Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing

• Published in: Cell reports (Cambridge) 2023-01, Vol.42 (1), p.111985-111985, Article 111985
• Main Authors: Tan, Huijuan, Luo, Wentao, Yan, Wei, Liu, Jie, Aizezi, Yalikunjiang, Cui, Ruixue, Tian, Ruijun, Ma, Jinbiao, Guo, Hongwei
• Format: Article
• Language: English
• Subjects: Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; CP: Molecular biology; DCL2/4; Gene Silencing; phase separation; RDR6; RNA Interference; RNA, Double-Stranded; RNA, Small Interfering - metabolism; SGS3; siRNA biogenesis; siRNA-body
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2022.111985

The generation of small interfering RNA (siRNA) involves many RNA processing components, including SUPPRESSOR OF GENE SILENCING 3 (SGS3), RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and DICER-LIKE proteins (DCLs). Nonetheless, how these components are coordinated to produce siRNAs is unclear. Here, we show that SGS3 forms condensates via phase separation in vivo and in vitro. SGS3 interacts with RDR6 and drives it to form siRNA bodies in cytoplasm, which is promoted by SGS3-targeted RNAs. Disrupting SGS3 phase separation abrogates siRNA body assembly and siRNA biogenesis, whereas coexpression of SGS3 and RDR6 induces siRNA body formation in tobacco and yeast cells. Dysfunction in translation and mRNA decay increases the number of siRNA bodies, whereas DCL2/4 mutations enhance their size. Purification of SGS3 condensates identifies numerous RNA-binding proteins and siRNA processing components. Together, our findings reveal that SGS3 phase separation-mediated formation of siRNA bodies is essential for siRNA production and gene silencing. [Display omitted] •SGS3 protein forms condensates through phase separation in vivo and in vitro•SGS3 interacts with RDR6 and RNAs and drives them to form siRNA bodies in cytoplasm•The properties of siRNA bodies are regulated by translation, mRNA decay, and DCL2/4•Several siRNA processing and RNA-binding proteins are identified in SGS3 condensates Tan et al. show that SGS3 undergoes liquid-liquid phase separation that drives RDR6, RNAs, RNA-binding proteins, and other siRNA processing components to form siRNA bodies in cytoplasm. The formation of SGS3/RDR6-containing condensates is necessary and sufficient for siRNA-mediated gene silencing, which is regulated by translation, mRNA decay, and DCL2/4 function.