RNA干涉技术及其应用

RNA干涉(RNA interference,RNA1)是由双链RNA导入而引起的转录后基因沉默,它可以作为一种有力的工具在多种有机体中抑制特异性基因的表达。文章简要介绍了RNA干涉的发现史、作用机制、特点及该项技术的用途。RNA1的作用机制可以分为起始阶段和效应阶段。双链RNA被Dicer消化成siRNAs(small interfermg RNAs),进一步形成RNA诱导沉默复合物(RNA-mduced silencmg complex,or RISC),在siRNAs的引导下切割靶mRNA。RNAi技术在疾病的基因治疗、功能基因组学及细胞信号通路分析等力面具有广阔的应用前景。     

A protein sensor for siRNA asymmetry

To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.     

R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway

The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.     

Argonaute2 is the catalytic engine of mammalian RNAi

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.     

RNAi技术及其应用

RNAi主要通过双链RNA(dsRNA)被核酸酶切割成21~23 nt的干涉性小的RNA,即siRNA,由siRNA介导识别并靶向切割同源性靶mRNA分子而实现。目前RNAi技术在基因功能和基因治疗等方面的研究有了广泛的应用,RNAi技术有望成为后基因组时代基因功能分析的有力工具。     

RNA干扰研究现状

RNA干扰(RNAi)是指生物体内利用双链RNA(dsRNA)诱导同源靶基因的mRNA特异性降解,从而导致转录后基因沉默的现象。其在抵抗病毒感染、抑制转座子活动、调控内源性基因表达等方面发挥重要作用。RNAi以其高特异性、高效性等显著优势将成为研究基因功能的全新手段。简要概括RNAi作用机制和siRNA技术的原理,同时也讨论了RNAi技术在其他领域,如在基因信号通路研究、高通量研究基因功能、基因治疗如肿瘤研究和疾病治疗等方面的应用。     

The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race

Increasingly complex networks of small RNAs act through RNA-interference (RNAi) pathways to regulate gene expression, to mediate antiviral responses, to organize chromosomal domains, and to restrain the spread of selfish genetic elements. Historically, RNAi has been defined as a response to double-stranded RNA. However, some small RNA species may not arise from double-stranded RNA precursors. Yet, like microRNAs and small interfering RNAs, such species guide Argonaute proteins to silencing targets through complementary base-pairing. Silencing can be achieved by corecruitment of accessory factors or through the activity of Argonaute itself, which often has endonucleolytic activity. As a specific and adaptive regulatory system, RNAi is used throughout eukarya, which indicates a long evolutionary history. A likely function of RNAi throughout that history is to protect the genome from both pathogenic and parasitic invaders.     

RNAi 3种分子加工机制研究进展

RNAi(RNA interference,RNA干扰)是一种高度保守的由小分子RNA介导的基因沉默过程。根据介导RNAi的小RNA长度以及结合Argonaute(AGO)蛋白家族成员的不同,将小RNA分为miRNA(microRNA)、siRNA(small interfering RNA)和piRNA(Piwi-interacting RNA)3类。根据近年来取得的研究进展,系统地阐述了这3类小RNA的基因组来源及其加工产生机制,归纳总结了这3类小RNA之间的区别和联系,并结合当前RNAi应用于疾病治疗存在的问题提出了自己的看法。RNAi机制的完善对于生物进化、生长发育及癌症等重大疑难疾病治疗具有重要的应用价值。     

飞蝗Argonaute1的分子特性及生物学功能

【目的】MicroRNAs(miRNAs)是一类长度约22 nt的非编码RNA,通过转录后调控的方式在多种生命活动中发挥重要功能。Argonaute1(AGO1)蛋白作为miRNA沉默复合物(miRNA silencing complex RISC)的重要组成部分,在miRNA调控通路中起着关键作用。论文旨在研究AGO1的生物学功能及其对飞蝗(Locusta moratoria)生长发育的影响,为探索昆虫miRNA的生物合成和农业害虫的有效控制提供理论依据。【方法】采用生物信息学方法在飞蝗转录组数据库中获得Lm AGO1 c DNA序列;使用在线蛋白翻译软件(Ex PASy)对Lm AGO1进行蛋白翻译,利用SMART分析Lm AGO1蛋白的功能结构域;选取家蚕(Bombyx mori)、果蝇(Drosophila melanogaster)和赤拟谷盗(Tribolium castaneum)等模式昆虫的同源序列与Lm AGO1氨基酸序列进行聚类分析,采用Phyml软件构建昆虫AGO蛋白的系统发育树;为了进一步研究Lm AGO1在飞蝗生长发育过程中的作用,使用T7 RiboMAX~(TM) Express RNAi System体外合成Lm AGO1的ds RNA,在飞蝗4龄第2天和5龄第2天若虫期连续两次注射ds RNA进行干扰,同时注射ds GFP作为对照。分别收集注射ds RNA后48 h和72 h的整虫样品提取RNA,反转录为c DNA。通过实时荧光定量PCR(RT-q PCR)检测Lm AGO1在不同时间点的干扰效率并观察虫体的发育表型。同时,为了检测Lm AGO1沉默是否会影响miRNA的生物合成,采用RT-q PCR对飞蝗体内5个高丰度miRNA表达进行定量分析。【结果】Lm AGO1蛋白含845个氨基酸,具有典型的AGO蛋白家族保守结构域,即位于213—348位点的PAZ结构域和502—804位点的PIWI结构域。聚类分析表明,Lm AGO1蛋白与其他昆虫的AGO1蛋白聚为一类。通过AGO1氨基酸序列同源比对结果显示Lm AGO1与模式昆虫果蝇、家蚕AGO1的氨基酸序列一致度高达82.2%和86.9%。RNAi结果表明,虫体注射ds Lm AGO1 48 h和72 h后,与对照组相比,Lm AGO1表达量均显著降低,干扰效率分别为88.1%和93.0%;进一步观察试虫生长发育的表型特征,与对照组相比,飞蝗4龄期注射dsL m AGO1后其生长发育并没有出现明显异常,待蜕皮发育至下一龄期(即5龄期)时,出现大量死亡,死亡率为89.3%;荧光定量PCR结果显示注射ds Lm AGO148 h后,飞蝗体内miRNA-252和miRNA-8的表达显著下降,干扰72 h后miRNA-7、let 7、miRNA-252、miRNA-8的表达均显著下降。【结论】飞蝗AGO1除参与RSIC的形成以外,还可能参与miRNA的剪切加工过程进而调控飞蝗的正常发育。     

Intersection of the RNA interference and X-inactivation pathways

In mammals, dosage compensation is achieved by X-chromosome inactivation (XCI) in the female. The noncoding Xist gene initiates silencing of the X chromosome, whereas its antisense partner Tsix blocks silencing. The complementarity of Xist and Tsix RNAs has long suggested a role for RNA interference (RNAi). Here, we report that murine Xist and Tsix form duplexes in vivo. During XCI, the duplexes are processed to small RNAs (sRNAs), most likely on the active X (Xa) in a Dicer-dependent manner. Deleting Dicer compromises sRNA production and derepresses Xist. Furthermore, without Dicer, Xist RNA cannot accumulate and histone 3 lysine 27 trimethylation is blocked on the inactive X (Xi). The defects are partially rescued by truncating Tsix. Thus, XCI and RNAi intersect, down-regulating Xist on Xa and spreading silencing on Xi.     

RNA干涉原理及其应用

RNA干涉是指外源dsRNA引发生物体内的基因的同源序列降解，从而表现出的基因转录后的沉默现象，它与植物中的共抑制和真菌中的基因压制可能具有相同的作用机制。在这一过程中，需要eIF2c类似蛋白因子、RNA螺旋酶、RNA依赖性RNA多聚酶、核糖核酸酶、ATP和转膜蛋白参与。RNA干涉可以用于功能基因组学研究，也可用于克服转基因生物的基因沉默现象，使外源基因在遗传改良生物中能更好地表达，还用于基因治疗，抑制有害基因的表达等。     

双链RNA激发的植物基因沉默及其在植物育种上的应用

在生物体内,存在大量的非编码RNA(ncRNA),小干扰RNA(siRNA)是其中一种,由双链RNA切割形成,能干扰基因表达,激发转录后的基因沉默。尽管双链RNA干扰是把基因"敲低"而不是"敲除",但它的高效和易操作性使之成为研究植物基因功能的有用工具,并通过转基因途径用于植物改良。与反义RNA技术和共抑制技术相比,RNA干扰对基因的沉默效率高,效果稳定。单基因RNA干扰就可调控多基因家族控制的农艺性状,而不必累积单基因突变。把病毒基因构建成反向重复结构转入植物体内,其转录出的RNA会通过分子内序列互补形成双链结构,激发转基因植物的RNA干扰机制,将入侵病毒的同源序列降解,使转基因植株获得对病毒的抗性。RNA干扰型抗病毒转基因植株中,转病毒基因的mRNA不存在或存在量很少,也不会翻译成有功能的病毒蛋白质,因此不存在病毒RNA重组、异源包装及协生作用的潜在生物风险,具有较高的生物安全性。     

Functional genomic analysis of RNA interference in C. elegans

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.