Two polyphyletic Piwis with Ago-like functions silence somaticgenes at the chromatin level. Piwi function in Paramecium

Most sRNA biogenesis mechanisms involve either RNAseIII cleavage or ping-pong amplification by different Piwi proteins harboring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate \textit{Paramecium} needs both, Dicer activity and two Ptiwi proteins. This mechanism involves primary siRNAs produced by non-translatable transgenes and secondary siRNAs from endogenous remote loci. Our data does not indicate any signatures from ping-pong amplification but a preference for 21nt read overlaps resulting from 23nt Dicer cuts. We show that Ptiwi13 and 14 do not mutually exclusively load primary and secondary siRNAs but have instead similar loading preferences for antisense 5'-U-23nt siRNAs. Both Ptiwis show in addition a general preference for Uridine-rich sRNAs along the entire sRNA length. Our data indicates both, Ptiwis and 2'-O-methylation to contribute to strand selection of Dicer cleaved siRNAs. As both Ptiwis show differential subcellular localization, Ptiwi13 in the cytoplasm and Ptiwi14 in the vegetative macronucleus, we conclude that cytosolic and nuclear silencing factors are necessary for efficient chromatin silencing. This unexpected function of two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways.