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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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Expression and dynamic localization pattern of a Ptiwi09-GFP fusion during autogamy. Pictures numbered 1 show DAPI staining; pictures numbered 2 and 3 show GFP fluorescence within the cell body and at the cell surface, respectively. Arrowheads indicate the different types of nuclei: red, old MAC before fragmentation; white, MICs and their meiotic products; yellow, new MAC anlagen. A, vegetative cell during division; B, beginning of meiosis I; C, meiosis I; D, beginning of meiosis II; E, skein formation; F, fragmentation of the old MAC; G, new MAC development; H, karyonide after the first vegetative division.
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Figure 6: Expression and dynamic localization pattern of a Ptiwi09-GFP fusion during autogamy. Pictures numbered 1 show DAPI staining; pictures numbered 2 and 3 show GFP fluorescence within the cell body and at the cell surface, respectively. Arrowheads indicate the different types of nuclei: red, old MAC before fragmentation; white, MICs and their meiotic products; yellow, new MAC anlagen. A, vegetative cell during division; B, beginning of meiosis I; C, meiosis I; D, beginning of meiosis II; E, skein formation; F, fragmentation of the old MAC; G, new MAC development; H, karyonide after the first vegetative division.

Mentions: To study the subcellular localization of Ptiwi09p, a GFP fusion was constructed by inserting the GFP coding sequence into the PTIWI09 gene, upstream of the PAZ domain (after codon 116). Expression of the fusion gene was under the control of the natural PTIWI09 up- and downstream sequences. After microinjection of the construct into the MAC of vegetative cells, no fluorescence could be detected during vegetative growth of transformed clones (Figure 6A). When autogamy was induced by starvation, GFP fluorescence first appeared in the cytoplasm (Figure 6B). It then accumulated to some extent in the old MAC during the crescent stage (i.e. prophase of meiosis I, Figure 6C), but was always excluded from micronuclei and later from their meiotic products. Surprisingly, the GFP fusion also localized to basal bodies. This localization pattern remained essentially unchanged throughout meiosis I, meiosis II and skein formation (Figure 6D–E). When fragmentation of the old MAC was complete (Figure 6F), GFP fluorescence started to decrease at these locations and progressively relocalized to the new MACs as they developed, until all of the fusion protein was concentrated there (Figure 6G). Fluorescence finally faded away from the new MAC after the caryonidal division (Figure 6H).Figure 6.


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)

Expression and dynamic localization pattern of a Ptiwi09-GFP fusion during autogamy. Pictures numbered 1 show DAPI staining; pictures numbered 2 and 3 show GFP fluorescence within the cell body and at the cell surface, respectively. Arrowheads indicate the different types of nuclei: red, old MAC before fragmentation; white, MICs and their meiotic products; yellow, new MAC anlagen. A, vegetative cell during division; B, beginning of meiosis I; C, meiosis I; D, beginning of meiosis II; E, skein formation; F, fragmentation of the old MAC; G, new MAC development; H, karyonide after the first vegetative division.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105430&req=5

Figure 6: Expression and dynamic localization pattern of a Ptiwi09-GFP fusion during autogamy. Pictures numbered 1 show DAPI staining; pictures numbered 2 and 3 show GFP fluorescence within the cell body and at the cell surface, respectively. Arrowheads indicate the different types of nuclei: red, old MAC before fragmentation; white, MICs and their meiotic products; yellow, new MAC anlagen. A, vegetative cell during division; B, beginning of meiosis I; C, meiosis I; D, beginning of meiosis II; E, skein formation; F, fragmentation of the old MAC; G, new MAC development; H, karyonide after the first vegetative division.
Mentions: To study the subcellular localization of Ptiwi09p, a GFP fusion was constructed by inserting the GFP coding sequence into the PTIWI09 gene, upstream of the PAZ domain (after codon 116). Expression of the fusion gene was under the control of the natural PTIWI09 up- and downstream sequences. After microinjection of the construct into the MAC of vegetative cells, no fluorescence could be detected during vegetative growth of transformed clones (Figure 6A). When autogamy was induced by starvation, GFP fluorescence first appeared in the cytoplasm (Figure 6B). It then accumulated to some extent in the old MAC during the crescent stage (i.e. prophase of meiosis I, Figure 6C), but was always excluded from micronuclei and later from their meiotic products. Surprisingly, the GFP fusion also localized to basal bodies. This localization pattern remained essentially unchanged throughout meiosis I, meiosis II and skein formation (Figure 6D–E). When fragmentation of the old MAC was complete (Figure 6F), GFP fluorescence started to decrease at these locations and progressively relocalized to the new MACs as they developed, until all of the fusion protein was concentrated there (Figure 6G). Fluorescence finally faded away from the new MAC after the caryonidal division (Figure 6H).Figure 6.

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
Related in: MedlinePlus