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
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:In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. We previously reported that dephosphorylation of the HP1-like protein Pdd1p is required for the formation of heterochromatin bodies during the process of programmed DNA elimination in the ciliated protozoan Tetrahymena. Here, we show that the heterochromatin body component Jub4p is required for Pdd1p phosphorylation, heterochromatin body formation and DNA elimination. Moreover, our analyses of unphosphorylatable Pdd1p mutants demonstrate that Pdd1p phosphorylation is required for heterochromatin body formation and DNA elimination, while it is dispensable for local heterochromatin assembly. Therefore, both phosphorylation and the following dephosphorylation of Pdd1p are necessary to facilitate the formation of heterochromatin bodies. We suggest that Jub4p-mediated phosphorylation of Pdd1p creates a chromatin environment that is a prerequisite for subsequent heterochromatin body assembly and DNA elimination. New macronuclei (MACs) of exconjugants were isolated from wild-type and various mutant cells at 12 hpm (hours post-mixing), sheared chromatin was immunoprecipitated andprecipitated DNA was analyzed by high-throughput sequencing
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.