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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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(A) Deletion of Su(Ste) repeats or aubsting-1 lead to a more than 200-fold increase of the Stellate protein level. Different quantities of lysates from testes of Su(Ste)-deficient flies were used for western analysis with antibodies against the Stellate protein and actin. (B) X-gal staining of wild-type and Su(Ste)-deficient testes, carrying transgenic reporters Ste703-lacZ or Ste134-lacZ. (C) Fold increase of Ste-lacZ mRNA level (light bars) and β-gal activity (dark bars) in testes of Su(Ste)-deficient males relatively to the wild-type ones. (D–F) S2 cell culture was transfected by plasmids encoding a Ste-lacZ reporter construct (D), or the GFP gene (E) or a lacZ reporter construct (F) driven by a heat-shock promoter and one of dsRNAs [GFP, Su(Ste), Ste or lacZ in (D) and GFP or Su(Ste) in (E) and (F)]. Decrease of abundance of mRNA (D–F) and β-gal activity (D and F) owing to transfection by homologous dsRNAs is shown.
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Figure 3: (A) Deletion of Su(Ste) repeats or aubsting-1 lead to a more than 200-fold increase of the Stellate protein level. Different quantities of lysates from testes of Su(Ste)-deficient flies were used for western analysis with antibodies against the Stellate protein and actin. (B) X-gal staining of wild-type and Su(Ste)-deficient testes, carrying transgenic reporters Ste703-lacZ or Ste134-lacZ. (C) Fold increase of Ste-lacZ mRNA level (light bars) and β-gal activity (dark bars) in testes of Su(Ste)-deficient males relatively to the wild-type ones. (D–F) S2 cell culture was transfected by plasmids encoding a Ste-lacZ reporter construct (D), or the GFP gene (E) or a lacZ reporter construct (F) driven by a heat-shock promoter and one of dsRNAs [GFP, Su(Ste), Ste or lacZ in (D) and GFP or Su(Ste) in (E) and (F)]. Decrease of abundance of mRNA (D–F) and β-gal activity (D and F) owing to transfection by homologous dsRNAs is shown.

Mentions: Western assay with anti-Stellate antibodies reveals that Stellate protein level in Su(Ste)-deficient testes increases much more dramatically (>200-fold) than the Stellate mRNA level (20- to 50-fold) (Figures 1 and 3A). Similar results we obtained using transgenic flies carrying Ste-lacZ reporter constructs driven by the Stellate promoter (Ste703-lacZ or Ste134-lacZ constructs contain the whole or 101 nt of Stellate promoter and first 141 or 33 nt of transcribed Stellate sequences, respectively). Deletion of Su(Ste) repeats exerts an insignificant effect on Ste-lacZ mRNA abundance (no more than 1.5-fold), but causes 4–7-fold increase of β-gal activity (Figure 3B and C). Slight increase of Ste-lacZ expression as compared to the endogenous Stellate genes level may be attributed to the structure of Ste-lacZ constructs, which contain incomplete Stellate sequences.Figure 3.


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)

(A) Deletion of Su(Ste) repeats or aubsting-1 lead to a more than 200-fold increase of the Stellate protein level. Different quantities of lysates from testes of Su(Ste)-deficient flies were used for western analysis with antibodies against the Stellate protein and actin. (B) X-gal staining of wild-type and Su(Ste)-deficient testes, carrying transgenic reporters Ste703-lacZ or Ste134-lacZ. (C) Fold increase of Ste-lacZ mRNA level (light bars) and β-gal activity (dark bars) in testes of Su(Ste)-deficient males relatively to the wild-type ones. (D–F) S2 cell culture was transfected by plasmids encoding a Ste-lacZ reporter construct (D), or the GFP gene (E) or a lacZ reporter construct (F) driven by a heat-shock promoter and one of dsRNAs [GFP, Su(Ste), Ste or lacZ in (D) and GFP or Su(Ste) in (E) and (F)]. Decrease of abundance of mRNA (D–F) and β-gal activity (D and F) owing to transfection by homologous dsRNAs is shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2691822&req=5

Figure 3: (A) Deletion of Su(Ste) repeats or aubsting-1 lead to a more than 200-fold increase of the Stellate protein level. Different quantities of lysates from testes of Su(Ste)-deficient flies were used for western analysis with antibodies against the Stellate protein and actin. (B) X-gal staining of wild-type and Su(Ste)-deficient testes, carrying transgenic reporters Ste703-lacZ or Ste134-lacZ. (C) Fold increase of Ste-lacZ mRNA level (light bars) and β-gal activity (dark bars) in testes of Su(Ste)-deficient males relatively to the wild-type ones. (D–F) S2 cell culture was transfected by plasmids encoding a Ste-lacZ reporter construct (D), or the GFP gene (E) or a lacZ reporter construct (F) driven by a heat-shock promoter and one of dsRNAs [GFP, Su(Ste), Ste or lacZ in (D) and GFP or Su(Ste) in (E) and (F)]. Decrease of abundance of mRNA (D–F) and β-gal activity (D and F) owing to transfection by homologous dsRNAs is shown.
Mentions: Western assay with anti-Stellate antibodies reveals that Stellate protein level in Su(Ste)-deficient testes increases much more dramatically (>200-fold) than the Stellate mRNA level (20- to 50-fold) (Figures 1 and 3A). Similar results we obtained using transgenic flies carrying Ste-lacZ reporter constructs driven by the Stellate promoter (Ste703-lacZ or Ste134-lacZ constructs contain the whole or 101 nt of Stellate promoter and first 141 or 33 nt of transcribed Stellate sequences, respectively). Deletion of Su(Ste) repeats exerts an insignificant effect on Ste-lacZ mRNA abundance (no more than 1.5-fold), but causes 4–7-fold increase of β-gal activity (Figure 3B and C). Slight increase of Ste-lacZ expression as compared to the endogenous Stellate genes level may be attributed to the structure of Ste-lacZ constructs, which contain incomplete Stellate sequences.Figure 3.

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