AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
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DOI：
10.1093/nar/gkz690
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发表时间：
2019-10-10
影响因子：
14.9
通讯作者：
Havelda, Zoltan
中科院分区：
文献类型：
--
作者：
Dalmadi, Agnes;Gyula, Peter;Havelda, Zoltan
RNA干扰(RNAi)是由20-24个核苷酸长的非编码调控(S)小RNA，如微RNA(Mi)和小干扰(Si)RNA通过ArgAerte(AGO)蛋白的作用而介导的。对植物粗提物大小分离的sRNA池进行高通量测序表明，大多数典型的miRNAs与高分子量RNA诱导的沉默复合体与Ago1(HMW RISC)共迁移有关。相反，大多数24个核苷酸长的siRNAs与AGO4(LMW RISC)共迁移的低分子量复合体相关。有趣的是，我们鉴定了一大组细胞质sRNA，包括成熟的miRNA序列，位于与蛋白质未结合的sRNA相对应的低分子范围内。通过比较RISC负载和蛋白质未结合的miRNAs池，我们发现了负载效率非常不同的miRNAs。选择的miRNAs在瞬时和转基因系统中的表达证实了它们的负载能力的改变，这意味着这一过程受与不同miRNA前体相关的信息控制。我们还表明，AGO蛋白的可用性是决定miRNAs负载效率的限制因素。我们的数据揭示了调控检查点的存在，该检查点决定了各种miRNAs的RISC加载效率，方法是只将产生的miRNAs的子集分类到具有生物活性的RISCs中。
RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of cytoplasmic sRNAs, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Expression of selected miRNAs in transient and transgenic systems validated their altered loading abilities implying that this process is controlled by information associated with the diverse miRNA precursors. We also showed that the availability of AGO proteins is a limiting factor determining the loading efficiency of miRNAs. Our data reveal the existence of a regulatory checkpoint determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.