Analyses of Early- and Late-scnRNAs of Tetrahymena
Ontology highlight
ABSTRACT:
ORGANISM(S): Tetrahymena thermophila
PROVIDER: GSE68455 | GEO | 2015/06/15
SECONDARY ACCESSION(S): PRJNA282767
REPOSITORIES: GEO
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Project description:The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5' end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5' uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3' terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo.
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Project description:The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5' end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5' uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3' terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo. We compared Twi1p-loaded scnRNAs to scnRNAs before they have been loaded into Twi1p by deep sequencing to understand how the two processes, the production of siRNAs by Dicer and the loading of siRNAs into Argonaute, shape the population of siRNAs in vivo.
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Project description:The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scnRNAs) of approximately 28-30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and non-homogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and by the selective degradation of scnRNAs in the parental macronucleus.
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Project description:Ciliates undergo developmentally programmed genome elimination, in which small RNAs direct the removal of target DNA segments, including transposable elements. At each sexual generation, the development of the macronucleus (MAC) requires massive and reproducible elimination of a large proportion of the germline micronuclear (MIC) genome, leading to a highly streamlined somatic MAC genome. 25-nt long scnRNAs are produced from the entire germline MIC genome during meiosis, and this initial complex small RNA population is then transported to the maternal MAC, where selection of scnRNAs corresponding to germline (MIC)-specific sequences is thought to take place. Selected scnRNAs, loaded onto the PIWI protein Ptiwi09, guide the deposition of histone H3 post-translational modifications (H3K9me3 and H3K27me3) onto transposable elements in the developing macronucleus, ultimately triggering their specific elimination. How germline-specific MIC scnRNAs are selected remains to be determined. Here, we provide important mechanistic insights into the scnRNA selection pathway by identifying a Paramecium homolog of Gametocyte specific factor 1 (Gtsf1) as essential for the selective degradation of scnRNAs corresponding to retained somatic MAC sequences. Consistently, we also show that Gstf1 is exclusively localized in the maternal macronucleus, where scnRNA selection is presumed to occur, and associates with the scnRNA-binding protein Ptiwi09. Furthermore, Gtsf1 is necessary for DNA elimination and correct H3K9me3 and H3K27me3 localization in the new developing macronucleus, demonstrating that the scnRNA selection process is important for genome elimination. We propose that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with nascent RNA transcribed from the maternal MAC genome, similarly to the mechanism suggested for microRNA target-directed degradation in metazoans.
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Project description:Ciliates undergo developmentally programmed genome elimination, in which small RNAs direct the removal of target DNA segments, including transposable elements. At each sexual generation, the development of the macronucleus (MAC) requires massive and reproducible elimination of a large proportion of the germline micronuclear (MIC) genome, leading to a highly streamlined somatic MAC genome. 25-nt long scnRNAs are produced from the entire germline MIC genome during meiosis, and this initial complex small RNA population is then transported to the maternal MAC, where selection of scnRNAs corresponding to germline (MIC)-specific sequences is thought to take place. Selected scnRNAs, loaded onto the PIWI protein Ptiwi09, guide the deposition of histone H3 post-translational modifications (H3K9me3 and H3K27me3) onto transposable elements in the developing macronucleus, ultimately triggering their specific elimination. How germline-specific MIC scnRNAs are selected remains to be determined. Here, we provide important mechanistic insights into the scnRNA selection pathway by identifying a Paramecium homolog of Gametocyte specific factor 1 (Gtsf1) as essential for the selective degradation of scnRNAs corresponding to retained somatic MAC sequences. Consistently, we also show that Gstf1 is exclusively localized in the maternal macronucleus, where scnRNA selection is presumed to occur, and associates with the scnRNA-binding protein Ptiwi09. Furthermore, Gtsf1 is necessary for DNA elimination and correct H3K9me3 and H3K27me3 localization in the new developing macronucleus, demonstrating that the scnRNA selection process is important for genome elimination. We propose that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with nascent RNA transcribed from the maternal MAC genome, similarly to the mechanism suggested for microRNA target-directed degradation in metazoans.
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