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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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Transgenes bearing ZAM or Idefix sequences placed in an antisense orientation are not targeted by the repression.Expression of sensor transgenes carrying ZAM or Idefix fragments inserted in a sense and an antisense orientation. The genomic structure of the so-called pGFP-Zenv, pGFP-ZenvAS, pGFP-IdU, pGFP-IdUAS transgenes are depicted on the left. The orientation of the fragment is indicated by an arrow. The repression mechanism is able to discriminate between sense and antisense targeted sequences. In an S/S genetic background, only transgenes with ZAM and Idefix in an antisense orientation are correctly expressed. Clear fluorescence due to GFP expression is detected in the ovarian follicles of pGFP-ZenvAS and pGFP-IdUAS transgenes, as illustrated on the right.
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pone-0001526-g003: Transgenes bearing ZAM or Idefix sequences placed in an antisense orientation are not targeted by the repression.Expression of sensor transgenes carrying ZAM or Idefix fragments inserted in a sense and an antisense orientation. The genomic structure of the so-called pGFP-Zenv, pGFP-ZenvAS, pGFP-IdU, pGFP-IdUAS transgenes are depicted on the left. The orientation of the fragment is indicated by an arrow. The repression mechanism is able to discriminate between sense and antisense targeted sequences. In an S/S genetic background, only transgenes with ZAM and Idefix in an antisense orientation are correctly expressed. Clear fluorescence due to GFP expression is detected in the ovarian follicles of pGFP-ZenvAS and pGFP-IdUAS transgenes, as illustrated on the right.

Mentions: A next set of experiments was performed with similar sensor GFP transgenes, but in which the fragments of ZAM and Idefix were inserted in the opposite orientation. Specifically, the 720 bp fragment within the third ORF of ZAM and the 456 bp fragment corresponding to the 5′ UTR of Idefix were tested in these experiments. These transgenes were denoted pGFP-ZenvAS and pGFP-IdUAS, respectively (Fig. 3). When transcribed, the resulting transgenes gave rise to transcripts which were antisense with respect to the endogenous ZAM or Idefix genomic RNAs. The ability of GFP to be expressed in the different lines was then assayed by introducing the actin-Gal4 transcription driver by crossing. While no fluorescence was observed when the expression of the (sense-strand) pGFP-Zenv or pGFPIdU transgenes was assayed, strong GFP fluorescence was detected in ovarian somatic tissues of the three independent (antisense) transgenic lines established with either pGFP-ZenvAS or pGFP-IdUAS (Fig 3 A and B). The intensity of the fluorescence was very similar to that observed with pGFP transgenes containing no ZAM or Idefix sequences, indicating that no silencing was exerted on these sensor transgenes.


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)

Transgenes bearing ZAM or Idefix sequences placed in an antisense orientation are not targeted by the repression.Expression of sensor transgenes carrying ZAM or Idefix fragments inserted in a sense and an antisense orientation. The genomic structure of the so-called pGFP-Zenv, pGFP-ZenvAS, pGFP-IdU, pGFP-IdUAS transgenes are depicted on the left. The orientation of the fragment is indicated by an arrow. The repression mechanism is able to discriminate between sense and antisense targeted sequences. In an S/S genetic background, only transgenes with ZAM and Idefix in an antisense orientation are correctly expressed. Clear fluorescence due to GFP expression is detected in the ovarian follicles of pGFP-ZenvAS and pGFP-IdUAS transgenes, as illustrated on the right.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001526-g003: Transgenes bearing ZAM or Idefix sequences placed in an antisense orientation are not targeted by the repression.Expression of sensor transgenes carrying ZAM or Idefix fragments inserted in a sense and an antisense orientation. The genomic structure of the so-called pGFP-Zenv, pGFP-ZenvAS, pGFP-IdU, pGFP-IdUAS transgenes are depicted on the left. The orientation of the fragment is indicated by an arrow. The repression mechanism is able to discriminate between sense and antisense targeted sequences. In an S/S genetic background, only transgenes with ZAM and Idefix in an antisense orientation are correctly expressed. Clear fluorescence due to GFP expression is detected in the ovarian follicles of pGFP-ZenvAS and pGFP-IdUAS transgenes, as illustrated on the right.
Mentions: A next set of experiments was performed with similar sensor GFP transgenes, but in which the fragments of ZAM and Idefix were inserted in the opposite orientation. Specifically, the 720 bp fragment within the third ORF of ZAM and the 456 bp fragment corresponding to the 5′ UTR of Idefix were tested in these experiments. These transgenes were denoted pGFP-ZenvAS and pGFP-IdUAS, respectively (Fig. 3). When transcribed, the resulting transgenes gave rise to transcripts which were antisense with respect to the endogenous ZAM or Idefix genomic RNAs. The ability of GFP to be expressed in the different lines was then assayed by introducing the actin-Gal4 transcription driver by crossing. While no fluorescence was observed when the expression of the (sense-strand) pGFP-Zenv or pGFPIdU transgenes was assayed, strong GFP fluorescence was detected in ovarian somatic tissues of the three independent (antisense) transgenic lines established with either pGFP-ZenvAS or pGFP-IdUAS (Fig 3 A and B). The intensity of the fluorescence was very similar to that observed with pGFP transgenes containing no ZAM or Idefix sequences, indicating that no silencing was exerted on these sensor transgenes.

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