Molecular insight into theDrosophilapiRNA pathway network through a combination of systematic protein interaction screening and structural prediction

Abstract

PIWI-interacting RNAs (piRNAs) are small, non-coding RNAs that are critical for genome integrity and fertility in animals. In complexes with PIWI proteins, they mediate the silencing of transposons and other genes at both transcriptional and post-transcriptional levels. While numerous additional proteins are known to be essential for piRNA biogenesis and function inDrosophilaand other animals, their molecular and mechanistic functions have remained largely unknown. To improve our molecular understanding of theDrosophilapiRNA pathway, we used a cell culture-based protein-protein interaction assay, called ReLo, and performed 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. We detected all previously reported interactions for which crystal structures have been determined, some of which are dependent on symmetric arginine dimethylation (sDMA), and several interactions previously identified by co-immunoprecipitation experiments. In addition, we identified previously unknown interactions. Through additional ReLo interaction testing and structural modeling using AlphaFold-Multimer, we have characterized six protein complexes at the molecular and structural levels, namely the Vreteno-BoYb, Vreteno-SoYb, Tejas-Spindle E, Tejas-Maelstrom, Tejas-Krimper complexes, as well as the complex composed of Armi and the Gasz-Daed heterodimer. 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.