Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants.
Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants.
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DOI：
10.1073/pnas.2102889118
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发表时间：
2021-08-03
影响因子：
11.1
通讯作者：
Iwakawa HO
中科院分区：
文献类型：
--
作者：
Sakurai Y;Baeg K;Lam AYW;Shoji K;Tomari Y;Iwakawa HO
RNA 依赖性 RNA 聚合酶 (RDR) 合成双链 RNA (dsRNA) 是次级小干扰 RNA (siRNA) 生物发生的关键步骤。然而，RDR 如何特异性地将初级小 RNA 的靶标转化为 dsRNA 中间体仍不清楚。在这里，我们开发了一种体外系统，该系统概括了在植物中具有重要生理意义的次级 siRNA 的产生。利用该系统，我们发现四种植物因子的组合可促进 RDR6 物理募集至目标 RNA。此外，我们发现通过去除poly(A)尾可以增强RDR6的dsRNA合成，这是通过在另一个带有适当错配的小RNA结合位点进行切割来实现的。我们的数据阐明了植物中二次 siRNA 生物发生所必需的分子事件。由初级小 RNA 靶向触发的次级小干扰 RNA (siRNA) 的产生对于许多生物体的正常发育和抗病毒防御至关重要。 RNA 依赖性 RNA 聚合酶 (RDR) 是该途径的关键因素。然而，RDR 如何特异性地将初级小 RNA 的靶标转化为双链 RNA (dsRNA) 中间体仍不清楚。在这里，我们开发了一个体外系统，可以剖析反式作用 siRNA 产生的分子机制，反式作用 siRNA 是一类在器官发育和应激反应中发挥作用的植物次级 siRNA。我们发现 dsRNA 结合蛋白、基因沉默抑制因子 3 的组合；假定的核 RNA 输出因子、SILENCING DEFECTIVE5、初级小 RNA 和 Argonaute 是 RDR6 物理募集至靶 RNA 所必需的。 RDR6 的 dsRNA 合成通过去除 Poly(A) 尾而大大增强，这可以通过在带有适当错配的第二个小 RNA 结合位点处进行切割来实现。重要的是，当第二个靶位点碱基配对的互补性太强时，小RNA-Argonaute复合物仍保留在切割位点，从而阻止RDR6启动dsRNA合成。我们的数据强调了二次 siRNA 生物发生中双小 RNA 靶向的光明和黑暗的一面。
Double-stranded RNA (dsRNA) synthesis by RNA-dependent RNA polymerase (RDR) is a critical step in secondary small interfering RNA (siRNA) biogenesis. However, how RDR specifically converts the targets of primary small RNAs into dsRNA intermediates remains unclear. Here, we developed an in vitro system that recapitulates the production of secondary siRNAs that are physiologically important in plants. Leveraging this system, we showed that a combination of four plant factors promotes physical recruitment of RDR6 to the target RNA. Moreover, we found that dsRNA synthesis by RDR6 is enhanced by the removal of the poly(A) tail, which is achieved by cleavage at another small RNA-binding site bearing appropriate mismatches. Our data elucidate the molecular events necessary for secondary siRNA biogenesis in plants. Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA–Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.
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DOI：
10.1038/nrg3355
发表时间：
2013-02
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
--
影响因子：
11.6
作者：
Howell, Miya D.;Fahlgren, Noah;Carrington, James C.
通讯作者：
Carrington, James C.
影响因子：
14.9
作者：
de Felippes FF;Marchais A;Sarazin A;Oberlin S;Voinnet O
通讯作者：
Voinnet O
影响因子：
9.2
作者：
Fahlgren, Noah;Montgomery, Talowa A.;Carrington, James C.
通讯作者：
Carrington, James C.
影响因子：
4.8
作者：
Curaba, Julien;Chen, Xuemei
通讯作者：
Chen, Xuemei