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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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Schematic representation of the roles of Ptiwi proteins in sRNA pathways. SiRNA pathway (left): In vegetative cells, dsRNA may be formed by the pairing of sense and antisense aberrant transcripts during transgene-induced silencing, or imported from the food vacuoles during dsRNA-induced silencing. Primary siRNAs generated through the action of Dcr1 and different RdRPs (38) are then be loaded onto Ptiwi13 and/or Ptiwi14, resulting in cleavage of homologous mRNAs at least in the case of dsRNA-induced silencing. In that case, further RdRP activity using the mRNA as a template would generate antisense secondary siRNAs which may be loaded onto Ptiwi12 and Ptiwi15 to amplify the silencing response. ScnRNA pathway (right): promiscuous bidirectional transcription of the MAC genome occurs at low levels during vegetative growth. Upon meiosis, bidirectional transcription of the MIC genome forms dsRNA which is cleaved into scnRNA duplexes by Dcl2 and Dcl3. The guide strands of scnRNAs are then loaded onto Ptiwi01 and Ptiwi09 in the cytoplasm and transported to the old MAC where they would scan nascent transcripts with the help of the Nowa1 and 2 proteins. ScnRNAs able to find a perfect match would be sequestered or degraded, so that only those homologous to MIC-specific sequences would later be available to target epigenetic modifications on these sequences in the developing new MAC, eventually leading to their elimination.
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Figure 7: Schematic representation of the roles of Ptiwi proteins in sRNA pathways. SiRNA pathway (left): In vegetative cells, dsRNA may be formed by the pairing of sense and antisense aberrant transcripts during transgene-induced silencing, or imported from the food vacuoles during dsRNA-induced silencing. Primary siRNAs generated through the action of Dcr1 and different RdRPs (38) are then be loaded onto Ptiwi13 and/or Ptiwi14, resulting in cleavage of homologous mRNAs at least in the case of dsRNA-induced silencing. In that case, further RdRP activity using the mRNA as a template would generate antisense secondary siRNAs which may be loaded onto Ptiwi12 and Ptiwi15 to amplify the silencing response. ScnRNA pathway (right): promiscuous bidirectional transcription of the MAC genome occurs at low levels during vegetative growth. Upon meiosis, bidirectional transcription of the MIC genome forms dsRNA which is cleaved into scnRNA duplexes by Dcl2 and Dcl3. The guide strands of scnRNAs are then loaded onto Ptiwi01 and Ptiwi09 in the cytoplasm and transported to the old MAC where they would scan nascent transcripts with the help of the Nowa1 and 2 proteins. ScnRNAs able to find a perfect match would be sequestered or degraded, so that only those homologous to MIC-specific sequences would later be available to target epigenetic modifications on these sequences in the developing new MAC, eventually leading to their elimination.

Mentions: We have shown that Ptiwi13 is required for both transgene- and dsRNA-induced silencing, while Ptiwi14 appears to be specifically involved in the former, and Ptiwi12 and 15 in the latter. Although both silencing methods were previously shown to result in the accumulation of ∼23-nt siRNAs Figure 7 (34,35), which appear to depend on the Dicer gene DCR1 (36), the present results indicate that the mechanisms involved are at least partially distinct. Interestingly, the same conclusion was drawn in a study of the roles of P. tetraurelia RdRPs, which showed that the two processes require different RdRP genes and that the associated siRNAs are of different types (38). Confirming the results of a previous small-scale sequencing study (36), it was found that dsRNA-induced siRNAs comprise two distinct classes: ∼23-nt primary siRNAs that are processed from both strands of the ingested dsRNA, and a faster migrating class (∼22 nt) with a strictly antisense polarity, believed to represent secondary siRNAs produced by an RdRP activity from the targeted mRNA. In contrast, only ∼23-nt siRNAs were found to accumulate in transgene-induced silencing; however, these were shown to differ from dsRNA-induced primary siRNAs in that they carry a modification of the 3′-terminal ribose (38). It is, therefore, tempting to speculate about the Ptiwi proteins that bind these different types of siRNAs.Figure 7.


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)

Schematic representation of the roles of Ptiwi proteins in sRNA pathways. SiRNA pathway (left): In vegetative cells, dsRNA may be formed by the pairing of sense and antisense aberrant transcripts during transgene-induced silencing, or imported from the food vacuoles during dsRNA-induced silencing. Primary siRNAs generated through the action of Dcr1 and different RdRPs (38) are then be loaded onto Ptiwi13 and/or Ptiwi14, resulting in cleavage of homologous mRNAs at least in the case of dsRNA-induced silencing. In that case, further RdRP activity using the mRNA as a template would generate antisense secondary siRNAs which may be loaded onto Ptiwi12 and Ptiwi15 to amplify the silencing response. ScnRNA pathway (right): promiscuous bidirectional transcription of the MAC genome occurs at low levels during vegetative growth. Upon meiosis, bidirectional transcription of the MIC genome forms dsRNA which is cleaved into scnRNA duplexes by Dcl2 and Dcl3. The guide strands of scnRNAs are then loaded onto Ptiwi01 and Ptiwi09 in the cytoplasm and transported to the old MAC where they would scan nascent transcripts with the help of the Nowa1 and 2 proteins. ScnRNAs able to find a perfect match would be sequestered or degraded, so that only those homologous to MIC-specific sequences would later be available to target epigenetic modifications on these sequences in the developing new MAC, eventually leading to their elimination.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Schematic representation of the roles of Ptiwi proteins in sRNA pathways. SiRNA pathway (left): In vegetative cells, dsRNA may be formed by the pairing of sense and antisense aberrant transcripts during transgene-induced silencing, or imported from the food vacuoles during dsRNA-induced silencing. Primary siRNAs generated through the action of Dcr1 and different RdRPs (38) are then be loaded onto Ptiwi13 and/or Ptiwi14, resulting in cleavage of homologous mRNAs at least in the case of dsRNA-induced silencing. In that case, further RdRP activity using the mRNA as a template would generate antisense secondary siRNAs which may be loaded onto Ptiwi12 and Ptiwi15 to amplify the silencing response. ScnRNA pathway (right): promiscuous bidirectional transcription of the MAC genome occurs at low levels during vegetative growth. Upon meiosis, bidirectional transcription of the MIC genome forms dsRNA which is cleaved into scnRNA duplexes by Dcl2 and Dcl3. The guide strands of scnRNAs are then loaded onto Ptiwi01 and Ptiwi09 in the cytoplasm and transported to the old MAC where they would scan nascent transcripts with the help of the Nowa1 and 2 proteins. ScnRNAs able to find a perfect match would be sequestered or degraded, so that only those homologous to MIC-specific sequences would later be available to target epigenetic modifications on these sequences in the developing new MAC, eventually leading to their elimination.
Mentions: We have shown that Ptiwi13 is required for both transgene- and dsRNA-induced silencing, while Ptiwi14 appears to be specifically involved in the former, and Ptiwi12 and 15 in the latter. Although both silencing methods were previously shown to result in the accumulation of ∼23-nt siRNAs Figure 7 (34,35), which appear to depend on the Dicer gene DCR1 (36), the present results indicate that the mechanisms involved are at least partially distinct. Interestingly, the same conclusion was drawn in a study of the roles of P. tetraurelia RdRPs, which showed that the two processes require different RdRP genes and that the associated siRNAs are of different types (38). Confirming the results of a previous small-scale sequencing study (36), it was found that dsRNA-induced siRNAs comprise two distinct classes: ∼23-nt primary siRNAs that are processed from both strands of the ingested dsRNA, and a faster migrating class (∼22 nt) with a strictly antisense polarity, believed to represent secondary siRNAs produced by an RdRP activity from the targeted mRNA. In contrast, only ∼23-nt siRNAs were found to accumulate in transgene-induced silencing; however, these were shown to differ from dsRNA-induced primary siRNAs in that they carry a modification of the 3′-terminal ribose (38). It is, therefore, tempting to speculate about the Ptiwi proteins that bind these different types of siRNAs.Figure 7.

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