RNA-dependent RNA polymerases in the black-legged tick produce Argonaute-dependent small RNAs and regulate genes

Small regulatory RNAs (sRNAs) are involved in anti-viral defense and gene regulation. Although RNA-dependent RNA Polymerases (RdRPs) play essential roles in sRNA biogenesis in nematodes, plants and fungi, their involvement in other animals remains controversial. We identify abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs) expressed in the tick ISE6 cell line. The RdRP-dependent sRNAs are mainly derived from sense and antisense strands of RNA polymerase III-transcribed genes and repetitive elements. Knockdown of AGO/RdRP causes miregulation of protein-coding genes.These results demonstrate 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 exploit RNAi for pest control. For small RNA analysis, 9 small RNA libraries of ISE6 cells depleted of small RNA factors and 1 control sample were used. To study the chemical structures of 5’ and 3’ terminal nucleotides of tick sRNAs, oxidized sRNA library and 5'TriP enriched library were used. For total RNAseq analysis, three sets of knockdown experiments(knock down Ago-16, RdRP1 or RdRP3) were performed independently, with one set of control.