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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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The repression machinery controlling ZAM and Idefix acts post-transcriptionally, before translation.A: Transcripts from the pGFP-ZU transgene were examined in northern blot experiments. A typical result is shown in A. GFP transcripts revealed by a riboprobe complementary to GFP mRNAs are detected in the U line and not in the S line. Actin is used as a loading control. B: Northern blots and quantification based on three northern blot experiments performed on flies containing pGFP-IdU and pGFP-IdUAS transgenes. Their structures are presented above the graph. No GFP transcripts synthesized from the pGFP-IdU transgene are detected by the GFP riboprobe in an S background, whereas their amount is high in a U background. An even higher amount of GFP transcripts is observed in an S or U background when the Idefix fragment is inserted in the opposite orientation (pGFP-IdUAS transgenes). C and D- RNase protection assays reveal the presence of small RNAs (20 to 30 nt long) that are homologous to ZAM and Idefix. These RNAs are detected in S lines and, at a much lower level, in the U line. Small RNAs homologous to the antisense strand of the 5′UTR of ZAM are presented in C. 20 to 30 nt long antisense strand RNAs (−) homologous to the 5′UTR or the gag gene of Idefix are detected. Sense strands (+) are absent or present in very small amounts. A typical experiment is presented in D. Signs (+) and (−) indicate respectively sense-strand and anti-sense strand RNAs of ZAM or Idefix revealed by the riboprobes.
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pone-0001526-g004: The repression machinery controlling ZAM and Idefix acts post-transcriptionally, before translation.A: Transcripts from the pGFP-ZU transgene were examined in northern blot experiments. A typical result is shown in A. GFP transcripts revealed by a riboprobe complementary to GFP mRNAs are detected in the U line and not in the S line. Actin is used as a loading control. B: Northern blots and quantification based on three northern blot experiments performed on flies containing pGFP-IdU and pGFP-IdUAS transgenes. Their structures are presented above the graph. No GFP transcripts synthesized from the pGFP-IdU transgene are detected by the GFP riboprobe in an S background, whereas their amount is high in a U background. An even higher amount of GFP transcripts is observed in an S or U background when the Idefix fragment is inserted in the opposite orientation (pGFP-IdUAS transgenes). C and D- RNase protection assays reveal the presence of small RNAs (20 to 30 nt long) that are homologous to ZAM and Idefix. These RNAs are detected in S lines and, at a much lower level, in the U line. Small RNAs homologous to the antisense strand of the 5′UTR of ZAM are presented in C. 20 to 30 nt long antisense strand RNAs (−) homologous to the 5′UTR or the gag gene of Idefix are detected. Sense strands (+) are absent or present in very small amounts. A typical experiment is presented in D. Signs (+) and (−) indicate respectively sense-strand and anti-sense strand RNAs of ZAM or Idefix revealed by the riboprobes.

Mentions: The trans-silencing phenomenon described above was analysed through GFP expression of the transgenes. To determine whether this silencing acted at the translational level or at the RNA level, we looked for GFP RNA encoded by the transgenic lines. Total RNA was extracted from ovaries of transgenic lines in either the S or U genetic backgrounds, and northern blots were performed. The nylon filters were probed first with a riboprobe corresponding to the GFP gene and second to one corresponding to the actin gene as a loading control. Typical results are presented in Figure 4. GFP transcripts synthesized from the pGFP-ZU transgene were not detected in S lines, or only at a very low level, whereas they were abundant in U lines (Figure 4A). Similar results were obtained when transgenes bearing Idefix sequences were analysed (Fig. 4B). The quantitative analysis of the transcripts as observed on northern blots is also presented in Fig. 4B. Further, while no GFP RNA was detected in S lines in which the transgene encoded a transcript containing a sense-strand sequence of Idefix, GFP RNA was clearly detected in S lines in which the transgene included an antisense sequence of Idefix (Fig. 4B). In this context, the amount of GFP RNA was appreciably higher than that detected from transgenes with Idefix in the sense orientation in U flies.


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)

The repression machinery controlling ZAM and Idefix acts post-transcriptionally, before translation.A: Transcripts from the pGFP-ZU transgene were examined in northern blot experiments. A typical result is shown in A. GFP transcripts revealed by a riboprobe complementary to GFP mRNAs are detected in the U line and not in the S line. Actin is used as a loading control. B: Northern blots and quantification based on three northern blot experiments performed on flies containing pGFP-IdU and pGFP-IdUAS transgenes. Their structures are presented above the graph. No GFP transcripts synthesized from the pGFP-IdU transgene are detected by the GFP riboprobe in an S background, whereas their amount is high in a U background. An even higher amount of GFP transcripts is observed in an S or U background when the Idefix fragment is inserted in the opposite orientation (pGFP-IdUAS transgenes). C and D- RNase protection assays reveal the presence of small RNAs (20 to 30 nt long) that are homologous to ZAM and Idefix. These RNAs are detected in S lines and, at a much lower level, in the U line. Small RNAs homologous to the antisense strand of the 5′UTR of ZAM are presented in C. 20 to 30 nt long antisense strand RNAs (−) homologous to the 5′UTR or the gag gene of Idefix are detected. Sense strands (+) are absent or present in very small amounts. A typical experiment is presented in D. Signs (+) and (−) indicate respectively sense-strand and anti-sense strand RNAs of ZAM or Idefix revealed by the riboprobes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211404&req=5

pone-0001526-g004: The repression machinery controlling ZAM and Idefix acts post-transcriptionally, before translation.A: Transcripts from the pGFP-ZU transgene were examined in northern blot experiments. A typical result is shown in A. GFP transcripts revealed by a riboprobe complementary to GFP mRNAs are detected in the U line and not in the S line. Actin is used as a loading control. B: Northern blots and quantification based on three northern blot experiments performed on flies containing pGFP-IdU and pGFP-IdUAS transgenes. Their structures are presented above the graph. No GFP transcripts synthesized from the pGFP-IdU transgene are detected by the GFP riboprobe in an S background, whereas their amount is high in a U background. An even higher amount of GFP transcripts is observed in an S or U background when the Idefix fragment is inserted in the opposite orientation (pGFP-IdUAS transgenes). C and D- RNase protection assays reveal the presence of small RNAs (20 to 30 nt long) that are homologous to ZAM and Idefix. These RNAs are detected in S lines and, at a much lower level, in the U line. Small RNAs homologous to the antisense strand of the 5′UTR of ZAM are presented in C. 20 to 30 nt long antisense strand RNAs (−) homologous to the 5′UTR or the gag gene of Idefix are detected. Sense strands (+) are absent or present in very small amounts. A typical experiment is presented in D. Signs (+) and (−) indicate respectively sense-strand and anti-sense strand RNAs of ZAM or Idefix revealed by the riboprobes.
Mentions: The trans-silencing phenomenon described above was analysed through GFP expression of the transgenes. To determine whether this silencing acted at the translational level or at the RNA level, we looked for GFP RNA encoded by the transgenic lines. Total RNA was extracted from ovaries of transgenic lines in either the S or U genetic backgrounds, and northern blots were performed. The nylon filters were probed first with a riboprobe corresponding to the GFP gene and second to one corresponding to the actin gene as a loading control. Typical results are presented in Figure 4. GFP transcripts synthesized from the pGFP-ZU transgene were not detected in S lines, or only at a very low level, whereas they were abundant in U lines (Figure 4A). Similar results were obtained when transgenes bearing Idefix sequences were analysed (Fig. 4B). The quantitative analysis of the transcripts as observed on northern blots is also presented in Fig. 4B. Further, while no GFP RNA was detected in S lines in which the transgene encoded a transcript containing a sense-strand sequence of Idefix, GFP RNA was clearly detected in S lines in which the transgene included an antisense sequence of Idefix (Fig. 4B). In this context, the amount of GFP RNA was appreciably higher than that detected from transgenes with Idefix in the sense orientation in U flies.

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