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

International Journal of Molecular Sciences

Ken E. Olson¹  Sundar Ganesan²  Apostolos G. Gittis²  Paola Carolina Valenzuela Leon²  Gaurav Shrivastava²  Adeline E. Williams²  Ines Martin-Martin²  Eric Calvo² 

[1] Center for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA;Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA;

关键词: Piwi4;    Aedes aegypti;    piRNA;    RNA interference;    RNAi;    Piwi;   

DOI  :  10.3390/ijms222312733

来源: DOAJ

【 摘 要 】

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 (K_D = 1.7 ± 0.8 μM and K_D 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.

【 授权许可】

Unknown