Molecular insight into the network of Drosophila cytoplasmic piRNA pathway proteins through a combination of systematic interaction screening and structural prediction
piRNA-bound PIWI proteins mediate the silencing of transposons at both the transcriptional and post-transcriptional levels, processes that are critical for genome integrity and fertility in animals. While numerous additional proteins are known to be essential for piRNA biogenesis and function in Drosophila and other animals, their molecular and mechanistic functions have remained largely unknown. To improve our molecular understanding of the Drosophila piRNA pathway, we used a cell culture-based protein-protein interaction assay called ReLo to perform a systematic pairwise interaction screen involving 22 factors operating in the cytoplasm, including PIWI proteins, Tudor domain-containing proteins (TDRDs), RNA helicases, and mitochondrial surface proteins. Through additional ReLo interaction testing and structural modeling using AlphaFold-Multimer, we have characterized six protein complexes at the molecular and structural levels. We believe that the results of this screen and our methodological approach are likely to guide future research into the molecular mechanisms underlying piRNA biogenesis and function.