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- Canran Feng
- Kyosuke Torimaru
- Mandy Yu Theng Lim
- Li-Ling Chak
- Kosuke Tsuji
- Tetsuya Tanaka
- Junko Iida
- Katsutomo Okamura
Small regulatory RNAs (sRNAs) are involved in anti-viral defense and gene regulation. Although RNA-dependent RNA Polymerases (RdRPs) are known to produce sRNA in nematodes, plants and fungi, whether they play roles in sRNA biogenesis in other animals remains controversial. In this study, we study sRNAs in the ISE6 cell line, which is derived from the black-legged tick, an important vector of human and animal pathogens. We identify abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs). RdRP-dependent sRNAs are mainly derived from sense and antisense strands of RNA polymerase III-transcribed genes and repetitive elements. Unlike C. elegans sRNA pathways, 5’-tri-phosphorylated sRNAs are not detected, suggesting that the tick pathways are distinct from the pathways known in worms. Knockdown of one of the RdRPs unexpectedly results in downregulation of a subset of viral transcripts, in contrast to their upregulation by AGO knockdown. Furthermore, we show that knockdown of AGO/RdRP causes misregulation of protein-coding genes including RNAi-related genes, suggesting feedback regulation. Luciferase assays demonstrate that one of the RdRP-regulated genes, the MEK1 ortholog IscDsor1 is regulated through its 3’UTR, where a putative sRNA target site resides. These results provide evidence that arachnid RdRPs are important sRNA biogenesis factors, and the discovery of novel pathways underscores the importance of characterizing sRNA biogenesis in various organisms to understand virus-vector interactions and to exploit RNAi for pest control.
RNA-dependent RNA Polymerases (RdRPs) are essential for biogenesis of small regulatory RNAs (sRNAs) in many organisms such as plants and fungi, but its general importance in animals besides nematodes remains controversial because experimental evidence is lacking. By using a tick cell line, we demonstrate that RdRP-dependent sRNAs are abundantly expressed and tick RdRPs regulate gene expression. These results indicate that ticks have unexpectedly complex sRNA biogenesis pathways that are essential for proper gene regulation. Because ticks are important vectors of human and animal pathogens, and their sRNA pathways control tick-borne viruses, studying the novel tick sRNA pathways may provide important information for a better understanding of vector-virus interactions and for developing RNA-based pesticides by utilizing tick’s RNA silencing mechanisms.