In ciliates, Piwi-bound small RNAs are involved in massive DNA elimination that takes place during sexual development. In Paramecium, we know so far of two classes of small RNAs involved in the excision of germline-limited DNA such as IESs and transposons. Scan RNAs (scnRNAs) are involved in the first round of the DNA elimination process. The later arising iesRNAs originate from eliminated IESs and are required to complete the DNA elimination.
We provide experimental evidence that Ptiwi01 and Ptiwi09 are essential for the scnRNA pathway and the elimination of maternally-controlled IESs. In addition we demonstrate that Ptiwi10 and Ptiwi11 are required for iesRNA-specific DNA elimination. Using deep sequencing we show that in both pathways the Ptiwi proteins select guide RNA strands from dsRNA Dicer cleavage products. Sequencing of DNA from different Ptiwi knock-downs also demonstrates that only one of the two small RNA pathways affects the excision of transposons.
Additionally we show that the late expressed Ptiwi10 and Ptiwi11, among many other proteins, are expressed from the new macronucleus and that their transcription depends on IES excision. We propose that the excision of IESs from proteins expressed in the new MAC may function as a checkpoint that maintains developmental timing.
In addition we are characterizing the remaining development-specific Ptiwi proteins to get a more detailed view of the complex role of small RNA-Ptiwi interactions in Paramecium development.