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Article

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

1
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
2
Center for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA
*
Authors to whom correspondence should be addressed.
Academic Editor: Michail Kotsyfakis
Int. J. Mol. Sci. 2021, 22(23), 12733; https://doi.org/10.3390/ijms222312733
Received: 31 October 2021 / Revised: 22 November 2021 / Accepted: 23 November 2021 / Published: 25 November 2021
(This article belongs to the Special Issue Molecular Biology of Disease Vectors)
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 arthropod-borne 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 3′ ends of piRNAs, bound to mature (3′ 2′ O-methylated) and unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 μM and KD 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. View Full-Text
Keywords: Piwi4; Aedes aegypti; piRNA; RNA interference; RNAi; Piwi; NLS; arbovirus; mosquito Piwi4; Aedes aegypti; piRNA; RNA interference; RNAi; Piwi; NLS; arbovirus; mosquito
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MDPI and ACS Style

Williams, A.E.; Shrivastava, G.; Gittis, A.G.; Ganesan, S.; Martin-Martin, I.; Valenzuela Leon, P.C.; Olson, K.E.; Calvo, E. Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization. Int. J. Mol. Sci. 2021, 22, 12733. https://doi.org/10.3390/ijms222312733

AMA Style

Williams AE, Shrivastava G, Gittis AG, Ganesan S, Martin-Martin I, Valenzuela Leon PC, Olson KE, Calvo E. Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization. International Journal of Molecular Sciences. 2021; 22(23):12733. https://doi.org/10.3390/ijms222312733

Chicago/Turabian Style

Williams, Adeline E., Gaurav Shrivastava, Apostolos G. Gittis, Sundar Ganesan, Ines Martin-Martin, Paola C. Valenzuela Leon, Ken E. Olson, and Eric Calvo. 2021. "Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization" International Journal of Molecular Sciences 22, no. 23: 12733. https://doi.org/10.3390/ijms222312733

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