Hl48 modulates argonaute 2 to enhance RNA interference in ticks

Background Ticks are major vectors of human and animal pathogens, and the development of novel, species-specific control strategies is urgently needed. RNA interference (RNAi) holds promise as a targeted approach for tick control; however, its practical application has been limited by variable efficiency and an incomplete understanding of the underlying regulatory mechanisms in these arthropods. Methods Using transcriptomic screening, molecular cloning, and functional genomics approaches, we identified and characterized Hl48, a previously uncharacterized ~48 kDa protein in Haemaphysalis longicornis ( H. longicornis ). Loss-of-function (RNAi) and gain-of-function (RNA activation, RNAa) strategies were employed to assess the role of Hl48 in RNAi regulation in vivo . Mechanistic investigations included dual-luciferase reporter assays, co-immunoprecipitation (Co-IP), molecular docking, site-directed mutagenesis, and surface plasmon resonance (SPR) analysis. Results Hl48 expression was significantly upregulated in response to RNA virus infection or exogenous dsRNA stimulation, but not to protozoan infection, functionally linking it to the RNAi pathway. Silencing of Hl48 substantially impaired RNAi efficiency against multiple target genes, whereas its activation enhanced RNAi responsiveness both in vivo and ex vivo . Mechanistically, Hl48 regulates the RNAi pathway through a dual mode of action: it directly interacts with the PIWI domain of Argonaute 2 (AGO2), the catalytic core of the RNA-induced silencing complex (RISC), and concurrently promotes transcriptional upregulation of ago2 . Key interaction residues (Thr28 and Glu93) were identified and validated by mutagenesis. Conclusion These findings establish Hl48 as a key positive regulator of the RNAi pathway in ticks, revealing a previously unrecognized layer of RNAi control in arthropods. This work provides a mechanistic foundation for the development of enhanced RNA-based strategies for tick-borne disease control.