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 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.

Highlights

  • The P-element-induced wimpy testis (PIWI)-interacting RNA pathway is an RNA interference (RNAi) mechanism that is traditionally known to silence transposable elements (TEs) that can integrate into the germline genome and threaten its integrity [1,2,3,4]. PIWI-interacting RNA (piRNA), 23–30 nucleotide small RNAs, bind Piwi proteins, a subfamily of the Argonautes. piRNA-bound Piwis assemble into piRNA-induced silencing complexes, where effector Piwis are targeted to complementary RNA substrates [5,6,7]

  • The aegypti Piwi4 (AePiwi4) PAZ model suggests that the protein contains hydrophobic regions buried within a flexible protein structure, allowing AePiwi4 to bind long 3 m piRNAs

  • We found that the resulting eGFP nuclear intensity sums outside of the nuclear surfaces was significantly higher for cells transfected with the eGFP construct as compared to cells transfected with SV40NLS-eGFP (p = 0.006), AePiwi4NLS-EGFP (p = 0.01), or AePiwi4Nterminal-eGFP (p = 0.05)

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Summary

Introduction

The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is an RNA interference (RNAi) mechanism that is traditionally known to silence transposable elements (TEs) that can integrate into the germline genome and threaten its integrity [1,2,3,4]. piRNAs, 23–30 nucleotide (nt) small RNAs (sRNAs), bind Piwi proteins, a subfamily of the Argonautes. piRNA-bound Piwis assemble into piRNA-induced silencing complexes (piRISCs), where effector Piwis are targeted to complementary RNA substrates [5,6,7]. Silencing AePiwi depleted 3 2 O-methylated (mature) Sindbis virus (SINV)-specific vsiRNAs and vpiRNAs and increased SINV, dengue (DENV2), and chikungunya (CHIKV) virus replication in infected cells [32]. This phenotype was recapitulated in DENV2-infected A. aegypti mosquitoes, where silencing AePiwi increased infectious virus titers 5–10 days post-infection (dpi) [32]. AePiwi has been consistently associated with long (28–30 nt), mature 3 2 O-methylated ( termed “3 m”) piRNAs, and it was found in both the cytoplasmic and nuclear fractions in an embryonic Aedes aegypti cell line (Aag2) [26,32,39]. We found that subtle structural differences across Piwi proteins may have important impacts on preferential RNA-binding behaviors and subcellular localization

Biophysical Properties of AePiwi4 by Structural Modeling and Alignment
Materials and Methods
Cloning
Recombinant Protein Purification
Western Blot
Mosquito Rearing
Sequence Alignment
Site Directed Mutagenesis
4.11. Circular Dichroism
4.13. Mouse Polyclonal Antibody Production
4.14. Mass Spectrometry
4.16. HEK293 Cell Culture and Transfection
4.17. Statistics
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