Project description:To study the role of chromatin remodeler during programmed genome reorganization in Paramecium tetraurelia. Paramecium ISWI1 was tagged with 3 FLAG and HA at its C-terminal. The recombinant plasmid was transformed into Paramecium and used for co-immunoprecipitation and Mass spectrometry studies to identify novel interacting partners of Paramecium ISWI1 protein that modulates the elimination of transposable elements.
Project description:The normally virulent type-I RH parasite is rendered avirulent when lacking ROP5. The avirulent phenotype is a consequence of interaction with the host innate immune system. We sought to understand if ROP5 alters host gene expression in order to escape host defenses. We saw no gene expression differences between host cells infected with wt (RH?ku80) or RH?ku80?rop5 parasites. We have included uninfected HFF samples that were harvested in parallel with the infected samples. Host gene expression in response to infection with Toxoplasma gondii. Two independent samples per sample type. Three sample types: HFF infected with RH?ku80, HFF infected with RH?ku80?rop5, and uninfected HFF.
Project description:The normally virulent type-I RH parasite is rendered avirulent when lacking ROP5. The avirulent phenotype is a consequence of interaction with the host innate immune system. We sought to understand if ROP5 alters host gene expression in order to escape host defenses. We saw no gene expression differences between host cells infected with wt (RH∆ku80) or RH∆ku80∆rop5 parasites. We have included uninfected HFF samples that were harvested in parallel with the infected samples.
Project description:Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics
Project description:DNA-PK is a heterotrimeric complex that consists of Ku70 (XRCC6), Ku80 (XRCC5) and DNA-PKcs (PRKDC) subunits. DNA-PK complex is a major player in DNA double strand break (DSB) repair via non-homologous end joining pathway. This process requires all of DNA-PK subunits. Ku70/Ku80 heterodimers firstly bind to DNA-ends at DSB, that increase affinity of DNA-PKcs to DNA-ends. Recruitment of DNA-PKcs subunit to DSB leads to phosphorylation events near DSB, recruitment of another NHEJ-related genes that restore DNA integrity. However, today a lot of evidence demonstrate participation of DNA-PK components in other cellular process, e.g. cytosolic DNA sensing, apoptosis regulation, cellular movement and adhesion. It is important to note that not all subunits of DNA-PK complex are necessary for these process. This demonstrate the independent functions of DNA-PK subunits. Here we for the first time using NGS-sequencing analyzed the transcriptional changes in HEK293T cells under depletion of Ku70, Ku80 or DNA-PKcs to characterize the independent functions of each subunit.
Project description:Signal amplification of the initial small RNA trigger is important to ensure the silencing of repetitive transposable elements (TEs). Curiously, secondary small RNA biogenesis occurs by various mechanisms that are coupled with distinct steps of TE silencing in different eukaryotes, such as nucleolytic cleavage of TE transcripts, recruitment of RNA-dependent RNA polymerase, and heterochromatin-directed transcription. How such a variety of small RNA amplification mechanisms has evolved has not been thoroughly elucidated to date. Ciliated protozoa perform small RNA-directed programmed DNA elimination of thousands of TE-related internal eliminated sequences (IESs) in the newly developed somatic nucleus. In the ciliate Paramecium, secondary small RNAs are produced after primary small RNAs induce the excision of IESs. To examine whether such post-excision production of secondary small RNAs is conserved, we investigate the causality between the excision of IESs and the biogenesis of secondary small RNAs in another ciliate, Tetrahymena. We show that secondary small RNAs accumulate at least a few hours before their derived IESs are excised and that DNA excision is dispensable for their biogenesis in this ciliate. Therefore, unlike the situation in Paramecium, small RNA amplification occurs prior to IES excision in Tetrahymena. This study reveals remarkable mechanistic diversity of secondary small RNA biogenesis mechanisms, even among ciliates showing similar DNA elimination processes, and thus raises the possibility that the evolution of TE-targeting small RNA amplification can be traced by investigating the DNA elimination mechanisms of ciliates.
