Molecular Characterizations and Functional Analyses of LmR2D2 in the Locusta migratoria siRNA Pathway

Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21 similar to 23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved posttranscriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Agog) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in Drosophila. However, the involvement of R2D2 in the siRNA pathway of Locusta migratoria is still unknown. In the present study, we identified an LmR2D2 gene from the transcriptome of L. migratoria. It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of LmR2D2 indicated that its transcript level was stable in third-instar nymphs of L. migratoria, whereas the tissue-dependent expression profile exhibited high levels of expression of LmR2D2 in the testis and ovary. When LmR2D2 was silenced by RNAi, the RNAi efficiency against Lm beta-tubulin as a marker gene was significantly diminished, as indicated by the 37.7% increased Lm beta-tubulin transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of L. migratoria.

Automated summary

The study identified an LmR2D2 gene in Locusta migratoria, encoding a protein with two dsRNA-binding domains. LmR2D2 plays an important role in the siRNA pathway, and its expression levels are stable during development in Locusta migratoria, with higher levels in the testis and ovary. Silencing LmR2D2 led to reduced RNAi efficiency against a marker gene, supporting its essential role in siRNA binding in vitro.