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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 PTIWI01-03-09 silencing on transposon elimination and maternal inheritance of macronuclear deletions. (A) Reversion of the trichocyst non-discharge phenotype in post-autogamous progeny of cells carrying a macronuclear deletion of the ND7 gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated. Reversion (+) indicates amplification of the ND7 gene in the new MACs. (B) Dot-blot quantification of the Sardine transposon and of the A surface antigen gene in post-autogamous progeny of cells carrying a macronuclear deletion of the A gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated (see Supplementary Figure S5 for dot-blot image). The signals obtained with the Sardine and A-gene probes were normalized with those of a mitochondrial DNA probe.
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Figure 5: Effects of PTIWI01-03-09 silencing on transposon elimination and maternal inheritance of macronuclear deletions. (A) Reversion of the trichocyst non-discharge phenotype in post-autogamous progeny of cells carrying a macronuclear deletion of the ND7 gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated. Reversion (+) indicates amplification of the ND7 gene in the new MACs. (B) Dot-blot quantification of the Sardine transposon and of the A surface antigen gene in post-autogamous progeny of cells carrying a macronuclear deletion of the A gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated (see Supplementary Figure S5 for dot-blot image). The signals obtained with the Sardine and A-gene probes were normalized with those of a mitochondrial DNA probe.

Mentions: We further tested the role of the PTIWI03-01-09 group of genes in the imprecise DNA elimination mechanism that is responsible for the deletion of MIC transposable elements during MAC development (67) and for maternally inherited deletions of non-essential cellular genes, which can be induced experimentally (34). A cell line reproducibly deleting the the A51 surface antigen and ND7 genes from the MAC genome at each sexual generation was fed dsRNAs for each of these genes alone, or in all possible combinations, for about 4 divisions prior to autogamy. Reversion of the ND7 MAC deletion in post-autogamous cell populations was assessed by phenotypic testing: in all silencing combinations involving either PTIWI01 or PTIWI09, but not in the PTIWI03 single silencing nor in the unsilenced control, the occurrence of trichocyst discharge indicated that the ND7 gene was at least partially maintained in the new MAC (Figure 5A). Total DNA from the same post-autogamous cell populations was then loaded on a dot-blot, which was successively hybridized with probes specific for the Sardine transposon, for the A51 gene, and for mitochondrial DNA as a loading control (Supplementary Figure S5). Quantification of the signals revealed that, as observed with ND7, the MAC deletion of the A51 gene reverted in all silencing combinations involving either PTIWI01 or PTIWI09; the relatively modest effect of the PTIWI09 single silencing could be due to a lower efficiency of silencing in that test since the PTIWI01-03 and PTIWI03-09 double silencing tests gave very similar results (Figure 5B). In contrast, elimination of the Sardine transposon was impaired only in silencing combinations involving both PTIWI01 and PTIWI09, as observed for IES excision.Figure 5.


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 PTIWI01-03-09 silencing on transposon elimination and maternal inheritance of macronuclear deletions. (A) Reversion of the trichocyst non-discharge phenotype in post-autogamous progeny of cells carrying a macronuclear deletion of the ND7 gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated. Reversion (+) indicates amplification of the ND7 gene in the new MACs. (B) Dot-blot quantification of the Sardine transposon and of the A surface antigen gene in post-autogamous progeny of cells carrying a macronuclear deletion of the A gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated (see Supplementary Figure S5 for dot-blot image). The signals obtained with the Sardine and A-gene probes were normalized with those of a mitochondrial DNA probe.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Effects of PTIWI01-03-09 silencing on transposon elimination and maternal inheritance of macronuclear deletions. (A) Reversion of the trichocyst non-discharge phenotype in post-autogamous progeny of cells carrying a macronuclear deletion of the ND7 gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated. Reversion (+) indicates amplification of the ND7 gene in the new MACs. (B) Dot-blot quantification of the Sardine transposon and of the A surface antigen gene in post-autogamous progeny of cells carrying a macronuclear deletion of the A gene and submitted during autogamy to dsRNA-induced silencing of PTIWI genes, as indicated (see Supplementary Figure S5 for dot-blot image). The signals obtained with the Sardine and A-gene probes were normalized with those of a mitochondrial DNA probe.
Mentions: We further tested the role of the PTIWI03-01-09 group of genes in the imprecise DNA elimination mechanism that is responsible for the deletion of MIC transposable elements during MAC development (67) and for maternally inherited deletions of non-essential cellular genes, which can be induced experimentally (34). A cell line reproducibly deleting the the A51 surface antigen and ND7 genes from the MAC genome at each sexual generation was fed dsRNAs for each of these genes alone, or in all possible combinations, for about 4 divisions prior to autogamy. Reversion of the ND7 MAC deletion in post-autogamous cell populations was assessed by phenotypic testing: in all silencing combinations involving either PTIWI01 or PTIWI09, but not in the PTIWI03 single silencing nor in the unsilenced control, the occurrence of trichocyst discharge indicated that the ND7 gene was at least partially maintained in the new MAC (Figure 5A). Total DNA from the same post-autogamous cell populations was then loaded on a dot-blot, which was successively hybridized with probes specific for the Sardine transposon, for the A51 gene, and for mitochondrial DNA as a loading control (Supplementary Figure S5). Quantification of the signals revealed that, as observed with ND7, the MAC deletion of the A51 gene reverted in all silencing combinations involving either PTIWI01 or PTIWI09; the relatively modest effect of the PTIWI09 single silencing could be due to a lower efficiency of silencing in that test since the PTIWI01-03 and PTIWI03-09 double silencing tests gave very similar results (Figure 5B). In contrast, elimination of the Sardine transposon was impaired only in silencing combinations involving both PTIWI01 and PTIWI09, as observed for IES excision.Figure 5.

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