Limits...
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.

Show MeSH

Related in: MedlinePlus

The effect of Su(Ste) repeats deletion or aub mutation on spliced and non-spliced Stellate mRNA abundance in the testes of Drosophila melanogaster. (A) Deletion of Su(Ste) repeats (compared with wild type) and the aubsting-1 (homozygous flies compared with heterozygous ones) lead to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts abundance (dark bars), but not nonspliced transcripts abundance (light bars). Quantitative RT-PCR was done using primers detecting either nonspliced (pairs of primers designated as eu non or het non in the text) or a sum of spliced and nonspliced Stellate transcripts (designated as eu sum or het sum). The quantity of spliced transcripts was calculated by subtracting the nonspliced transcript quantity from the sum. We were able to detect euchromatic and heterochromatic Stellate transcripts separately, since the 3′ end nucleotides of the primers were complementary to variants of single nucleotide polymorphisms, which distinguish types of Stellate genes (see Supplementary Data). The middle four bars show the effect of Su(Ste) deletion causing derepression of Stellate genes in the fly strain carrying a transgenic construct with six heterochromatic Stellate genes in the euchromatin of chromosome 3. (B) PCR analysis with plasmids carrying eu- or heterochromatic Stellate genes or a Su(Ste) repeat confirms primers specificity. (C) Verification of localization of detected Stellate copies. The diagram shows values, which correspond to the results of dividing the Stellate/Adh ratio in salivary gland DNA by the Stellate/Adh ratio in total DNA. This test confirms that het non and het sum or eu non and eu sum primers detect Stellate copies located in the hetero- or euchromatin, correspondingly. Using of fly strain with six heterocromatic Stellate genes in the euchromatin leads to increasing of the values for primers specific to heterochromatic Stellate genes confirming validity of the test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691822&req=5

Figure 1: The effect of Su(Ste) repeats deletion or aub mutation on spliced and non-spliced Stellate mRNA abundance in the testes of Drosophila melanogaster. (A) Deletion of Su(Ste) repeats (compared with wild type) and the aubsting-1 (homozygous flies compared with heterozygous ones) lead to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts abundance (dark bars), but not nonspliced transcripts abundance (light bars). Quantitative RT-PCR was done using primers detecting either nonspliced (pairs of primers designated as eu non or het non in the text) or a sum of spliced and nonspliced Stellate transcripts (designated as eu sum or het sum). The quantity of spliced transcripts was calculated by subtracting the nonspliced transcript quantity from the sum. We were able to detect euchromatic and heterochromatic Stellate transcripts separately, since the 3′ end nucleotides of the primers were complementary to variants of single nucleotide polymorphisms, which distinguish types of Stellate genes (see Supplementary Data). The middle four bars show the effect of Su(Ste) deletion causing derepression of Stellate genes in the fly strain carrying a transgenic construct with six heterochromatic Stellate genes in the euchromatin of chromosome 3. (B) PCR analysis with plasmids carrying eu- or heterochromatic Stellate genes or a Su(Ste) repeat confirms primers specificity. (C) Verification of localization of detected Stellate copies. The diagram shows values, which correspond to the results of dividing the Stellate/Adh ratio in salivary gland DNA by the Stellate/Adh ratio in total DNA. This test confirms that het non and het sum or eu non and eu sum primers detect Stellate copies located in the hetero- or euchromatin, correspondingly. Using of fly strain with six heterocromatic Stellate genes in the euchromatin leads to increasing of the values for primers specific to heterochromatic Stellate genes confirming validity of the test.

Mentions: To elucidate whether the repression of Stellate genes by Su(Ste) piRNAs occurs at the transcriptional or post-transcriptional level, we estimated the effect of Su(Ste) deletion or the aubsting-1 mutation on the abundance of spliced and nonspliced Stellate transcripts in testes by quantitative RT-PCR. Deletion of the Su(Ste) locus or aubsting-1 lead to the disappearance of Su(Ste) piRNAs (14,15). If Su(Ste) piRNAs suppressed transcription of Stellate genes, the loss of silencing would cause a comparable increase of spliced and newly transcribed nonspliced transcripts quantity. We found that both Su(Ste) deletion and aubsting-1 lead to an insignificant increase of nonspliced transcript amount, while the level of spliced transcripts is dramatically increased (10–50 times) (Figure 1A). Thus, we consider posttranscriptional mRNA degradation as the predominant mechanism of Stellate silencing.Figure 1.


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)

The effect of Su(Ste) repeats deletion or aub mutation on spliced and non-spliced Stellate mRNA abundance in the testes of Drosophila melanogaster. (A) Deletion of Su(Ste) repeats (compared with wild type) and the aubsting-1 (homozygous flies compared with heterozygous ones) lead to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts abundance (dark bars), but not nonspliced transcripts abundance (light bars). Quantitative RT-PCR was done using primers detecting either nonspliced (pairs of primers designated as eu non or het non in the text) or a sum of spliced and nonspliced Stellate transcripts (designated as eu sum or het sum). The quantity of spliced transcripts was calculated by subtracting the nonspliced transcript quantity from the sum. We were able to detect euchromatic and heterochromatic Stellate transcripts separately, since the 3′ end nucleotides of the primers were complementary to variants of single nucleotide polymorphisms, which distinguish types of Stellate genes (see Supplementary Data). The middle four bars show the effect of Su(Ste) deletion causing derepression of Stellate genes in the fly strain carrying a transgenic construct with six heterochromatic Stellate genes in the euchromatin of chromosome 3. (B) PCR analysis with plasmids carrying eu- or heterochromatic Stellate genes or a Su(Ste) repeat confirms primers specificity. (C) Verification of localization of detected Stellate copies. The diagram shows values, which correspond to the results of dividing the Stellate/Adh ratio in salivary gland DNA by the Stellate/Adh ratio in total DNA. This test confirms that het non and het sum or eu non and eu sum primers detect Stellate copies located in the hetero- or euchromatin, correspondingly. Using of fly strain with six heterocromatic Stellate genes in the euchromatin leads to increasing of the values for primers specific to heterochromatic Stellate genes confirming validity of the test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: The effect of Su(Ste) repeats deletion or aub mutation on spliced and non-spliced Stellate mRNA abundance in the testes of Drosophila melanogaster. (A) Deletion of Su(Ste) repeats (compared with wild type) and the aubsting-1 (homozygous flies compared with heterozygous ones) lead to an increase of spliced euchromatic (eu) and heterochromatic (hetero) Stellate transcripts abundance (dark bars), but not nonspliced transcripts abundance (light bars). Quantitative RT-PCR was done using primers detecting either nonspliced (pairs of primers designated as eu non or het non in the text) or a sum of spliced and nonspliced Stellate transcripts (designated as eu sum or het sum). The quantity of spliced transcripts was calculated by subtracting the nonspliced transcript quantity from the sum. We were able to detect euchromatic and heterochromatic Stellate transcripts separately, since the 3′ end nucleotides of the primers were complementary to variants of single nucleotide polymorphisms, which distinguish types of Stellate genes (see Supplementary Data). The middle four bars show the effect of Su(Ste) deletion causing derepression of Stellate genes in the fly strain carrying a transgenic construct with six heterochromatic Stellate genes in the euchromatin of chromosome 3. (B) PCR analysis with plasmids carrying eu- or heterochromatic Stellate genes or a Su(Ste) repeat confirms primers specificity. (C) Verification of localization of detected Stellate copies. The diagram shows values, which correspond to the results of dividing the Stellate/Adh ratio in salivary gland DNA by the Stellate/Adh ratio in total DNA. This test confirms that het non and het sum or eu non and eu sum primers detect Stellate copies located in the hetero- or euchromatin, correspondingly. Using of fly strain with six heterocromatic Stellate genes in the euchromatin leads to increasing of the values for primers specific to heterochromatic Stellate genes confirming validity of the test.
Mentions: To elucidate whether the repression of Stellate genes by Su(Ste) piRNAs occurs at the transcriptional or post-transcriptional level, we estimated the effect of Su(Ste) deletion or the aubsting-1 mutation on the abundance of spliced and nonspliced Stellate transcripts in testes by quantitative RT-PCR. Deletion of the Su(Ste) locus or aubsting-1 lead to the disappearance of Su(Ste) piRNAs (14,15). If Su(Ste) piRNAs suppressed transcription of Stellate genes, the loss of silencing would cause a comparable increase of spliced and newly transcribed nonspliced transcripts quantity. We found that both Su(Ste) deletion and aubsting-1 lead to an insignificant increase of nonspliced transcript amount, while the level of spliced transcripts is dramatically increased (10–50 times) (Figure 1A). Thus, we consider posttranscriptional mRNA degradation as the predominant mechanism of Stellate silencing.Figure 1.

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