figure 2C). We used a riboprobe detecting the sum of Su(Ste) piRNAs, or an oligonucleotide probe complementary to the individual Su(Ste)-4 piRNA, which was shown to be the most abundant among testes piRNAs immunoprecipitated with the Aub protein (11). In the nuclear fraction, Su(Ste) piRNAs are only 2-fold less abundant than in the cytoplasm (Figure 2D). Western analysis revealed the presence of the Aub protein also both in the nuclear and cytoplasmic fractions (Figure 2C). We also performed immunostaining to examine the localization of Aub in whole testes (Figure 2F) and isolated nuclei (Figure 2G). In the spermatocytes Aub is detected as a bright perinuclear ring (Figure 2F) representing the so-called nuage structure (29). In the isolated nuclei it was weaker, but a significant signal remained to be co-localized with the lamina (Figure 2G). Taking into account Aub co-localization with the lamina, we suggest that Aub is localized not only in the perinuclear organelle nuage, but also on the inner side of the nuclear membrane. Thus, fractionation experiments and immunostaining indicate that the piRNA–Aub complex is located both in the nucleus and cytoplasm.Figure 2.


Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster.

Kotelnikov RN, Klenov MS, Rozovsky YM, Olenina LV, Kibanov MV, Gvozdev VA - Nucleic Acids Res. (2009)

Degradation of Stellate mRNA by the piRNA machinery occurs both in the nucleus and the cytoplasm. (A) Deletion of Su(Ste) repeats leads to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts quantity both in the nuclear (n) and cytoplasmic (c) fractions. (B) Estimation of the purity of nuclear and cytoplasmic fractions. Upper rows: northern analysis with probes complementary to mitochondrial methionine tRNA (mt tRNA M) and cytoplasmic lysine tRNA (tRNA K) in nuclear (n) and cytoplasmic (c) fractions. Lower rows: western analysis with antibodies against nuclear lamin, cytoplasmic γ-tubulin and membrane marker calnexin proteins. (C) Su(Ste) piRNAs and the Aub protein are found both in nuclear and cytoplasmic fractions. Upper row: northern analysis with a riboprobe complementary to a pool of Su(Ste) piRNAs. Middle row: northern analysis with a DNA oligonucleotide complementary to a unique Su(Ste)-4 piRNA. Lower row: Western analysis with antibodies against Aub. (D) Amount (%) of mitochondrial methionine tRNA (from B) and Su(Ste) piRNAs (from C) in the nuclear fraction as compared to the cytoplasm. (E) Northern analysis confirms the proportionality of the hybridization signal to the amount of loaded RNA (methionine tRNA probe). (F) Localization of Aub in Drosophila testis. Testes were immunostained with anti-Aub (shown in red) and anti-lamin (shown in green). Specificity of anti-Aub was verified by immunostaining of testes of aubHN/aubQC42 (–/–) trans-heterozygous mutant flies. Scale bars: 10 µm. (G) Aub co-localizes with lamina in the nuclei. Nuclear fraction was immunostained with anti-Aub and anti-lamin. A dot-like signal of Aub co-localizes with lamina.
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Figure 2: Degradation of Stellate mRNA by the piRNA machinery occurs both in the nucleus and the cytoplasm. (A) Deletion of Su(Ste) repeats leads to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts quantity both in the nuclear (n) and cytoplasmic (c) fractions. (B) Estimation of the purity of nuclear and cytoplasmic fractions. Upper rows: northern analysis with probes complementary to mitochondrial methionine tRNA (mt tRNA M) and cytoplasmic lysine tRNA (tRNA K) in nuclear (n) and cytoplasmic (c) fractions. Lower rows: western analysis with antibodies against nuclear lamin, cytoplasmic γ-tubulin and membrane marker calnexin proteins. (C) Su(Ste) piRNAs and the Aub protein are found both in nuclear and cytoplasmic fractions. Upper row: northern analysis with a riboprobe complementary to a pool of Su(Ste) piRNAs. Middle row: northern analysis with a DNA oligonucleotide complementary to a unique Su(Ste)-4 piRNA. Lower row: Western analysis with antibodies against Aub. (D) Amount (%) of mitochondrial methionine tRNA (from B) and Su(Ste) piRNAs (from C) in the nuclear fraction as compared to the cytoplasm. (E) Northern analysis confirms the proportionality of the hybridization signal to the amount of loaded RNA (methionine tRNA probe). (F) Localization of Aub in Drosophila testis. Testes were immunostained with anti-Aub (shown in red) and anti-lamin (shown in green). Specificity of anti-Aub was verified by immunostaining of testes of aubHN/aubQC42 (–/–) trans-heterozygous mutant flies. Scale bars: 10 µm. (G) Aub co-localizes with lamina in the nuclei. Nuclear fraction was immunostained with anti-Aub and anti-lamin. A dot-like signal of Aub co-localizes with lamina.
Mentions: It was shown that siRNA-mediated degradation of transcripts may occur in the cytoplasm (25,26) and in the nucleus (27,28), but it is poorly understood where piRNA-mediated degradation takes place. We examined the effect of Su(Ste) deletion on Stellate transcript abundance in the nuclei and the cytoplasm separately. Nuclear and cytoplasmic fractions were obtained from lysates of Su(Ste)-deficient and wild-type testes. Quantitative RT-PCR revealed that Su(Ste) deletion leads to 38- and 13-fold increase of spliced heterochromatic Stellate mRNA amount in the cytoplasmic and nuclear fraction, respectively (Figure 2A). A similar result was obtained for euchromatic Stellate mRNA. The purity of the fractions was evaluated by western analysis using antibodies against lamin and γ-tubulin, which are nuclear and cytoplasmic proteins, respectively (Figure 2B). According to the procedure of fractionation nuclear fraction should be free of nuclear membranes (21). In order to control this, western analysis with an antibody against the endoplasmic reticulum membrane marker calnexin protein was done (Figure 2B). Contamination of the nuclear fraction by the cytoplasm was also estimated by northern analysis with a probe complementary to cytoplasmic lysine tRNA and mitochondrial methionine tRNA (Figure 2B). According to both western and northern analyses, the extent of nuclear fraction contamination by the cytoplasm does not exceed 10% (Figure 2D). Thus, the observed significant accumulation of Stellate transcripts in the nuclear fraction can not be explained by cytoplasmic contamination. We conclude that Stellate mRNA degradation occurs both in the nucleus and the cytoplasm. This result suggests that the complex of the Aub protein and Su(Ste) piRNAs performing silencing of Stellate genes (11,14) may be found both in the nuclear and cytoplasmic fractions. Using northern hybridization, we detected Su(Ste) piRNAs in both fractions (Figure 2C). We used a riboprobe detecting the sum of Su(Ste) piRNAs, or an oligonucleotide probe complementary to the individual Su(Ste)-4 piRNA, which was shown to be the most abundant among testes piRNAs immunoprecipitated with the Aub protein (11). In the nuclear fraction, Su(Ste) piRNAs are only 2-fold less abundant than in the cytoplasm (Figure 2D). Western analysis revealed the presence of the Aub protein also both in the nuclear and cytoplasmic fractions (Figure 2C). We also performed immunostaining to examine the localization of Aub in whole testes (Figure 2F) and isolated nuclei (Figure 2G). In the spermatocytes Aub is detected as a bright perinuclear ring (Figure 2F) representing the so-called nuage structure (29). In the isolated nuclei it was weaker, but a significant signal remained to be co-localized with the lamina (Figure 2G). Taking into account Aub co-localization with the lamina, we suggest that Aub is localized not only in the perinuclear organelle nuage, but also on the inner side of the nuclear membrane. Thus, fractionation experiments and immunostaining indicate that the piRNA–Aub complex is located both in the nucleus and cytoplasm.Figure 2.

Bottom Line: We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction.Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of beta-gal activity.In cell culture, exogenous Su(Ste) dsRNA dramatically decreases beta-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics of Cell, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.

ABSTRACT
Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA-Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of beta-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases beta-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation.

Show MeSH
Related in: MedlinePlus