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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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Phylogenetic analysis and expression profiles of PTIWI genes. (A) Phylogenetic tree based on an alignment of deduced protein sequences. For the PTIWI04 pseudogene, a virtual protein sequence (PTIWI04cor) was created by correcting nonsense and frameshift mutations, using the PTIWI05 paralogue. Bootstrap values are indicated. The scale bar indicates the branch length corresponding to 0.5 substitution per site (inferred using the WAG model). The residues found at the positions of the slicer catalytic triad in each protein are shown on the right. (B) Expression profiles during the life cycle, as determined from NimbleGen microarray data (see ‘Materials and Methods’ section). The histograms show averages, over 3 or 4 biological replicates, of the fractions of cells in the different cytological stages (depicted and colour-coded below) during vegetative growth and at five different time points during mass autogamy. The graphs on the right show the variations in expression levels (arbitrary units, divided by 100) for all PTIWI genes and for the NOWA1-2, DCL2-3, and PGM (the putative endonuclease) genes. Error bars represent the standard errors computed from the 3 or 4 biological replicates. Graph scales were adapted to the expression levels of gene groups.
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Figure 1: Phylogenetic analysis and expression profiles of PTIWI genes. (A) Phylogenetic tree based on an alignment of deduced protein sequences. For the PTIWI04 pseudogene, a virtual protein sequence (PTIWI04cor) was created by correcting nonsense and frameshift mutations, using the PTIWI05 paralogue. Bootstrap values are indicated. The scale bar indicates the branch length corresponding to 0.5 substitution per site (inferred using the WAG model). The residues found at the positions of the slicer catalytic triad in each protein are shown on the right. (B) Expression profiles during the life cycle, as determined from NimbleGen microarray data (see ‘Materials and Methods’ section). The histograms show averages, over 3 or 4 biological replicates, of the fractions of cells in the different cytological stages (depicted and colour-coded below) during vegetative growth and at five different time points during mass autogamy. The graphs on the right show the variations in expression levels (arbitrary units, divided by 100) for all PTIWI genes and for the NOWA1-2, DCL2-3, and PGM (the putative endonuclease) genes. Error bars represent the standard errors computed from the 3 or 4 biological replicates. Graph scales were adapted to the expression levels of gene groups.

Mentions: Expression data were obtained from single-channel NimbleGen microarrays covering all 39 642 annotated genes, with six different 50-mer probes per gene. Raw signals were processed using the standard RMA method (55). This includes a first step of background subtraction for each array, followed by between-array normalization which was carried out using the normalizeBetweenArrays function from the limma package (56). The latter step adjusts signals so that expression values have similar distributions across all arrays considered in the analysis (for the autogamy time course, a total of 21 microarrays including 3 or 4 biological replicates for each of the six time points). The expression level of each gene was taken as the median signal from the six probes, averaged over the biological replicates of each time point. The vegetative time point (Veg) is the average from four mass cultures containing only log-phase cells showing no sign of meiosis. Autogamy was induced by letting cultures starve; because cells enter autogamy from a fixed point of the cell cycle, which is not synchronized in vegetatively growing cultures, there is a minimal asynchrony of ∼6 h (one cell cycle) in the progression through the different cytological stages. The meiosis time point (Mei) is the average of four samples containing 20–39% of cells undergoing meiosis, and little or no fragmentation of the old MAC (see histograms in Figure 1B). The ‘Mei+F’ samples contained a similar proportion of meiotic cells (20–29%), but also 37–43% of cells with a fragmented old MAC. ‘Dev1′ samples contained 35–56% of cells with fragmented old MACs, and 35–51% of cells that already contained clearly visible new MACs (anlagen). ‘Dev2′ samples contained 73–98% of cells with visible anlagen, and the ‘Dev3′ samples were extracted ∼9 h after ‘Dev2′ samples. Microarray platform and data analyses have been described in more detail elsewhere (57) and are publicly available at the Gene Expression Omnibus database (58) under accession numbers GSE17996, GSE17997, GSE17998 and GSE18002.Figure 1.


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)

Phylogenetic analysis and expression profiles of PTIWI genes. (A) Phylogenetic tree based on an alignment of deduced protein sequences. For the PTIWI04 pseudogene, a virtual protein sequence (PTIWI04cor) was created by correcting nonsense and frameshift mutations, using the PTIWI05 paralogue. Bootstrap values are indicated. The scale bar indicates the branch length corresponding to 0.5 substitution per site (inferred using the WAG model). The residues found at the positions of the slicer catalytic triad in each protein are shown on the right. (B) Expression profiles during the life cycle, as determined from NimbleGen microarray data (see ‘Materials and Methods’ section). The histograms show averages, over 3 or 4 biological replicates, of the fractions of cells in the different cytological stages (depicted and colour-coded below) during vegetative growth and at five different time points during mass autogamy. The graphs on the right show the variations in expression levels (arbitrary units, divided by 100) for all PTIWI genes and for the NOWA1-2, DCL2-3, and PGM (the putative endonuclease) genes. Error bars represent the standard errors computed from the 3 or 4 biological replicates. Graph scales were adapted to the expression levels of gene groups.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Phylogenetic analysis and expression profiles of PTIWI genes. (A) Phylogenetic tree based on an alignment of deduced protein sequences. For the PTIWI04 pseudogene, a virtual protein sequence (PTIWI04cor) was created by correcting nonsense and frameshift mutations, using the PTIWI05 paralogue. Bootstrap values are indicated. The scale bar indicates the branch length corresponding to 0.5 substitution per site (inferred using the WAG model). The residues found at the positions of the slicer catalytic triad in each protein are shown on the right. (B) Expression profiles during the life cycle, as determined from NimbleGen microarray data (see ‘Materials and Methods’ section). The histograms show averages, over 3 or 4 biological replicates, of the fractions of cells in the different cytological stages (depicted and colour-coded below) during vegetative growth and at five different time points during mass autogamy. The graphs on the right show the variations in expression levels (arbitrary units, divided by 100) for all PTIWI genes and for the NOWA1-2, DCL2-3, and PGM (the putative endonuclease) genes. Error bars represent the standard errors computed from the 3 or 4 biological replicates. Graph scales were adapted to the expression levels of gene groups.
Mentions: Expression data were obtained from single-channel NimbleGen microarrays covering all 39 642 annotated genes, with six different 50-mer probes per gene. Raw signals were processed using the standard RMA method (55). This includes a first step of background subtraction for each array, followed by between-array normalization which was carried out using the normalizeBetweenArrays function from the limma package (56). The latter step adjusts signals so that expression values have similar distributions across all arrays considered in the analysis (for the autogamy time course, a total of 21 microarrays including 3 or 4 biological replicates for each of the six time points). The expression level of each gene was taken as the median signal from the six probes, averaged over the biological replicates of each time point. The vegetative time point (Veg) is the average from four mass cultures containing only log-phase cells showing no sign of meiosis. Autogamy was induced by letting cultures starve; because cells enter autogamy from a fixed point of the cell cycle, which is not synchronized in vegetatively growing cultures, there is a minimal asynchrony of ∼6 h (one cell cycle) in the progression through the different cytological stages. The meiosis time point (Mei) is the average of four samples containing 20–39% of cells undergoing meiosis, and little or no fragmentation of the old MAC (see histograms in Figure 1B). The ‘Mei+F’ samples contained a similar proportion of meiotic cells (20–29%), but also 37–43% of cells with a fragmented old MAC. ‘Dev1′ samples contained 35–56% of cells with fragmented old MACs, and 35–51% of cells that already contained clearly visible new MACs (anlagen). ‘Dev2′ samples contained 73–98% of cells with visible anlagen, and the ‘Dev3′ samples were extracted ∼9 h after ‘Dev2′ samples. Microarray platform and data analyses have been described in more detail elsewhere (57) and are publicly available at the Gene Expression Omnibus database (58) under accession numbers GSE17996, GSE17997, GSE17998 and GSE18002.Figure 1.

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