Analysis of Piwi proteins involved in Dicer-dependent pathways leading to the elimination of germline-specific DNA

Piwi proteins and their associated piRNAs protect eukaryotic germlines against the spread of transposable elements. In the ciliate Paramecium, these proteins and small RNAs (sRNAs) guide the developmentally programmed elimination of germline-limited genomic regions, including transposons and transposon-derived DNA segments called Internal Eliminated Sequences (IESs). Two distinct sRNA pathways have been identified as involved in this process: the scnRNAs, which bound to two Piwi proteins, Ptiwi01 and Ptiwi09, and the iesRNAs, whose binding partners we have now identified as the previously unclassified Ptiwi10 and Ptiwi11. We show that scnRNAs and iesRNAs bind their Piwi proteins in a mutually exclusive manner, which together with distinct Dicer-like proteins for these sRNA classes, results in separate biogenesis pathways. We also demonstrate that the gene encoding Ptiwi10 is transcribed from the developing nucleus and that its transcription depends on IES excision, suggesting a possible checkpoint control mechanism in forming the new somatic genome.