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Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling.

Bouhouche K, Gout JF, Kapusta A, Bétermier M, Meyer E - Nucleic Acids Res. (2011)

Bottom Line: We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle.Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus.Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie de l'Ecole Normale Supérieure, CNRS UMR8197, INSERM U1024, 46 rue d'Ulm, 75005 Paris, France.

ABSTRACT
Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

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Effects of PTIWI01 and PTIWI09 silencing on IES excision. (A) Total DNA was extracted after autogamy from cells grown on Klebsiella (no silencing) or silenced for ND7 or for genes of the PTIWI01-03-09 group, as indicated, and PCR amplified using pairs of primers located in the flanking sequences of IESs 51G4404 and 51G2832. The excised version (mac) can always be amplified from the fragments of the old MAC, while unexcised copies (mic) can be detected only when they accumulate in the new MACs. (B) Total DNA samples were extracted at different time points (h) from the same autogamy time courses as in Figure 3, and semi-quantitative PCR analyses were performed for 6 IESs with one primer in the IES and the other in the flanking sequence. The gel is shown only for IES 51G4404 (see Supplementary Figure S4 for other gel images). The histograms represent the amounts of unexcised IES copies, after normalization with the intensity of a control PCR amplifying the macronuclear TMP1b gene. 51G4404, 51G2832 and 51A2591 are maternally controlled IESs, while 51G1413, 51A6435 and 51A4578 are not (43). The status of 51B3931 is unknown.
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Figure 4: Effects of PTIWI01 and PTIWI09 silencing on IES excision. (A) Total DNA was extracted after autogamy from cells grown on Klebsiella (no silencing) or silenced for ND7 or for genes of the PTIWI01-03-09 group, as indicated, and PCR amplified using pairs of primers located in the flanking sequences of IESs 51G4404 and 51G2832. The excised version (mac) can always be amplified from the fragments of the old MAC, while unexcised copies (mic) can be detected only when they accumulate in the new MACs. (B) Total DNA samples were extracted at different time points (h) from the same autogamy time courses as in Figure 3, and semi-quantitative PCR analyses were performed for 6 IESs with one primer in the IES and the other in the flanking sequence. The gel is shown only for IES 51G4404 (see Supplementary Figure S4 for other gel images). The histograms represent the amounts of unexcised IES copies, after normalization with the intensity of a control PCR amplifying the macronuclear TMP1b gene. 51G4404, 51G2832 and 51A2591 are maternally controlled IESs, while 51G1413, 51A6435 and 51A4578 are not (43). The status of 51B3931 is unknown.

Mentions: Different assays were used to examine genome rearrangements in the developing new MACs after silencing of these genes. IES excision was first tested by PCR on small amounts of DNA extracted from post-autogamous cell populations, using pairs of primers located in the flanking sequences of two different IESs. PCR products from MIC DNA cannot be detected in such assays because of the high MAC:MIC ploidy ratio (200:1); in addition to the IES-excised products that are always amplified from the old MAC, IES-containing products are observed only if unexcised IES copies accumulate in the developing new MACs. This was the case only in post-autogamous cells from the PTIWI01-09 and PTIWI01-09-03 silencing combinations (Figure 4A), suggesting lethality was indeed due to defects in genome rearrangements.Figure 4.


Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling.

Bouhouche K, Gout JF, Kapusta A, Bétermier M, Meyer E - Nucleic Acids Res. (2011)

Effects of PTIWI01 and PTIWI09 silencing on IES excision. (A) Total DNA was extracted after autogamy from cells grown on Klebsiella (no silencing) or silenced for ND7 or for genes of the PTIWI01-03-09 group, as indicated, and PCR amplified using pairs of primers located in the flanking sequences of IESs 51G4404 and 51G2832. The excised version (mac) can always be amplified from the fragments of the old MAC, while unexcised copies (mic) can be detected only when they accumulate in the new MACs. (B) Total DNA samples were extracted at different time points (h) from the same autogamy time courses as in Figure 3, and semi-quantitative PCR analyses were performed for 6 IESs with one primer in the IES and the other in the flanking sequence. The gel is shown only for IES 51G4404 (see Supplementary Figure S4 for other gel images). The histograms represent the amounts of unexcised IES copies, after normalization with the intensity of a control PCR amplifying the macronuclear TMP1b gene. 51G4404, 51G2832 and 51A2591 are maternally controlled IESs, while 51G1413, 51A6435 and 51A4578 are not (43). The status of 51B3931 is unknown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 4: Effects of PTIWI01 and PTIWI09 silencing on IES excision. (A) Total DNA was extracted after autogamy from cells grown on Klebsiella (no silencing) or silenced for ND7 or for genes of the PTIWI01-03-09 group, as indicated, and PCR amplified using pairs of primers located in the flanking sequences of IESs 51G4404 and 51G2832. The excised version (mac) can always be amplified from the fragments of the old MAC, while unexcised copies (mic) can be detected only when they accumulate in the new MACs. (B) Total DNA samples were extracted at different time points (h) from the same autogamy time courses as in Figure 3, and semi-quantitative PCR analyses were performed for 6 IESs with one primer in the IES and the other in the flanking sequence. The gel is shown only for IES 51G4404 (see Supplementary Figure S4 for other gel images). The histograms represent the amounts of unexcised IES copies, after normalization with the intensity of a control PCR amplifying the macronuclear TMP1b gene. 51G4404, 51G2832 and 51A2591 are maternally controlled IESs, while 51G1413, 51A6435 and 51A4578 are not (43). The status of 51B3931 is unknown.
Mentions: Different assays were used to examine genome rearrangements in the developing new MACs after silencing of these genes. IES excision was first tested by PCR on small amounts of DNA extracted from post-autogamous cell populations, using pairs of primers located in the flanking sequences of two different IESs. PCR products from MIC DNA cannot be detected in such assays because of the high MAC:MIC ploidy ratio (200:1); in addition to the IES-excised products that are always amplified from the old MAC, IES-containing products are observed only if unexcised IES copies accumulate in the developing new MACs. This was the case only in post-autogamous cells from the PTIWI01-09 and PTIWI01-09-03 silencing combinations (Figure 4A), suggesting lethality was indeed due to defects in genome rearrangements.Figure 4.

Bottom Line: We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle.Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus.Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie de l'Ecole Normale Supérieure, CNRS UMR8197, INSERM U1024, 46 rue d'Ulm, 75005 Paris, France.

ABSTRACT
Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

Show MeSH