Both LmDicer-1 and two LmDicer-2s participate in siRNA-mediated RNAi pathway and contribute to high gene silencing efficiency in Locusta migratoria
Both LmDicer-1 and two LmDicer-2s participate in siRNA-mediated RNAi pathway and contribute to high gene silencing efficiency in Locusta migratoria
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LmDicer-1 和两个 LmDicer-2 均参与 siRNA 介导的 RNAi 途径，并有助于飞蝗的高基因沉默效率
DOI：
10.1016/j.ibmb.2022.103865
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发表时间：
2022-11-09
影响因子：
3.8
通讯作者：
Zhang，Jianzhen
中科院分区：
文献类型：
--
作者：
Gao，Lu;Wang，Yanli;Zhang，Jianzhen
切丁酶属于一类大的RNase III多结构域核糖核酸酶，并且是RNA干扰（RNAi）途径的中心组分。在昆虫中，Dicer-2已知在小干扰RNA（siRNA）介导的RNAi途径中切割长双链RNA（dsRNA）。而Dicer-1在miRNA介导的RNAi途径中负责切割前体microRNA（pre 28 miRNAs）。在本研究中，我们在飞蝗中鉴定了一个LmDicer-1和两个LmDicer-2（LmDicer-2a和LmDicer-2b）基因。RNAi实验结果表明，敲除Dicergenes后，对靶基因Lmβ-Tubulin的RNAi效率降低，提示所有Dicergenes都参与了siRNA介导的RNAi途径。在沉默每个LmDicergene后，对dsLmβ-Tubulin产生的siRNA进行序列分析，结果显示定位到dsLmβ-Tubulin的siRNA模式没有显著差异。这一结果表明所有三种LmDicer都能够从双链RNA产生siRNA。然后利用大肠杆菌表达系统构建了由不同结构域组成的重组蛋白，并将重组蛋白与dsLmβ-Tubulin共孵育，发现重组Dicer蛋白能成功切割dsLmβ-Tubulin。然而，缺乏dsRBD结构域的LmDicer-2a-R失去活性，表明dsRBD结构域对于Dicer功能至关重要。此外，这些蛋白在果蝇S2细胞中的过表达提高了RNAi效率。我们的siRNA亲和层析和LC-MS/MS分析鉴定了LmDicer-2a、LmDicer-2b、LmR 2D 2、LmAgo 2a、LmAgo 1、LmStaufen和LmTARBP 2为RNA诱导沉默复合物的组分。综上所述，这些数据表明LmDicer-1和两个LmDicer-2都参与siRNA介导的RNAi途径，并且可能有助于在L中的高RNAi效率。迁徙者。
Dicers belong to a class of large RNase III multidomain ribonucleases and are central components of the RNA interference (RNAi) pathways. In insects, Dicer-2 has been known to cleave long double-stranded RNA (dsRNA) in small interfering RNA (siRNA)-mediated-RNAi pathway. However, Dicer-1 is responsible for cleaving precursor microRNAs (pre28 miRNAs) in miRNA-mediated RNAi pathway. In this study, we identified oneLmDicer-1and twoLmDicer-2(LmDicer-2aandLmDicer-2b) genes in Locusta migratoria. The RNAi of RNAi assay showed that knockdown of each of theDicergenes reduced RNAi efficiency against a target gene (Lmβ-Tubulin), suggesting that all these genes participated in the siRNA-mediated RNAi pathway. Sequence analyses of the siRNAs generated from dsLmβ-Tubulinafter silencing eachLmDicergene showed no significant difference in the pattern of siRNAs mapped to dsLmβ-Tubulin. This result indicated that all the three LmDicers are capable of generating siRNAs from the dsRNA. We then generated recombinant proteins consisting of different domains usingEscherichia coliexpression system and incubated each recombinant protein with dsLmβ-Tubulin.We found that the recombinant Dicer proteins successfully cleaved dsLmβ-Tubulin. However, LmDicer-2a-R lacking dsRBD domain lost activity, suggesting that dsRBD domain is critical for Dicer function. Furthermore, overexpression of these proteins inDrosophilaS2 cells improved RNAi efficiency. Our siRNA affinity chromatography and LC-MS/MS analysis identified LmDicer-2a, LmDicer-2b, LmR2D2, LmAgo2a, LmAgo1, LmStaufen and LmTARBP2 as constituents of RNA-induced silencing complex. Taken together, these data show that both LmDicer-1 and two LmDicer-2s all participate in siRNA-mediated RNAi pathway and likely contribute to high RNAi efficiency inL. migratoria.