Tschopp Marie-Aude, Iki Taichiro, Brosnan Christopher A, Jullien Pauline E, Pumplin Nathan
Department of Biology, ETH Zürich, 8092 Zürich, Switzerland.
Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
RNA. 2017 May;23(5):782-797. doi: 10.1261/rna.059519.116. Epub 2017 Feb 23.
Small RNAs play an important role in regulating gene expression through transcriptional and post-transcriptional gene silencing. Biogenesis of small RNAs from longer double-stranded (ds) RNA requires the activity of dicer-like ribonucleases (DCLs), which in plants are aided by dsRNA binding proteins (DRBs). To gain insight into this pathway in the model plant , we searched for interactors of DRB4 by immunoprecipitation followed by mass spectrometry-based fingerprinting and discovered DRB7.1. This interaction, verified by reciprocal coimmunoprecipitation and bimolecular fluorescence complementation, colocalizes with markers of cytoplasmic siRNA bodies and nuclear dicing bodies. In vitro experiments using tobacco BY-2 cell lysate (BYL) revealed that the complex of DRB7.1/DRB4 impairs cleavage of diverse dsRNA substrates into 24-nucleotide (nt) small interfering (si) RNAs, an action performed by DCL3. DRB7.1 also negates the action of DRB4 in enhancing accumulation of 21-nt siRNAs produced by DCL4. Overexpression of DRB7.1 in altered accumulation of siRNAs in a manner reminiscent of mutant plants, suggesting that DRB7.1 can antagonize the function of DRB4 in siRNA accumulation in vivo as well as in vitro. Specifically, enhanced accumulation of siRNAs from an endogenous inverted repeat correlated with enhanced DNA methylation, suggesting a biological impact for DRB7.1 in regulating epigenetic marks. We further demonstrate that RNase three-like (RTL) proteins RTL1 and RTL2 cleave dsRNA when expressed in BYL, and that this activity is impaired by DRB7.1/DRB4. Investigating the DRB7.1-DRB4 interaction thus revealed that a complex of DRB proteins can antagonize, rather than promote, RNase III activity and production of siRNAs in plants.
小RNA通过转录和转录后基因沉默在调控基因表达中发挥重要作用。从较长的双链（ds）RNA生成小RNA需要类Dicer核糖核酸酶（DCL）的活性，在植物中，双链RNA结合蛋白（DRB）会协助DCL发挥作用。为深入了解模式植物中的这一途径，我们通过免疫沉淀结合基于质谱的指纹图谱技术寻找DRB4的相互作用蛋白，并发现了DRB7.1。这种相互作用通过相互免疫沉淀和双分子荧光互补得到验证，它与细胞质小干扰RNA（siRNA）小体和细胞核切割小体的标记物共定位。使用烟草BY - 2细胞裂解物（BYL）进行的体外实验表明，DRB7.1/DRB4复合物会损害多种dsRNA底物切割成24个核苷酸（nt）的小干扰（si）RNA的过程，这一过程由DCL3执行。DRB7.1还会抵消DRB4在增强DCL4产生的21 - nt siRNA积累方面的作用。在 中过表达DRB7.1会改变siRNA的积累，其方式类似于 突变体植物，这表明DRB7.1在体内和体外都能拮抗DRB4在siRNA积累中的功能。具体而言，来自内源性反向重复序列的siRNA积累增强与DNA甲基化增强相关，这表明DRB7.1在调节表观遗传标记方面具有生物学影响。我们进一步证明，RNase三型（RTL）蛋白RTL1和RTL2在BYL中表达时会切割dsRNA，并且这种活性会被DRB7.1/DRB4削弱。因此，对DRB7.1 - DRB4相互作用的研究表明，DRB蛋白复合物在植物中会拮抗而非促进RNase III活性和siRNA的产生。
