Characterization of RNAi Components Involved in Endogenous and Exogenous RNAi Pathways in Paramecium tetraurelia

eng: RNAi by Feeding in Paramecium tetraurelia has been used in molecular biology labs around the world. But apart from the enzymes involved in this pathway, little is known about their actual functions. In this thesis, the various components of the RNAi machinery have been studied to shed light on their involvement during feeding-associated siRNA biogenesis.It was shown that the two RdRPs involved in the pathway, RdRP1 and RdRP2, interact with the exogenous dsRNA trigger and use it as a template to generate new RNA strands which are then further processed by Dicer1. This represents the first example of RdRPs within an exogenously triggered RNAi pathway being responsible for primary siRNA production and interacting with the exogenous trigger RNA directly. Beside RdRPs, the function of Ptiwi proteins within the RNAi by Feeding pathway as well as the endogenous RNAi pathway was analyzed. While the three Ptiwis, Ptiwi12, Ptiwi13 and Ptiwi15, have been associated with the pathway in previous publications, this work showed that Ptiwi14 is also involved in this pathway, loading both primary siRNAs and secondary siRNAs, while the aforementioned three Ptiwis only associate with primary siRNAs. This increases the likelihood of the RNAi by Feeding pathway in Paramecium tetraurelia influencing the cellular chromatin organization, as already described in other species like C. elegans.Additionally, this work analyzed the two paramecium specific proteins Pds1 and Pds2, so far only described as involved in the pathway, but not characterized at all. Pds1 localizes in the cytoplasm of the cell and remains elusive, while Pds2 was localized in cellular membranes, harboring potential to be involved in dsRNA uptake.Last, untemplated nucleotides added to primary siRNAs produced in the RNAi by Feeding pathway were analyzed. This led to the hypothesis of poly-uridylation serving as an RNA degradation signal, supporting the degradation of the passenger strand from the primary siRNA duplex.