Hl48 modulates argonaute 2 to enhance RNA interference in ticks

Area of Science:

• Molecular Biology
• Entomology
• RNA Interference

Background:

• Ticks are significant vectors of pathogens, necessitating novel control methods.
• RNA interference (RNAi) shows promise for tick control but faces challenges due to variable efficiency and poorly understood regulatory mechanisms.
• Developing species-specific tick control is crucial for public health and animal welfare.

Purpose of the Study:

• To identify and characterize novel regulatory proteins involved in the RNAi pathway in *Haemaphysalis longicornis*.
• To elucidate the function and mechanism of the identified protein, Hl48, in regulating RNAi efficiency.
• To explore the potential of Hl48 as a target for enhancing RNA-based tick control strategies.

Main Methods:

• Transcriptomic screening, molecular cloning, and functional genomics were used to identify and characterize Hl48.
• Loss-of-function (RNAi) and gain-of-function (RNA activation, RNAa) strategies were employed to assess Hl48's role in vivo.
• Mechanistic studies included dual-luciferase reporter assays, co-immunoprecipitation (Co-IP), molecular docking, mutagenesis, and surface plasmon resonance (SPR).

Main Results:

• Hl48 expression increased with RNA virus infection or dsRNA stimulation, indicating its involvement in the RNAi pathway.
• Silencing Hl48 reduced RNAi efficiency, while activating it enhanced RNAi responsiveness.
• Hl48 directly interacts with Argonaute 2 (AGO2) and upregulates *ago2* transcription, demonstrating a dual regulatory mechanism.

Conclusions:

• Hl48 is identified as a key positive regulator of the RNAi pathway in ticks.
• This study reveals a novel layer of RNAi regulation in arthropods.
• The findings provide a mechanistic basis for developing improved RNA-based strategies for tick-borne disease control.