RNAi机器

小分子非编码RNA,以不同于蛋白质调控因子的全新方式,通过RNA干扰(RNAi),调控mRNA稳定性、蛋白质合成、染色质的组织和基因组的结构.由于可方便地施加和释放控制,RNAi已被广泛作为通过沉默作用研究基因功能的手段,并正在被应用于一些重大疾病的诊治.小分子RNA沉默作用的发挥依赖于一个由多种蛋白质因子组成的RNAi机器.在过去的几年中,对RNAi机器中重要蛋白质因子的结构功能,及它们在小向导RNA指导下沉默基因表达的分子机制等方面的研究都取得了诸多突破性的进展.     

Dynamics of gene silencing by RNA interference

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.     

Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency

In RNA interference (RNAi), guide RNAs direct RNA-induced silencing complexes (RISC) to their mRNA targets, thus enabling the cleavage that leads to gene silencing. We describe a strong inverse correlation between the degree of guide-RNA secondary structure formation and gene silencing by small interfering (si)RNA. Unstructured guide strands mediate the strongest silencing whereas structures with base-paired ends are inactive. Thus, the availability of terminal nucleotides within guide structures determines the strength of silencing. A to G and C to U base exchanges, which involve wobble base-pairing with the target but preserve complementarity, turned inactive into active guide structures, thereby expanding the space of functional siRNAs. Previously observed base degenerations among mature micro (mi)RNAs together with the data presented here suggest a crucial role of the guide-RNA structures in miRNA action. The analysis of the effect of the secondary structures of guide-RNA sequences on RNAi efficiency provides a basis for better understanding RNA silencing pathways and improving the design of siRNAs.     

The effects of spacer sequences on silencing efficiency of plant RNAi vectors

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.     

RNA干扰(RNAi)文库研究进展

RNAi是由双链RNA(dsRNA)引发的转录后基因沉默现象,由dsRNA产生的小分子siRNA会导致生物体内同源转录产物特异性降解,是基因表达调控的重要方式之一。目前RNAi技术已发展成为遗传分析强有力的工具,在基因功能分析鉴定方面发挥越来越大的作用。构建大规模的RNAi文库进而转变成RNAi突变体库是功能基因组学研究的重要手段,因此如何利用简单经济的方法构建特定物种的高效RNAi文库就成为关键问题。综述了目前构建RNAi文库的不同方法以及每种构建方法的优点和存在的不足,为不同研究目的的RNAi文库的构建提供参考。     

Inputs and outputs for chromatin-targeted RNAi

Plant gene silencing is targeted to transposons and repeated sequences by small RNAs from the RNA interference (RNAi) pathway. Like classical RNAi, RNA-directed chromatin silencing involves the cleavage of double-stranded RNA by Dicer endonucleases to create small interfering RNAs (siRNAs), which bind to the Argonaute protein. The production of double-stranded RNA (dsRNA) must be carefully controlled to prevent inappropriate silencing. A plant-specific RNA polymerase IV (Pol IV) initiates siRNA production at silent heterochromatin, but Pol IV-independent mechanisms for making dsRNA also exist. Downstream of siRNA biogenesis, multiple chromatin marks might be targeted by Argonaute-siRNA complexes, yet mechanisms of chromatin modification remain poorly understood. Genomic studies of siRNA target loci promise to reveal novel biological functions for chromatin-targeted RNAi.     

RNA binding proteins in the RNA interference phenomenon

The ability of short RNAs (21-27 nucleotides) to silence genes containing homologous nucleotide sequences is related to RNA silencing. The pathways of short RNAs (siRNA and microRNA) biogenesis from their precursors, double stranded and hairpin RNAs respectively, are briefly reviewed. The functioning of specific RNA binding domains found for the first time in the proteins operating in RNA interference (RNAi) is considered. The interactions of these domains with the earlier well known RNA binding modules in RNAi proteins are described.     

Argonaute蛋白与小RNA的作用机制

RNA干扰(RNA interference, RNAi)是在植物、动物、线虫、真菌以及昆虫等生物体中普遍存在的通过双链RNA(double strand RNA, dsRNA)诱导的抑制同源基因表达的一种保守的调控机制.小分子RNA通过特异性地识别结合RNA诱导的沉默复合体（RNA-induced silencing complex, RISC）对目标mRNA的表达在转录和翻译水平进行抑制.作为RISC的重要组成成分,Argonaute蛋白（Ago）发挥了至关重要的作用.为了进一步阐明Ago蛋白在RNA干扰中对小分子RNA的作用机制,本文介绍了Ago蛋白的结构、分类及其在RNA干扰机制中的作用,并着重阐述了目前已知的植物Ago蛋白对小分子RNA的几种作用机制,以及目前研究发现的Ago蛋白的功能作用,从而更进一步证实Ago蛋白对小分子RNA的作用是一个复杂的过程.     

细胞核内RNA干扰途径研究进展

RNA干扰(RNAi)是双链RNA分子在mRNA水平关闭相应序列基因表达的过程。RNAi的分子机制及功能仍然有待深入研究与阐明,但已经在基因组结构与功能研究中得到广泛运用。我们简要综述RNAi的作用机制,及已发现的细胞核内干扰途径——RNA指导的DNA甲基化、异染色质、DNA切除和减数分裂性沉默等相关研究进展。     

Branched, tripartite-interfering RNAs silence multiple target genes with long guide strands

Structural modifications could provide classical small interfering RNA (siRNA) structure with several advantages, including reduced off-target effects and increased silencing activity. Thus, RNA interference (RNAi)-triggering molecules with diverse structural modifications have been investigated by introducing variations on duplex length and overhang structure. However, most of siRNA structural variants are based on the linear duplex structure. In this study, we introduce a branched, non-linear tripartite-interfering RNA (tiRNA) structure that could induce silencing of multiple target genes. Surprisingly, the gene silencing by tiRNA structure does not require Dicer-mediated processing into smaller RNA units, and the 38-nt-long guide strands can trigger specific gene silencing through the RNAi machinery in mammalian cells. tiRNA also shows improved gene silencing potency over the classical siRNA structure when complexed with cationic delivery vehicles due to the enhanced intracellular delivery. These results demonstrate that tiRNA is a novel RNA nanostructure for executing multi-target gene silencing with increased potency, which could be utilized as a structural platform to develop efficient anticancer or antiviral RNAi therapeutics.     

Environmental RNA interference

The discovery of RNA interference (RNAi), the process of sequence-specific gene silencing initiated by double-stranded RNA (dsRNA), has broadened our understanding of gene regulation and has revolutionized methods for genetic analysis. A remarkable property of RNAi in the nematode Caenorhabditis elegans and in some other multicellular organisms is its systemic nature: silencing signals can cross cellular boundaries and spread between cells and tissues. Furthermore, C. elegans and some other organisms can also perform environmental RNAi: sequence-specific gene silencing in response to environmentally encountered dsRNA. This phenomenon has facilitated significant technological advances in diverse fields including functional genomics and agricultural pest control. Here, we describe the characterization and current understanding of environmental RNAi and discuss its potential applications.     

RNA干扰与植物抗病毒

RNA干扰是多种生物体内由双链RNA介导的同源mRNA降解现象,是植物体内天然的抗病毒机制。然而病毒在长期进化过程中也获得了通过编码沉默抑制蛋白来对抗植物体RNAi系统的能力。本文对RNA干扰过程、病毒编码的沉默抑制蛋白及利用干扰技术进行抗病毒基因工程研究进行简要综述。     

RNA干涉在纤毛虫中的研究进展

RNA干涉是dsRNA介导的基因沉默现象,本文简要介绍了其作用的机制和生物学意义,重点阐述了RNA干涉在原生动物纤毛虫中的发现与应用,比较了RNA干涉与纤毛虫大核基因组重排机理的异同,并对RNA干涉在纤毛虫中传输的技术途径-RNAi喂饲法的原理也做了详细的介绍。