Project Summary/Abstract Transposable elements (TEs), also known as jumping genes are mobile DNA elements that move from one genomic location to the other in the genome. The activity of TEs can cause major changes in genome structure and must be restricted to prevent developmental defects, aging, neurodegenerative diseases and cancer. TE mobility is silenced during germline development to prevent genome changes from being passed on to the progeny. In the germline, piRNA pathway silences transposons. The primary function of the piRNA pathway is mediated by PIWI clade proteins which in Drosophila are represented by Piwi, Aubergine (Aub) and Argonaute 3 (Ago3) proteins. These proteins bind piRNAs (~26 nucleotides in length) and target TEs through sequence-specific complementarity. All three proteins have non-redundant function in silencing TE. TE silencing in the germline is compartmentalized. Piwi is nuclear and is needed for transcriptional silencing of TEs. Aub and Ago3 are cytoplasmic and are needed for post-transcriptional silencing of TEs. Majority of piRNAs are made by Aub and Ago3 involved ping-pong amplification that occurs in the nuage, a RNA-rich perinuclear granule. In the germline cells, Aub and Ago3 participate in ping-pong cycle to produce piRNAs and these piRNAs are loaded onto Piwi; however, neither the mechanism nor the proteins involved in coupling piRNA biogenesis with piRNA loading onto Piwi are known. Aub and Ago3 need to interact for piRNA biogenesis; however the mechanism by which Aub and Ago3 interact is not known. A seamless network needs to exist between the nuclear pore complex (NPC) and the nuage to ensure that Piwi-piRNA complexes assembled in the nuage translocate into the nucleus and silence transposons; however the mechanism by which the NPC might regulate Piwi nuclear function is elusive. Our preliminary data shows for the first time that Nup358, a key component of cytoplasmic filaments of the NPC, interacts with Piwi and is required for a) Piwi's entry into the nucleus, b) TE silencing, c) loading of piRNAs onto Piwi, d) piRNA biogenesis, and e) Aub-Ago3 interaction. These data suggest that Nup358 is a key player in piRNA pathway and by characterizing how Nup358 regulates piRNA pathway, we will reveal significant insights into how the NPC promotes piRNA biogenesis, TE silencing and genome stability. The scientific premise of this proposal is that there is sufficient evidence that Nup358 is a key player in piRNA pathway, but the mechanism by which Nup358 achieves the same is elusive. Based on the preliminary data, we hypothesize that Nup358 recruits Piwi to the nuclear membrane and couples piRNA biogenesis with piRNA loading onto Piwi. To test this hypothesis, we will biochemically define Piwi-Nup358 interaction (Aim I) and unravel the mechanism by which Nup358 couples piRNA biogenesis with piRNA loading onto Piwi (Aim II).