Extensive research has characterized distinct exogenous RNAi pathways interfering in gene expression during vegetative growth of the unicellular model ciliate Paramecium. However, role of RNAi in endogenous transcriptome regulation, and environmental adaptation is unknown. Here, we describe the first genome-wide profiling of endogenous sRNAs in context of different transcriptomic states (serotypes). We developed a pipeline to identify, and characterize 2602 siRNA producing clusters (SRCs). Our data show no evidence that SRCs produce miRNAs, and in contrast to other species, no preference for strand specificity of siRNAs. Interestingly, most SRCs overlap coding genes and a separate group show siRNA phasing along the entire open reading frame, suggesting that the mRNA transcript serves as a source for siRNAs. Integrative analysis of siRNA abundance and gene expression levels revealed surprisingly that mRNA and siRNA show negative as well as positive associations. Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Importantly, most SRCs depend on both RDRs, reminiscent to primary siRNAs in the RNAi against exogenous RNA, indicating mechanistic overlaps between exogenous and endogenous RNAi contributing to flexible transcriptome adaptation.

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Most sRNA biogenesis mechanisms involve either RNAseIII cleavage or ping-pong amplification by different Piwi proteins harboring slicer activity. Here, we follow thequestion why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from endogenous remote loci. Our data does not indicate any signatures from ping-pongamplification but Dicer cleavage of long dsRNA. We show that Ptiwi13 and 14 have different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and Ptiwi14 loaded siRNAs show a 5′-U signature. 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. This unexpected function of two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. As both Ptiwis show differential subcellular localization, Ptiwi13 in the cytoplasm and Ptiwi14 in thevegetative macronucleus, we conclude that cytosolic and nuclear silencing factors are necessary for efficient chromatin silencing.