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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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Accumulation of scnRNAs during autogamy. Total RNA samples from a wild-type culture grown on Klebsiella (no silencing) and a culture submitted to dsRNA-induced silencing of PTIWI01 and PTIWI09 were extracted at different time points (hrs) of autogamy. The histograms show the fraction of cells in each cytological stage (same colour code as in Figure 1; meiosis was not documented by DAPI staining). The t = 0 time point was arbitrarily defined as the first sample in which ∼50% of cells had begun old MAC fragmentation. (A) Northern blot detection of PTIWI01 and PTIWI09 mRNAs. Signals were quantified and normalized with the 17S rRNA signal. (B) To examine scnRNA accumulation, RNA samples were 5′-end-labelled with T4 kinase and run on a 15% polyacrylamide–urea gel. The lower panel shows hybridization of the same membranes with a tRNA probe as a loading control. The size marker (M) shows the position of 20- and 30-nt RNAs.
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Figure 3: Accumulation of scnRNAs during autogamy. Total RNA samples from a wild-type culture grown on Klebsiella (no silencing) and a culture submitted to dsRNA-induced silencing of PTIWI01 and PTIWI09 were extracted at different time points (hrs) of autogamy. The histograms show the fraction of cells in each cytological stage (same colour code as in Figure 1; meiosis was not documented by DAPI staining). The t = 0 time point was arbitrarily defined as the first sample in which ∼50% of cells had begun old MAC fragmentation. (A) Northern blot detection of PTIWI01 and PTIWI09 mRNAs. Signals were quantified and normalized with the 17S rRNA signal. (B) To examine scnRNA accumulation, RNA samples were 5′-end-labelled with T4 kinase and run on a 15% polyacrylamide–urea gel. The lower panel shows hybridization of the same membranes with a tRNA probe as a loading control. The size marker (M) shows the position of 20- and 30-nt RNAs.

Mentions: Plasmids used for T7Pol-driven dsRNA production in silencing experiments were obtained by cloning PCR products from each gene (see Supplementary Table S1A) using plasmid L4440 and Escherichia coli strain HT115 DE3, as previously described (33). The ND7, ND169 and ICL7a dsRNA fragments covered positions 873–1269, 1450–1860 and 1–580 of the PTETG500020001, GSPATG00008337001 and GSPATG00021610001 gene models, respectively. Probes used for the northern blots in Figure 2 were the same fragments as used for dsRNA production in silencing experiments. Probes used in Figure 3 and Supplementary Figure S3 are indicated in Supplementary Table S1A; the GAPDH probe covered positions 1–640 of GSPATG00013616001. The PTIWI09-GFP fusion was constructed by inserting a homemade EGFP-coding sequence, optimized for Paramecium codon usage, after codon 116 of the PTIWI09 coding sequence. The fusion was under the control of natural PTIWI09 regulatory sequences (512 bp upstream of translation start and 241 bp downstream of translation stop).Figure 2.


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)

Accumulation of scnRNAs during autogamy. Total RNA samples from a wild-type culture grown on Klebsiella (no silencing) and a culture submitted to dsRNA-induced silencing of PTIWI01 and PTIWI09 were extracted at different time points (hrs) of autogamy. The histograms show the fraction of cells in each cytological stage (same colour code as in Figure 1; meiosis was not documented by DAPI staining). The t = 0 time point was arbitrarily defined as the first sample in which ∼50% of cells had begun old MAC fragmentation. (A) Northern blot detection of PTIWI01 and PTIWI09 mRNAs. Signals were quantified and normalized with the 17S rRNA signal. (B) To examine scnRNA accumulation, RNA samples were 5′-end-labelled with T4 kinase and run on a 15% polyacrylamide–urea gel. The lower panel shows hybridization of the same membranes with a tRNA probe as a loading control. The size marker (M) shows the position of 20- and 30-nt RNAs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105430&req=5

Figure 3: Accumulation of scnRNAs during autogamy. Total RNA samples from a wild-type culture grown on Klebsiella (no silencing) and a culture submitted to dsRNA-induced silencing of PTIWI01 and PTIWI09 were extracted at different time points (hrs) of autogamy. The histograms show the fraction of cells in each cytological stage (same colour code as in Figure 1; meiosis was not documented by DAPI staining). The t = 0 time point was arbitrarily defined as the first sample in which ∼50% of cells had begun old MAC fragmentation. (A) Northern blot detection of PTIWI01 and PTIWI09 mRNAs. Signals were quantified and normalized with the 17S rRNA signal. (B) To examine scnRNA accumulation, RNA samples were 5′-end-labelled with T4 kinase and run on a 15% polyacrylamide–urea gel. The lower panel shows hybridization of the same membranes with a tRNA probe as a loading control. The size marker (M) shows the position of 20- and 30-nt RNAs.
Mentions: Plasmids used for T7Pol-driven dsRNA production in silencing experiments were obtained by cloning PCR products from each gene (see Supplementary Table S1A) using plasmid L4440 and Escherichia coli strain HT115 DE3, as previously described (33). The ND7, ND169 and ICL7a dsRNA fragments covered positions 873–1269, 1450–1860 and 1–580 of the PTETG500020001, GSPATG00008337001 and GSPATG00021610001 gene models, respectively. Probes used for the northern blots in Figure 2 were the same fragments as used for dsRNA production in silencing experiments. Probes used in Figure 3 and Supplementary Figure S3 are indicated in Supplementary Table S1A; the GAPDH probe covered positions 1–640 of GSPATG00013616001. The PTIWI09-GFP fusion was constructed by inserting a homemade EGFP-coding sequence, optimized for Paramecium codon usage, after codon 116 of the PTIWI09 coding sequence. The fusion was under the control of natural PTIWI09 regulatory sequences (512 bp upstream of translation start and 241 bp downstream of translation stop).Figure 2.

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