The in vivo dsRNA Cleavage Has Sequence Preference in Insects

Area of Science:

• Molecular Biology
• Entomology
• Genetics

Background:

• Exogenous double-stranded RNA (dsRNA) triggers RNA interference (RNAi) in insects by being processed into small interfering RNA (siRNA).
• The specific sequence compositions and cleavage sites dictating dsRNA processing in vivo remain largely uncharacterized, hindering RNAi application optimization.

Purpose of the Study:

• To investigate sequence preferences and regularity in dsRNA processing into siRNA in vivo across different insect species.
• To identify specific nucleotide residues and sequence motifs involved in dsRNA cleavage and siRNA generation.

Main Methods:

• Injection of dsRNA (0.04 mg/g) into Asian corn borers and cotton bollworms.
• Analysis of in vivo generated siRNAs using RNA-sequencing (RNA-Seq).
• Site-directed mutagenesis of identified dsRNA cleavage sites (GGU) to assess impact on siRNA production.

Main Results:

• dsRNA processing in Asian corn borers and cotton bollworms showed a strong preference for GGU nucleotide residues at the 5' and 3' ends, creating a distinct siRNA peak.
• Mutating the GGU site significantly reduced siRNA production and abolished the characteristic siRNA peak.
• In contrast, the red flour beetle (Coleoptera) exhibited more diverse cleavage sites (e.g., AAG, GUG, GUU) with a conserved A/U base composition.

Conclusions:

• dsRNA in vivo processing into siRNA is not random but exhibits sequence preference and regularity in insects.
• The identified cleavage preferences, such as GGU in Lepidoptera and A/U-rich sites in Coleoptera, provide insights into insect-specific RNAi mechanisms.
• Understanding these cleavage regularities can enhance the efficacy and application of RNAi technology in pest control and functional genomics.