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锥虫中RNA干扰突变体的功能互补

Functional complementation of RNA interference mutants in trypanosomes.

作者信息

Rusconi Filippo, Durand-Dubief Mickaël, Bastin Philippe

机构信息

UMR5153 CNRS, USM0503 MNHN, U565 INSERM-57, rue Cuvier, P.B 26 - F-75231, Paris Cedex 05, France.

出版信息

BMC Biotechnol. 2005 Feb 9;5:6. doi: 10.1186/1472-6750-5-6.

Abstract

BACKGROUND

In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.

RESULTS

Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility.

CONCLUSIONS

This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.

摘要

背景

在许多真核细胞中,双链RNA(dsRNA)会触发RNA干扰(RNAi),即同源序列RNA的特异性降解。RNAi现在是反向遗传学项目的主要工具,包括大规模高通量筛选。最近的报道对RNAi的特异性提出了质疑,给基于RNAi的实验解释带来了问题。

结果

以原生动物布氏锥虫为模型,我们设计了一种功能互补试验,以确定RNAi时观察到的表型效应是由于靶向基因的特异性沉默所致。这应用于一个编码副鞭毛杆蛋白2(TbPFR2)的细胞骨架基因,其产物对鞭毛运动至关重要。我们通过表达一种带有标签的抗RNAi的TbPFR2来证明对通过靶向其非翻译区的dsRNA沉默的TbPFR2的互补作用,该蛋白可以在鞭毛中进行免疫定位。接下来,我们通过克氏锥虫的TbPFR2直系同源基因的异源表达,对通过靶向其编码序列的dsRNA沉默的TbPFR2进行了功能互补:鞭毛恢复了运动能力。

结论

这项工作表明,可以很容易地进行功能互补实验,以确定RNAi实验中观察到的表型效应确实是由于靶向基因的特异性沉默所致。此外,当在不适合RNAi的生物体中难以进行反向遗传学研究时,这里描述的结果特别有意义。此外,我们的策略应该为详细阐述其他生物体基因的功能剖析研究奠定坚实的基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8876/549545/53c0caebf82d/1472-6750-5-6-1.jpg

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