在. 的小干扰 RNA 扩增过程中，RDR6 通过 SGS3 和 SDE5 的协同招募
Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in .
机构信息
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8518, Japan;
Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan.
出版信息
Proc Natl Acad Sci U S A. 2021 Aug 24;118(34). doi: 10.1073/pnas.2102885118.
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 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.
小干扰 RNA（siRNA）通常从 RNA 诱导沉默复合物（RISC）切割的转录本中扩增而来，该复合物包含小 RNA（sRNA）和 Argonaute 蛋白。扩增的 siRNA 被称为次级 siRNA，对于增强靶基因抑制非常重要。在植物中，由包含 22 个核苷酸（nt）的 sRNA 和 Argonaute 1（AGO1）的 RISC 切割靶标会触发 siRNA 扩增。在这条途径中，由 RNA 依赖性 RNA 聚合酶 6（RDR6）将切割片段转化为双链 RNA（dsRNA），然后由 Dicer 样蛋白将 dsRNA 加工成 siRNA。由于非特异性 RDR6 募集会导致非靶标 siRNA 的产生，因此 RDR6 特异性募集到作为 dsRNA 形成模板的靶 RNA 至关重要。先前的研究表明，基因沉默抑制物 3（SGS3）结合并稳定与切割靶标相关的包含 22nt sRNA 的 AGO1 RISC，但 RDR6 如何募集到由包含 22nt sRNA 的 AGO1 RISC 切割的靶标尚不清楚。在这里，我们使用悬浮培养细胞制备的无细胞提取物，建立了一个体外次级 siRNA 产生系统，其中包含 22nt siRNA 的 AGO1-RISC 而不是包含 21nt siRNA 的 AGO1-RISC 诱导次级 siRNA 的产生。在该系统中，添加重组沉默缺陷 5（SDE5）蛋白可显著增强次级 siRNA 的产生。我们表明，RDR6 与 SGS3 和 SDE5 一起被包含 22nt siRNA 的 AGO1-RISC 募集到切割片段。SGS3-SDE5-RDR6 多组分识别系统和 poly(A) 尾巴抑制可能有助于确保 siRNA 扩增的特异性。
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