Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection.

Nieves Ayllón,Ondrej Hajdušek,Radek Šíma,Lesley Bell-Sakyi,Alejandro Cabezas-Cruz,Sabína Havlíková,Claudia Rückert,Victoria Naranjo,Pilar Alberdi,Ruth C Galindo,Boris Klempa,Mária Kazimírová,Katherine M Kocan,José De La Fuente,Petr Kopáček,Margarita Villar

doi:10.1371/journal.pone.0133038

Abstract

Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.

Highlights

RNA interference (RNAi) is conserved in eukaryotes with a variety of functions including immune response and gene regulation [1]
Ago2 and Dcr2 proteins are essential for the insect antiviral RNAi pathway while Ago1 and Dcr1 proteins are involved in the insect micro RNA pathway [3]
To rule out off-target effects, the experiment was repeated using another double-stranded RNAs (dsRNAs) targeting tick Tudor-staphylococcal nuclease homologues (SN) (SN instead of Tudor domain) and the results confirmed previous results using Tudor domain-targeted dsRNA (Table 1). These results demonstrated that Tudor-SN is involved in dsRNA-mediated RNAi in ticks and prompted the question of whether this protein is involved in tick innate immune responses and other cellular processes

Summary

Introduction

RNA interference (RNAi) is conserved in eukaryotes with a variety of functions including immune response and gene regulation [1]. Analysis of the RNAi pathways in different model organisms suggests that both Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are components of the basic RNAi machinery [2]. Tudor-SN has been implicated in eukaryotes in a variety of cellular processes such as transcription, processing of dsRNA, RNAi, splicing regulation and stress response [4,5,6]. RNAi has been evolutionarily conserved in eukaryotes, in some organisms such as Leishmania and Trypanosoma the RNAi pathways may be inactive through loss of Dicer and/ or Ago [7] or some components such as Tudor-SN may play a minor role [8]

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Results

Conclusion