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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 with a GFP reporter gene fused to a ZAM sequence act as sensors of the repression.The genomic structures of the transgenes pGFP-Zenv and pGFP-Idgag used in this study are presented at the tops of both panels: The grey boxes correspond to the UASt promoter, the dotted boxes to the GFP gene, and the white box to the env fragment of ZAM or the gag fragment of Idefix. Triangles indicate the FRT sites. Focal plane of the follicles dissected from a line in which the pGFP-Zenv transgene is driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Zenv transgene in an S genetic background before (A) or after (B) flp-recombinase action, or in a U genetic background before the flp treatment (C). GFP expression in the ovarioles of a transgenic line bearing the pGFP-Idgag transgene driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Idgag transgene in an S genetic background before (D) or after (E) flp-recombinase action, or in a U genetic background before the flp treatment (F). No GFP is detected in ovaries of the S lines. Its expression is recovered after the flp treatment or when the COM locus is mutated, as in the U genetic background.
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pone-0001526-g002: Transgenes with a GFP reporter gene fused to a ZAM sequence act as sensors of the repression.The genomic structures of the transgenes pGFP-Zenv and pGFP-Idgag used in this study are presented at the tops of both panels: The grey boxes correspond to the UASt promoter, the dotted boxes to the GFP gene, and the white box to the env fragment of ZAM or the gag fragment of Idefix. Triangles indicate the FRT sites. Focal plane of the follicles dissected from a line in which the pGFP-Zenv transgene is driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Zenv transgene in an S genetic background before (A) or after (B) flp-recombinase action, or in a U genetic background before the flp treatment (C). GFP expression in the ovarioles of a transgenic line bearing the pGFP-Idgag transgene driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Idgag transgene in an S genetic background before (D) or after (E) flp-recombinase action, or in a U genetic background before the flp treatment (F). No GFP is detected in ovaries of the S lines. Its expression is recovered after the flp treatment or when the COM locus is mutated, as in the U genetic background.

Mentions: Transgenes pZ475 and pZ499 both initiate transcription from the ZAM promoter at nucleotide 326. When these transgenes are transcribed, 149 and 173 nucleotides, respectively, of ZAM are present at the 5′ end of the transcripts (Fig. 1, table). By contrast, the ZAM promoter is absent in pZ310, and no ZAM sequence is transcribed. To determine whether the ZAM promoter or the presence of a ZAM fragment within a chimeric transcript is responsible for the differential expression of ZAM in the S and U lines, we designed constructs in which a GFP reporter gene, driven by the UASt promoter, is fused downstream of its coding sequence and upstream of the polyadenylation site to diverse fragments of ZAM (Fig 2 A). Transgenic lines were established and tested for GFP expression in the ovaries. A 720 bp fragment from within the third ORF of ZAM was tested. This fragment corresponds to the region encoding the Env protein, spanning nucleotides 6385 to 7105 of the ZAM sequence. This ZAM fragment was inserted in an orientation such that transcription of the transgene would give rise to mRNA corresponding to the sense-strand fragment of ZAM. Furthermore, the env fragment was flanked by FRT elements, which are targets for flp recombinase. Transgenic lines were established and denoted pGFP-Zenv. The expression of the UASt transgenes was induced by crossing with flies containing a ubiquitous somatic actin-Gal4 driver in the S genetic background. Data obtained are presented in Fig. 2 A, B and C.


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 with a GFP reporter gene fused to a ZAM sequence act as sensors of the repression.The genomic structures of the transgenes pGFP-Zenv and pGFP-Idgag used in this study are presented at the tops of both panels: The grey boxes correspond to the UASt promoter, the dotted boxes to the GFP gene, and the white box to the env fragment of ZAM or the gag fragment of Idefix. Triangles indicate the FRT sites. Focal plane of the follicles dissected from a line in which the pGFP-Zenv transgene is driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Zenv transgene in an S genetic background before (A) or after (B) flp-recombinase action, or in a U genetic background before the flp treatment (C). GFP expression in the ovarioles of a transgenic line bearing the pGFP-Idgag transgene driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Idgag transgene in an S genetic background before (D) or after (E) flp-recombinase action, or in a U genetic background before the flp treatment (F). No GFP is detected in ovaries of the S lines. Its expression is recovered after the flp treatment or when the COM locus is mutated, as in the U genetic background.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211404&req=5

pone-0001526-g002: Transgenes with a GFP reporter gene fused to a ZAM sequence act as sensors of the repression.The genomic structures of the transgenes pGFP-Zenv and pGFP-Idgag used in this study are presented at the tops of both panels: The grey boxes correspond to the UASt promoter, the dotted boxes to the GFP gene, and the white box to the env fragment of ZAM or the gag fragment of Idefix. Triangles indicate the FRT sites. Focal plane of the follicles dissected from a line in which the pGFP-Zenv transgene is driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Zenv transgene in an S genetic background before (A) or after (B) flp-recombinase action, or in a U genetic background before the flp treatment (C). GFP expression in the ovarioles of a transgenic line bearing the pGFP-Idgag transgene driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Idgag transgene in an S genetic background before (D) or after (E) flp-recombinase action, or in a U genetic background before the flp treatment (F). No GFP is detected in ovaries of the S lines. Its expression is recovered after the flp treatment or when the COM locus is mutated, as in the U genetic background.
Mentions: Transgenes pZ475 and pZ499 both initiate transcription from the ZAM promoter at nucleotide 326. When these transgenes are transcribed, 149 and 173 nucleotides, respectively, of ZAM are present at the 5′ end of the transcripts (Fig. 1, table). By contrast, the ZAM promoter is absent in pZ310, and no ZAM sequence is transcribed. To determine whether the ZAM promoter or the presence of a ZAM fragment within a chimeric transcript is responsible for the differential expression of ZAM in the S and U lines, we designed constructs in which a GFP reporter gene, driven by the UASt promoter, is fused downstream of its coding sequence and upstream of the polyadenylation site to diverse fragments of ZAM (Fig 2 A). Transgenic lines were established and tested for GFP expression in the ovaries. A 720 bp fragment from within the third ORF of ZAM was tested. This fragment corresponds to the region encoding the Env protein, spanning nucleotides 6385 to 7105 of the ZAM sequence. This ZAM fragment was inserted in an orientation such that transcription of the transgene would give rise to mRNA corresponding to the sense-strand fragment of ZAM. Furthermore, the env fragment was flanked by FRT elements, which are targets for flp recombinase. Transgenic lines were established and denoted pGFP-Zenv. The expression of the UASt transgenes was induced by crossing with flies containing a ubiquitous somatic actin-Gal4 driver in the S genetic background. Data obtained are presented in Fig. 2 A, B and C.

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