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In Drosophila melanogaster the COM locus directs the somatic silencing of two retrotransposons through both Piwi-dependent and -independent pathways.

Desset S, Buchon N, Meignin C, Coiffet M, Vaury C - PLoS ONE (2008)

Bottom Line: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements.Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus.They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique (CNRS), UMR6247-GReD, Clermont Université; INSERM, Faculté de Médecine, BP38, Clermont-Ferrand, France.

ABSTRACT

Background: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells.

Principal findings: Here, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus.

Conclusion: Our data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization.

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ZAM and Idefix are regulated by a PIWI-dependent pathway in the reproductive apparatus.In situ hybridization experiments reveal ZAM and Idefix expression in female gonads from third instar larvae. ZAM and Idefix transcripts are not detected in S flies with a wild-type piwi gene (left). As shown by the black staining, ZAM and Idefix mRNAs are detected in U flies with a wild-type piwi gene (middle). In S lines homozygous for the piwi3 allele, ZAM or Idefix transcripts are no longer repressed, and their transcription is visualised in gonads (right). Probes used in these experiments are indicated on the left.
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pone-0001526-g005: ZAM and Idefix are regulated by a PIWI-dependent pathway in the reproductive apparatus.In situ hybridization experiments reveal ZAM and Idefix expression in female gonads from third instar larvae. ZAM and Idefix transcripts are not detected in S flies with a wild-type piwi gene (left). As shown by the black staining, ZAM and Idefix mRNAs are detected in U flies with a wild-type piwi gene (middle). In S lines homozygous for the piwi3 allele, ZAM or Idefix transcripts are no longer repressed, and their transcription is visualised in gonads (right). Probes used in these experiments are indicated on the left.

Mentions: The Piwi protein has been shown to be involved in the rasiRNA pathway to maintain transposon silencing in the germline [1]. To test whether Piwi is also necessary for the repression of ZAM and Idefix, we first investigated the effect of the piwi3 mutation on the expression of endogenous ZAM and Idefix elements in the ovaries of S-line flies (Fig. 5). Because the morphology of homozygous piwi3 ovaries is severely affected in adult flies, it was impossible to investigate ZAM and Idefix expression in adult ovaries. We thus performed experiments in the gonads of third instar larvae. By in situ RNA analysis using strand-specific riboprobes for ZAM and Idefix, we found that both of the elements are expressed in female gonads of third instar larvae from the U line, as shown in a homozygous [U/U; piwi+/+] genetic background (Figure 5, middle). No staining corresponding to ZAM or Idefix RNA was ever detected in the gonads of larvae having the corresponding genotypes in the S line [S/S; piwi+/3] (not shown) or [S/S; piwi+/+] (Figure 5, left). By contrast, clear expression of ZAM and Idefix was observed in the homozygous genetic background [S/S; piwi3/3], displaying a pattern of expression similar to that detected in the U line (Fig. 5, right).


In Drosophila melanogaster the COM locus directs the somatic silencing of two retrotransposons through both Piwi-dependent and -independent pathways.

Desset S, Buchon N, Meignin C, Coiffet M, Vaury C - PLoS ONE (2008)

ZAM and Idefix are regulated by a PIWI-dependent pathway in the reproductive apparatus.In situ hybridization experiments reveal ZAM and Idefix expression in female gonads from third instar larvae. ZAM and Idefix transcripts are not detected in S flies with a wild-type piwi gene (left). As shown by the black staining, ZAM and Idefix mRNAs are detected in U flies with a wild-type piwi gene (middle). In S lines homozygous for the piwi3 allele, ZAM or Idefix transcripts are no longer repressed, and their transcription is visualised in gonads (right). Probes used in these experiments are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211404&req=5

pone-0001526-g005: ZAM and Idefix are regulated by a PIWI-dependent pathway in the reproductive apparatus.In situ hybridization experiments reveal ZAM and Idefix expression in female gonads from third instar larvae. ZAM and Idefix transcripts are not detected in S flies with a wild-type piwi gene (left). As shown by the black staining, ZAM and Idefix mRNAs are detected in U flies with a wild-type piwi gene (middle). In S lines homozygous for the piwi3 allele, ZAM or Idefix transcripts are no longer repressed, and their transcription is visualised in gonads (right). Probes used in these experiments are indicated on the left.
Mentions: The Piwi protein has been shown to be involved in the rasiRNA pathway to maintain transposon silencing in the germline [1]. To test whether Piwi is also necessary for the repression of ZAM and Idefix, we first investigated the effect of the piwi3 mutation on the expression of endogenous ZAM and Idefix elements in the ovaries of S-line flies (Fig. 5). Because the morphology of homozygous piwi3 ovaries is severely affected in adult flies, it was impossible to investigate ZAM and Idefix expression in adult ovaries. We thus performed experiments in the gonads of third instar larvae. By in situ RNA analysis using strand-specific riboprobes for ZAM and Idefix, we found that both of the elements are expressed in female gonads of third instar larvae from the U line, as shown in a homozygous [U/U; piwi+/+] genetic background (Figure 5, middle). No staining corresponding to ZAM or Idefix RNA was ever detected in the gonads of larvae having the corresponding genotypes in the S line [S/S; piwi+/3] (not shown) or [S/S; piwi+/+] (Figure 5, left). By contrast, clear expression of ZAM and Idefix was observed in the homozygous genetic background [S/S; piwi3/3], displaying a pattern of expression similar to that detected in the U line (Fig. 5, right).

Bottom Line: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements.Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus.They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique (CNRS), UMR6247-GReD, Clermont Université; INSERM, Faculté de Médecine, BP38, Clermont-Ferrand, France.

ABSTRACT

Background: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells.

Principal findings: Here, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus.

Conclusion: Our data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization.

Show MeSH