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. GRO-Seq and Examination of siRNAs in wild-type,nullisomic 4, EMA1 KO, and TWI1 KO Tetrahymena cells
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.
Project description:Microarray analyses were performed to compare the gene expression profiles of wild-type and several mutant strains of the ciliated protozoan Tetrahymena thermophila. Elimination of H3K4 methylation (hht2-K4Q) and knockout of either of the ubiquitylation enzymes (delta-UBC2 and delta-BRE1) affects a broader spectrum of genes than elimination of H2B ubiquitylation (htb1-K115R). Cells at mid-logarithmic growing phase (cell density of 200,000 cells/ml) were collected. Then total RNAs were extracted and hybridized.
Project description:Epigenetic inheritance of acquired traits is widespread among eukaryotes but how and to what extent such information is trans-generationally inherited is still unclear. The patterns of programmed DNA elimination in ciliates are epigenetically and trans-generationally inherited, and it has been proposed that small RNAs, which shuttle between the germline and the soma, regulate this epigenetic inheritance. In this study, we test the existence of such small RNA-mediated communication by epigenetically disturbing the pattern of DNA elimination in Tetrahymena. We show that the pattern of DNA elimination is indeed determined by the selective turnover of small RNAs, which is induced by the interaction between germline-derived small RNAs and the somatic genome. In addition, we show that DNA elimination of an element is regulated by small RNA-mediated communication with other eliminated elements. By contrast, no evidence obtained thus far supports the notion that transfer of epigenetic information from the soma to the germline, if any, regulates DNA elimination. Our results indicate that small RNA-mediated trans-nuclear and trans-element communication, in addition to unknown information in the germline genome, contributes to determining the pattern of DNA elimination.
Project description:Microarray analyses were performed to compare the gene expression profiles of wild-type and several mutant strains of the ciliated protozoan Tetrahymena thermophila. Elimination of H3K4 methylation (hht2-K4Q) and knockout of either of the ubiquitylation enzymes (delta-UBC2 and delta-BRE1) affects a broader spectrum of genes than elimination of H2B ubiquitylation (htb1-K115R).
Project description:Distinct classes of small RNAs are often selectively sorted to different Argonaute proteins. Various properties of small RNAs, such as length, terminal nucleotide, thermodynamic asymmetry and duplex mismatches, can impact sorting in different RNA silencing pathways in diverse eukaryotes. The developmentally regulated ~26-32 nt siRNAs, which are involved in programmed DNA elimination in Tetrahymena, show a strong bias for uracil at the 5' end. In this study, we analyzed loaded and unloaded populations of ~26-32 nt siRNAs by deep RNA sequencing. We show that the production process is the main determinant of size, whereas the 5' uracil bias is attributed not only to the process of loading siRNAs into the Argonaute protein Twi1p but also significantly to the initial processing of the siRNAs. We also show that both the loaded and the unloaded ~26-32 nt siRNAs have a strong bias for adenine as the 3rd base from the 3' terminus, suggesting that most of these siRNAs are direct Dicer products and little post-processing amplification of this class of siRNAs occurs. Further, we demonstrate that the siRNA-loading process in vivo can be deduced from the fraction of siRNAs with uracil as the first base. These findings provide biochemical bases for the attributes of ~26-32 nt siRNAs, which should help improve our understanding of their production and turnover in vivo. Examination of siRNA populations in wild-type and TWI1 KO Tetrahymena cells