*Article Aedes aegypti* **Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization**

**Adeline E. Williams 1,2 , Gaurav Shrivastava <sup>1</sup> , Apostolos G. Gittis <sup>1</sup> , Sundar Ganesan <sup>1</sup> , Ines Martin-Martin <sup>1</sup> , Paola Carolina Valenzuela Leon <sup>1</sup> , Ken E. Olson 2,\* and Eric Calvo 1,\***


**Abstract:** The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from *Drosophila* studies to maintain the integrity of the germline genome by silencing transposable elements (TE). *Aedes aegypti* mosquitoes, which are the key vectors of several arthropodborne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of *A. aegypti* Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 30 ends of piRNAs, bound to mature (30 2 0 O-methylated) and unmethylated RNAs with similar micromolar affinities (K<sup>D</sup> = 1.7 ± 0.8 µM and K<sup>D</sup> of 5.0 ± 2.2 µM, respectively; *p* = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.

**Keywords:** Piwi4; *Aedes aegypti*; piRNA; RNA interference; RNAi; Piwi; NLS; arbovirus; mosquito
