Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis.

Manabu Yoshikawa, Yong-Woon Han, Hirofumi Fujii, Shu Aizawa, Tatsuya Nishino, Masayuki Ishikawa

Proceedings of the National Academy of Sciences of the United States of America 2021 Aug 24

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Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA-containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA-containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA-containing AGO1-RISCs but not 21-nt siRNA-containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA-containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3-SDE5-RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.

Citation

Manabu Yoshikawa, Yong-Woon Han, Hirofumi Fujii, Shu Aizawa, Tatsuya Nishino, Masayuki Ishikawa. Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America. 2021 Aug 24;118(34)