Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.
Bottom Line: Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs.PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development.We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells.
Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland.Show MeSH
Related in: MedlinePlus
Mentions: Upon PTCAF-1 knockdown (PTCAF-1 KD) we observed a strong decrease of PTCAF-1 mRNA level in the early stages of autogamy (E) compared to control (Figure 1C). To assess whether PtCAF-1 might play a role in Paramecium sexual development, we tested the survival of sexual progeny after PTCAF-1 KD (Figure 2A). Sixty-seven percent of the PTCAF-1 KD cells in our survival test died during a time course of 3 days (up to 12 cell divisions) after the progeny cells were refed and allowed to divide vegetatively (Figure 2A). Twenty-seven percent of the cells did not divide at the normal rate of four divisions per 24 h and 6% showed no growth defects. In the control culture, 94% of the cells grew at normal rates while 6% died. In the positive control (NOWA1 KD), 97% of the cells died and 3% survived with a reduced growth rate. This result was confirmed by using RNAi construct targeting an alternative region of PTCAF-1 (Supplementary Figure S2A). No cell death or reduced division was observed for Paramecium cells grown vegetatively in silencing medium for more than 12 divisions. PTCAF-1 KD did not result in premature termination of development at an early stage as we were able to observe all the developmental stages and the final formation of new developing MACs (data not shown). We saw no structural irregularities during development and there was no notable differences in DNA content between the PTCAF-1 KD cells and the control cells. Our deep sequencing results suggest that the overall scnRNA levels that are detected at the early time point during development are not affected in PTCAF-1 KD compared to control (Figure 4B). This suggests that the expression and functions of Dcl2/3 and Ptiwi01/09 (responsible for the production and protection of scnRNAs) were not affected by PTCAF-1 KD. We confirmed by northern blot that the expression of PTIWI01 is not affected by PTCAF-1 KD (Figure 1C), and that the localization of Ptiwi09-GFP is unchanged by this knockdown (data not shown).
Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland.