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将Dicer鉴定为响应转染双链RNA的双链RNA结合和切割试剂的方案。

Protocol for identifying Dicer as dsRNA binding and cleaving reagent in response to transfected dsRNA.

作者信息

Dai Yunpeng, Wang Jiaxin, Zhang Jiaqi, Liu Xing, Sun Gang, Lu Jinfeng, Li Yang

机构信息

Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China.

CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

出版信息

STAR Protoc. 2025 Mar 21;6(1):103591. doi: 10.1016/j.xpro.2024.103591. Epub 2025 Jan 24.

DOI:10.1016/j.xpro.2024.103591
PMID:39862431
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11804107/
Abstract

Mammalian Dicer has been proved to be functional on double-stranded RNAs (dsRNAs) and involved in antiviral immunity or immune regulation. Here, we present a protocol for identifying Dicer as a dsRNA binding and cleaving factor to transfected dsRNA in cell lines, based on small RNA sequencing (RNA-seq) and dsRNA-immunoprecipitation (dsRNA-IP). We detail both experimental processes and analysis on small RNA-seq data. This protocol can be further applied to identify proteins that process dsRNA into small RNAs, like RNase L. For complete details on the use and execution of this protocol, please refer to Dai et al..

摘要

哺乳动物的Dicer已被证明对双链RNA（dsRNA）具有功能，并参与抗病毒免疫或免疫调节。在此，我们基于小RNA测序（RNA-seq）和双链RNA免疫沉淀（dsRNA-IP），提出了一种在细胞系中鉴定Dicer作为双链RNA结合和切割因子以作用于转染的双链RNA的方案。我们详细介绍了实验过程以及对小RNA-seq数据的分析。该方案可进一步应用于鉴定将双链RNA加工成小RNA的蛋白质，如核糖核酸酶L。有关本方案使用和执行的完整详细信息，请参考戴等人的研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/305c684b3e56/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/4f9a1f522cf1/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/c18c55b42794/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/3ab7f7b0eeca/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/305c684b3e56/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/4f9a1f522cf1/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/c18c55b42794/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/3ab7f7b0eeca/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fcc7/11804107/305c684b3e56/gr3.jpg

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本文引用的文献

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Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity.鼠循环细胞外囊泡含有病毒衍生的 siRNA，可在抗病毒免疫中发挥作用。
EMBO J. 2022 Jun 1;41(11):e109902. doi: 10.15252/embj.2021109902. Epub 2022 Mar 28.
3
Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2.
病毒来源的双链 RNA 的高效 Dicer 加工及其受 RIG-I 样受体 LGP2 的调节。
PLoS Pathog. 2021 Aug 3;17(8):e1009790. doi: 10.1371/journal.ppat.1009790. eCollection 2021 Aug.
4
The activation of antiviral RNA interference not only exists in neural progenitor cells but also in somatic cells in mammals.抗病毒 RNA 干扰的激活不仅存在于神经祖细胞中，也存在于哺乳动物的体细胞中。
Emerg Microbes Infect. 2020 Dec;9(1):1580-1589. doi: 10.1080/22221751.2020.1787798.
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RNA interference functions as an antiviral immunity mechanism in mammals.RNA 干扰作为哺乳动物抗病毒免疫机制发挥作用。
Science. 2013 Oct 11;342(6155):231-4. doi: 10.1126/science.1241911.
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Establishment, immortalisation and characterisation of pteropid bat cell lines.建立、永生化及特性鉴定翼手目蝙蝠细胞系。
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