Class4_RNAi-miRNA
科学网概观miRNA
科学网概观miRNA概观miRNAMicroRNAs(miRNAs)是一类非编码的小RNA分子，通过与靶RNA的3?UTR互补或部分互补结合，使其降解或介导其翻译抑制，参与细胞增殖、凋亡、分化、代谢、发育、肿瘤转移等多种生物学过程.miRNAs基因通常位于基因间或编码蛋白基因的内含子中，在核内由RNA聚合酶II或III转录产生具有特征性茎环结构的pri-miRNA,然后在Drosha-DGCR8复合体的作用下，剪接成70nt的pre-miRNA,它由exportin5由核内运到胞浆。
在胞浆内，pre-miRNA在Dicer酶作用下剪切成22bp的成熟双链miRNA,其中的一条链与RISC结合而参与基因转录后水平的调控。
根据miRBase数据库，目前所发现的miRNAs已超过700个，随着高通量测序的应用，将会有更多的新的miRNAs被发现。
可能近90%的人类基因受到miRNAs调控，然而，当过表达或抑制某一个miRNA时，在发生调变的众多基因当中寻找并鉴定其中起关键作用的靶基因仍然具有相当大的挑战。
目前鉴定miRNA靶基因的常用策略是利用生物信息学软件预测，结合基因芯片分析以及生物学实验方法来研究miRNA的功能及寻找其中起重要作用的靶基因。
此外，利用蛋白质质谱来寻找miRNA靶基因也成为一种新的途径。
microRNA的筛选在上千个miRNA中，哪些miRNA在特定的生物学功能起着关键的作用呢？这是研究miRNA功能所面临的首要问题。
miRNA基因技术可以快速有效的提供miRNA表达图谱。
通过比较正常样本与疾病样本中miRNA表达图谱的差异，寻找在生物学功能上起作用的miRNA。
该技术为临床肿瘤诊断提供了新的思路，也为miRNA作为肿瘤的标志物提供了依据。
然而基因芯片技术的结果是半定量的，且重复性较差,这就需要通过其它的实验方法来进一步验证。
miRNA的实验验证为了进一步验证miRNA在组织细胞内的表达，目前常用来检测miRNA的技术主要有以下几种：Northern杂交;原位杂交; Stem-loop 实时定量RT PCR.这三种技术各有利弊，可以相互结合应用，来反映细胞内miRNA的真实表达水平。
分子生物学RNA干扰(RNAi)
Craig Mello A professor of Molecular Medicine University of Massachusetts Medical School
In 1998: Fire & Mello in Nature 证实在RNAi 中，真正起作用的是dsRNA
表达与C. elegant worm unc-22基因同源的dsRNA的 细菌喂食线虫，则线虫表现出类似unc-22缺失的表型
•Dicer protein :
Kenneth Kemphues Professor of Genetics
Su Guo Cornell graduate student
Two-cell
Four-cell for distribution of Two-cell to visualize germline-specific mitotic spindles granules.
( The discovery of RNA-mediated interference )
In 1990: Dr. Jorgensen 共抑制现象(cosuppression)
Richard Jorgensen, PhD. Univ. of Arizona RNAi Innovator Awardee
Negative control
Endogenous mex-3 RNA
Injected with mex-3 antisense RNA
Injected with dsRNA corresponding to mex-3
Effects of mex-3 RNA interference on levels of the endogenous mRNA
小RNA测序测序介绍(含miRNA测序原理及案例分析)
9
样品要求
样品类型：完整且无污染的总RNA； 样品需求量（单次）：≥ 10 μg； 样品浓度：≥ 200 ng/μl； 样品纯度：OD260/280= 1.8~2.2、OD260/230≥ 2.0、28S:18S≥ 1.5、 RIN≥ 8.0、23S:16S≥ 1.5(此项要求只针对原核生物)； 提取总RNA时请不要使用Qiagen等公司的过柱试剂盒，也不要使用 LiCl沉淀； 如果是使用mirVana™ miRNA Isolation Kit等试剂盒回收的< 200 nt的 Small RNA（无法判断RNA的完整性），测序请提供浓度≥ 20 ng/µl、 总量≥ 1 μg的RNA样品。
Unann tags
比对区域
前后一定长度序列
1）二级结构; 2 ） Dicer 酶 切 位点; 3）能量。 预测
Pre-miRNA
5.(已知的与新的)miRNA的碱基偏好性
Tags TACGAAGTGCGGC… TGCGCAAGTATGT… TCAGCGAGTAGCT… … 不同长度的tags首位碱基偏好性
最终目的：从测序结果中筛除非miRNA的tags。
比对参考基因组
• 小RNA基因组比对结果统计。
Exon_sense Intron_ sense
tag
Intron_antisense exon_anti -sense
各染色体上 覆盖的tags
内含子
基因间区
基因区域上 覆盖的tags
重复序列上覆盖 的tags
8.miRNA靶基因相关分析
• 得到miRNA的靶基因后，可以用与RNA-Seq类似
的方法对这些基因的功能进行分析，如GO、 KEGG注释等；对于样品间差异miRNA，同样可 以对其靶基因进行GO、KEGG富集分析。
RNA干扰系统解决方案
RNA干扰系统解决方案张远涛 Ph.D.Application Specialist, MCB 8008100192-233 (北京） cnmcbsupport@2© 2008 Applied BiosystemsTraditional RNAs3© 2008 Applied BiosystemsWhat is RNA?Ribonucleic acidRibonucleotides (Ribose, base, & phosphate)TypesCoding: messenger RNA (mRNA) Non-coding:Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA) Small nucleolar RNA (snoRNA) Interference RNA (RNAi) Short interfering RNA (siRNA) Micro RNA (miRNA)4 © 2008 Applied Biosystems5© 2008 Applied BiosystemsRNAisiRNA是一种外源性的序列 高度保守，长度在21个碱 基左右的小分子双链RNA 由长链或发夹状dsRNA剪 切而成 通过与特异序列mRNA结合 降解目标mRNA6© 2008 Applied Biosystems为何研究siRNA？研究及验证基因的功能 了解生物信号传导途径 研究药物作用靶基因及其调控机制 具有重要的临床应用价值7© 2008 Applied BiosystemsRNAi的作用机制short-interfering RNA (siRNA) = ~21 bp dsRNA RISC = RNA Induced Silencing Complex8 © 2008 Applied Biosystems什么是siRNA?Sense Antisense• 双链RNA • 通常21 bp长，其3’端2个碱基 突出helicase• 其反义链与目标mRNA互补RISC• 设计要小心防止脱靶 • 通常化学合成，也可以通过体 外转录（IVT）的方式获得RISCRISCRISC9Target mRNA Applied Biosystems © 2008确保有效的RNAi 结果“Whither RNAi?" Nature Cell Biology. June 2003. 5(6) p.489-490.选择一段高度有效的siRNA序列 有效的转染方法 用尽可能低的siRNA浓度（减小毒性） 用第二个有效的siRNA去沉默同一个目标基因验证结果 检测RNAi效果 (在mRNA和蛋白水平) 对照●正对照siRNA (例如： GAPDH) •证明siRNA是有效的 •容易检测knockdown效果 负对照siRNA •作为实验条件的对照（不同组织，培养 •不靶向任何序列，作为对照表 条件等) 型 •作为由于转染方法引起基因表 达变化的对照© 2008 Applied Biosystems没处理的对照 监控转染方法的毒性10siRNA实验流程转染 siRNAs抽提RNA(RNAqueous®-4PCR or MagMAX™-96 Total RNA Isolation Kit)5’ 3’ 3’ 5’需要 • 每个基因需要2条以siRNA • 有效的转染方法 • 检测RNAi的效果 • 正负对照用qRT-PCR检测 Knockdown的效果负对照 siRNA Survivin siRNA检测表型负对照siRNA Survivin siRNA11© 2008 Applied BiosystemsAmbion and the RNAi WorkflowAlternate Methods pSilencer™ siRNA Vectors (plasmids) pSilencer™ 5.1 Retro Vectors pSilencer™ adeno Silencer® Construction Kit Silencer® Cocktail Kits (RNase III and Dicer) Silencer® Express KitssiRNA DesignsiRNA SynthesissiRNA DeliveryAssayBiological Impact Cell-Based Asays Protein Detection qRT-PCR12Transfection Chemical Synthesis siPORT™ NeoFX Silencer® Select Pre-designed siRNAs siPORT™ Amine Silencer® Select Validated siRNAs • TaqMan Gene siPORT™ Lipid Silencer® Select Controls siRNAs expression Assays Silencer® Transfection II Kit Custom Designed Silencer® Select siRNA • TaqMan Cell to Ct kits ® CellReady Transfection ® Pre-designed siRNAs Silencer Silencer Optimization Kit Silencer® Validated siRNAs Electroporation Silencer® Controls siRNAs siPORT™ Electroporation Buffer/Kit Custom Select siRNA Sythesis siPORTer™-96 Electroporation Chamber ® siRNA Applied Biosystems © 2008 Starter Kit SilencerMEGAscript RNAi Kit－用于dsRNA制备和纯化• 适用于线虫、果蝇和其它无 脊椎动物系统的dsRNA • 用于非哺乳动物系统成功进 行RNAi实验13© 2008 Applied Biosystems哺乳动物系统进行RNAi干涉的五种方法体外准备－Method #1: 化学合成Best for: 需要大量超纯确定序列的siRNA的研究，Not suitable for: 长期研究－Method #2: 体外转录Best for: 适合筛选有效的siRNA序列，或化学合成siRNA有困难时。
检测miRNA方法
检测miRNA方法
检测miRNA的方法主要有以下几种：
1. Northern blotting（北方印迹法）：通过RNA电泳分离和迁移，然后使用探针结合miRNA进行检测。
2. qRT-PCR（实时定量逆转录聚合酶链反应）：利用逆转录将miRNA转录为cDNA，然后使用PCR进行定量检测。
3. Microarray技术（芯片技术）：将已知的miRNA探针固定在芯片上，通过杂交检测待测miRNA。
4. Next-generation sequencing（下一代测序）：利用高通量测序技术，将小RNA转录本进行测序，然后通过比对和统计分析来确定miRNA的存在和表达水平。
5. 免疫细胞化学染色：利用抗体和荧光标记来检测miRNA的表达和定位。
6. 探针法：通过使用与目标miRNA互补的标记探针来检测miRNA。
这些方法各有优缺点，选择合适的方法需根据实验目的、预算、样本量等因素综合考虑。
miRNA研究方法
miRNA研究方法尽管早在1993年，科学家就发现了第一个microRNA，但直到2003年以后，这种小RNA分子的大作用才不断被发现。
研究显示：miRNA通过与转录本的相互作用，关闭或抑制基因的表达，影响了30%的基因。
miRNA在多个组织中，例如正常和肿瘤组织中差异表达。
因此，通过表达谱分析寻找疾病相关miRNA并进行发病机理研究，最终应用于肿瘤诊断和治疗，已经成为目前miRNA研究的重要方向。
在研究中，从深度测序、miRNA芯片到通过导入化学合成的mimics/antagomirs 等实现miRNA过表达和抑制，都是研究中常用的方法。
同时，在miRNA研究中，生物信息学分析扮演着越来越重要的角色。
下表介绍了miRNA研究和应用中科学家关注的主要科学问题以及目前常用的研究方法。
miRNA主要研究技术深度测序抽提分离小分子（例如18-30nt）RNA，通过RT-PCR扩增之后，利用solexa深度测序，并进行生物信息学分析，获得miRNA表达谱。
深度测序结合生物信息学分析手段，可以对海量数据进行分析，分别统计出已知的miRNA （miRNA-known）、新的miRNA（miRNA-new）以及可能的新miRNA（miRNA-cadidate new），并对新发现的miRNA进行靶基因分析，功能预测等。
通过深度测序的方法，可以发现新的miRNA，为进一步的深入研究奠定基础。
miRNA芯片利用miRNA芯片（例如Agilent miRNA芯片），可以高通量分析miRNA表达的时空特异性、不同样本（例如癌组织和癌旁组织）中miRNA的差异表达，进而进行把基因分析，功能注释、通路分析，网络分析等，以了解miRNA在疾病发生中的作用。
与深度测序不同，miRNA芯片针对已知miRNA进行研究。
筛选到的差异miRNA可以利用q-pCR进行验证。
q-PCR q-PCR技术可以用来检测miRNA以及其靶mRNA和相关mRNA等的检测，主要方法有茎环法和加尾法等。
体外分析microRNA功能
Analysis of microRNA effector functions in vitroBingbing Wang a ,John G.Doench b ,Carl D.Novinaa,c,*aCancer Immunology and AIDS,Dana-Farber Cancer Institute,and Department of Pathology,Harvard Medical School,Boston,MA 02115,USAbHarvard Institute of Proteomics,Harvard Medical School,Cambridge,MA 02141,USAcBroad Institute of MIT and Harvard,Cambridge,MA 02142,USAAccepted 13April 2007AbstractAnalyses of gene functions in vitro provide the means to dissect biological phenomena.Development of cell-free assays for transcrip-tion,splicing,and translation has yielded great insights into macromolecular interactions and functions required for these processes.This article discusses development of cell-free assays to test macromolecular interactions and activities required for microRNA effector func-tions.This chapter also compares in vitro analyses to complementary studies in cells and the technical considerations that permit molec-ular analysis of microRNA function in cell-free conditions.Ó2007Elsevier Inc.All rights reserved.Keywords:MicroRNA;siRNA;RNA interference;Translation;Cell-free;Gene silencing1.IntroductionmicroRNAs (miRNAs)are an evolutionarily ancient,endogenous class of non-coding RNAs implicated in numerous biological processes [1,2].In mammals,miR-NA-regulated gene expression has been implicated in chro-mosome segregation [3,4],limb morphogenesis [5],and differentiation [6,7].The potential importance of natural gene regulation by miRNAs is highlighted by the identifica-tion of dysregulated miRNA expression in numerous dis-eases,including several cancers (reviewed in [8]).Moreover,bioinformatic methods predict that approxi-mately one-third of all human genes may be targets of miRNAs [9,10]suggesting that miRNAs may govern vast gene regulatory networks.Despite their profound influence over gene expression,the mechanisms used by miRNAs to silence target gene expression are poorly understood.Though there are rare exceptions in mammals [11,12],miRNAs typically bind to the 30-UTR of target mRNAs with imperfectly complementary base pairing and lead totranslational repression of target mRNAs.miRNAs are loaded into an effector complex called the microribonucle-oprotein complex (miRNP),originally discovered by its association with multiple miRNAs and further character-ized to mediate gene silencing [13,14].miRNAs are funda-mentally related to a class of small RNAs,the short interfering RNAs (siRNAs),which are used in another gene silencing pathway,RNA interference (RNAi).siR-NAs,however,are incorporated into the RNA-induced silencing complex (RISC),typically bind to target mRNAs with perfectly complementary base pairing,and lead to cleavage and degradation of target mRNAs [15–17].Importantly,a miRNA can act as an siRNA to induce mRNA cleavage (i.e.RNAi),and an siRNA can act as a miRNA to induce translational gene silencing,simply by manipulating the degree of sequence complementarity between the small RNA and the mRNA target [14,18].Thus,an individual small RNA can lead to biochemically distinct mechanisms of gene silencing:reporter mRNAs with mismatches to the central nucleotides of the small RNA promote translational repression whereas mRNAs with exact sequence complementarity undergo mRNA cleavage.Several cell culture models have used chemically1046-2023/$-see front matter Ó2007Elsevier Inc.All rights reserved.doi:10.1016/j.ymeth.2007.04.003*Corresponding author.E-mail address:carl_novina@ (C.D.Novina)./locate/ymethMethods 43(2007)91–104synthesized,exogenously introduced siRNAs to interrogate the functions of miRNAs([18–22],and others).We have recently adapted one such cell-based system,utilizing the CXCR4siRNA,to an in vitro assay for miRNA-dependent translational repression[23].2.The factors involved in gene silencingA common feature of all characterized silencing effector complexes,such as the miRNP and RISC,is the presence of at least one member of the Argonaute(Ago)family of proteins[13,14,24–27].Ago proteins are defined by the presence of a PAZ(Piwi–Argonaute–Zwille)domain and a PIWI domain[28].The human genome encodes eight Ago proteins,which are further separated into the Ago and PIWI sub-families.Ago1,2,3,and4are closely related and have each been shown to bind to miRNAs[29].Fur-thermore,Ago2has been shown genetically[22]and bio-chemically[29]to be responsible for siRNA-directed mRNA cleavage.Structural studies of the Ago proteins indicate that Agos-1and-4lack the histidine residue required to coordinate Mg++[30]and consequently are dis-pensable for the mRNA cleavage reaction[22].Interest-ingly,Ago3contains the active site histidine residue but is still dispensable for the mRNA cleavage reaction[22].While Ago2is clearly the only Argonaute capable of performing RNAi,it is dispensable for translational repres-sion.Knockout mouse embryonicfibroblasts lacking Ago2 can no longer cleave mRNA targets perfectly complemen-tary to the CXCR4siRNA but can still translationally repress mRNA with imperfectly complementary CXCR4 target sites[22]suggesting that any or all of Agos-1,-3, and-4are responsible for miRNA-directed translational gene silencing.Indeed,it may be that any individual Ago is capable of translational repression,but that the activity is governed by other trans-associated factors in the miRNP complex.A unified view of the various proteins and associated activities that comprise either RISC or the miRNP has not yet emerged.For example,RISC activity has been found associated with complexes ranging from140kDa to500kDa,and as large as80S(reviewed in[31]).The Dro-sophila80S‘‘holo-RISC’’identified by Sontheimer and col-leagues demonstrates two levels of assembly and contains the RNAi proteins Dicer2,Ago2,VIG,TSN,and dFXR [32],as well as ribosomal components L5,L11and5S rRNA[33].Similarly,the miRNP was isolated as a550-kDa complex containing Gemin3and Gemin4[13,14]. For both the miRNP and RISC,many associated factors remain to be identified.With the exception of Ago2in RISC,the functional requirement for any of these proteins in gene silencing is not yet understood.3.Biochemical reconstitution of miRNA functionRecent data have presented conflicting views of the pre-cise mechanism of translational repression by miRNAs.Some reports suggest that miRNAs target actively translat-ing mRNAs[34,35]either by blocking translation initiation [20,23,36]or translation post-initiation[21,37,38].Other reports suggest that miRNAs target mRNAs in P-bodies [39,40]or stress granules[41],sites that do not support active translation.By selectively removing and reconstitut-ing different proteins in a controlled setting,cell-free reac-tions permit a precise molecular understanding of the mechanisms used by miRNAs to repress translation.Addi-tionally,cell-free reactions can clarify the molecular func-tions of the factors implicated in gene silencing.Biochemical reconstitution of miRNA-directed gene silencing in vitro is a prerequisite for determining functional requirements for miRNP-associated factors.In thefirst in vitro RNAi reaction performed in Drosophila embryo lysates,Tuschl,Zamore,Bartel and Sharp demonstrated dsRNA-mediated cleavage of the mRNA target comple-mentary to the input long dsRNA[16]in an ATP-depen-dent fashion[17].Other cell-free RNAi reactions that have been described include Drosophila[42]and human [43,44]Dicer cleavage of dsRNA and Drosha cleavage of primary miRNA transcripts[45–47].Thus,biochemical requirements are becoming well-defined for these processes but the requirements for miRNP-directed translational repression remain unknown.Recently,we described the first cell-free translational repression reaction that recapit-ulates known molecular hallmarks of miRNA function in cells[23].These assays permit analysis of the precise molec-ular mechanisms of miRNA function in translational repression in comparison to mRNA cleavage in vitro.Sev-eral assays are presented that permit molecular analysis of gene silencing in vitro.4.Preparation of reporter mRNAsThe reporter system used for in vitro analyses utilizes the same constructsfirst described in cell-based analyses of miRNA functions that elucidated and extended some of the early principles of miRNA targeting([18,19,21], Fig.1).One such hallmark of miRNA targeting recapitu-lated in vitro,discovered both experimentally and compu-tationally,is the requirement for perfect base pairing through the seed region of the miRNA:mRNA duplex, defined as nucleotides2–7from the50-end of the miRNA [19,48,49].While our original in vitro studies were per-formed by delivering the small RNA as a siRNA duplex, experiments using endogenous miRNAs are possible.This method has the possible advantage of more accurately rep-resenting genuine miRNA-directed repression without the effects of miRNA processing,per se,as the miRNA biogen-esis machinery may also participate in small RNA effector functions[32,50–52].Considerations for the use of endog-enous miRNAs to repress natural target mRNAs are dis-cussed below.For purposes of clarity in this report,we are defining small RNAs as siRNAs when they have perfect comple-mentarity to their mRNA target and as miRNAs when92 B.Wang et al./Methods43(2007)91–104they have imperfect complementarity to their mRNA tar-get.Small RNA can either be introduced as a duplex(i.e. as a canonical siRNA)or as a single strand(i.e.a canonical mature miRNA);the degree of complementarity to the tar-get,not the method of introduction,governs silencing activity in vitro.The CXCR4small RNA serves as the test small RNA while a GFP small RNA serves as the control. Test mRNAs contain binding sites for the CXCR4small RNA while control mRNAs lack binding sites,with the canonical‘‘wild-type’’site shown in Fig.1.One important consideration in choosing control small RNAs is that con-trol RNAs should demonstrate activity on other target mRNAs with sites for that control RNA.Controls that are important especially for analysis of miRNA activity at endogenous mRNA targets include the use of antagomirs.Antagomirs are antisense RNAs that bind to miRNAs to represses their natural function [53–56].Usually,antagomirs have one or more modifica-tions that increase the affinity of the antisense RNAs for their miRNAs.These modifications may be20O-methyl-ated RNA backbones,locked nucleic acid backbones,or other modifications available through several vendors (Dharmacon,Exiqon Gene linkä,Integrated DNA Tech-nologies,Sigma–Aldrich,and Qiagen).Often,antagomirs possess one of several substitutions that permit visualiza-tion in cells or promote transduction across membranes. These may be used for in vitro analyses,though the latter substitutions are considered unnecessary and may actually inhibit the efficiency of the antagomir in vitro.4.1.In vitro transcription of reporter plasmidsAlthough it is theoretically possible to perform coupled transcription-translation reactions to measure miRNA functions in vitro,the major advantage of performing these reactions separately is that the reactions may be uncoupled. Stated another way,performing in vitro transcription reac-tions and purifying the transcripts enables more controlled analyses of the translation step.Purified transcripts may be modified in a variety of ways and may also be added in dif-ferent amounts and at different times to gain insights into the thermodynamic and kinetic parameters governing miRNA function.Linearizing the plasmid reporters permits analysis of translational repression from defined mRNAs containing different ing mRNAs lacking30UTRs also tests the dependence of miRNA function upon cognate miRNA binding sites.1.Linearize pcDNA3.1-based(Invitrogen)plasmidswith EcoRV to generate zero,two,four or six miC-XCR4binding site reporters.2.Linearize pRL-TK-based(Promega)plasmidswith XhoI to generate the one siCXCR4binding site reporter and the RL0X internal control reporter.3.The restriction digestion reactions proceed at37°Cfor2h.4.Extract the linearized plasmids with phenol:chloro-form:isoamyl alcohol(25:24:1,pH8.0).5.Add DEPC-treated H20to the supernatant to afinalvolume of100l L.6.Add2.5volumes of100%ethanol and1/10volumesof3M NaOAC(pH5.5).7.Precipitate RNA pellets by storing the mixture atÀ80°C for1h.8.Resuspend RNA pellets in afinal volume of20l L ofDEPC-treated H2O.RNase-free conditions are important during this and all subsequent steps involv-ing mRNAs and miRNAs.9.Perform in vitro transcription with5l g of linearizedplasmids using the RiboMax T7Large-Scale RNA Production kit according to the manufacturer’s pro-tocols(Promega).10.Purify the RNAs using the RNeasy mini kit accord-ing to the manufacturer’s protocols(Qiagen).4.2.Capping in vitro transcriptsUnlike siRNA-directed mRNA cleavage,miRNA-direc-ted translational repression requires an mRNA cap for function in vitro[23].The issue of whether miRNAs repress translation initiation or post-initiation in cells hingespartly B.Wang et al./Methods43(2007)91–10493upon whether translation driven by viral internal ribosome entry sites(IRESes)could be repressed by miRNAs.It is difficult to determine whether uncapped mRNAs are capped after transfection of mRNAs into cells.One princi-ple advantage of testing mRNA cap-dependency of miRNA functions in vitro is that either capped or uncapped mRNAs may be added to translation repression reactions directly,circumventing the issue of whether viral IRESes accurately represent cellular translation.Indeed,the mRNA cap structure may be important for translational repression by miRNAs.Similar to results observed in cells [20],physiological mRNA caps are required for transla-tional repression by miRNAs in vitro[23].Cap analogs available in some translation kits do not promote transla-tional repression by miRNAs[23].The exact molecular mechanism of the mRNA cap dependency for miRNA function is still unknown.The recombinant version of the mRNA capping enzyme has been reported[57–59]and may be obtained commercially(Ambion).1.The capping reaction buffer is composed of75mMTris(pH7.9),9mM KCl,3.75mM DTT,1.88mM MgCl2,0.15mg/mL BSA,1mM GTP,and1mM S-adenosyl Methionine(Ambion).2.Add20pmol mRNA reporter per7-methyl G cap-ping reaction.3.Add capping enzyme to afinal volume of15l L.4.Incubate the capping reaction at37°C for1h.5.Terminate the reaction by placing the mixture on ice.6.Extract the capped mRNAs by adding phenol:chloro-form:isoamyl alcohol(25:24:1,pH6.0).7.Add2.5volumes of100%ethanol and1/10volumesof3M NaOAC(pH5.5).8.Precipitate RNAs by storing the mixture atÀ80°Cfor1h.9.Resuspend RNA pellets in DEPC-treated H2O to afinal volume of20l L.10.Quantitate RNAs by UV spectrophotometry.4.3.Polyadenylating in vitro transcriptsmiRNA-directed translational repression requires a poly(A)tail for function in vitro[23].Thefirst report of miRNA-directed translational repression in vitro described polyadenylation of transcripts to200adenosines because mRNAs in vivo are on average between200and250nucle-otides in length[60]and because200adenosines promote robust translational repression while shorter poly(A)tails demonstrate less activity.Transcripts are polyadenylated in vitro using recombinant,purified poly(A)polymerase (PAP;[61])which is commercially available(Ambion and USB).Calibrating activity of the enzyme will determine reaction conditions and duration of the polyadenylation reaction.The approximate length of the poly(A)tail and yield of the reaction can be assessed by preparing serial dilutions of RNAs of known lengths.In fact,it may even be advisable to reassess length of the mRNA after the in vitro translational repression assay.Though,it has not been tested in vitro,miRNAs have been reported to pro-mote deadenylation of target mRNAs in cells[62].Alternatively,mRNAs containing poly(A)tails may be generated through use of plasmids containing adenosine tracts.This method has the advantage that an accurate and reproducible number of adenosines may be consis-tently incorporated into target mRNAs without the need to continuously monitor and calibrate the polyadenylation reaction.However,one limitation to this method is the instability and poor expression of plasmids containing long tracts of adenosines.For this reason,the poly(A)polymer-ase method of polyadenylating mRNAs is recommended.1.Polyadenylation reaction buffer is composed of20mMTris–HCl(pH7.0),50mM KCl,0.7mM MnCl2,0.2mM EDTA,100l g/mL acetylated BSA,10%glyc-erol,0.5mM ATP.2.Add10pmol of7-methyl G capped-mRNA.3.Add600U PAP to afinal reaction volume of25l L.4.Incubate at37°C for5–30min.5.Terminate polyadenylation reactions using1l L of5mM EDTA.6.Monitor the polyadenylation reaction on1%agarose–formaldehyde e a0.28–6.58kb RNA marker (Promega)to assess migration of RNAs and thus extent of polyadenylation of reporter RNAs.4.4.Biotinylating the poly(A)tails of in vitro transcriptsOne very important variation on polyadenylation of transcripts in vitro is biotinylation of poly(A)tails[23].This method enables several different downstream analyses of miRNA functions(discussed below).Streptavidin precipi-tation of biotinylated poly(A)tails permits highly efficient recovery of transcripts.Thus,factors strongly associated with target RNAs may be separated from factors more transiently associated with target RNAs.Most impor-tantly,the presence of biotins in the poly(A)tail does not inhibit miRNA repression in the ratios described below [23].The ratio of biotinyl-adenosine to adenosine used in the following reactions may be modified to incorporate more or less biotin-A as required for each experiment.It is recommended that miRNA-dependent translational repression be reassessed at significantly higher levels of bio-tin in the poly(A)tail.1.Perform biotinylation reactions as described above using2.5nM biotin–ATP(Perkin–Elmer)added into reactionscontaining0.5mM ATP,20mM Tris–HCl(pH7.0), 50mM KCl,0.7mM MnCl2,0.2mM EDTA,100l g/ mL BSA,10%glycerol,with0.5mM ATP,5nM bio-tin–ATP(Perkin–Elmer),and600U PAP.Thus,on average,every poly(A)tail has one biotinyl-adenosine for every99adenosines.94 B.Wang et al./Methods43(2007)91–1042.Incubate reactions at37°C for30min.3.Terminate and monitor the progress of these reactions asdescribed above.5.In vitro mRNA translation assayOurfirst attempts to develop a miRNA-dependent translational repression reaction were performed in rabbit reticulocyte lysate(RRL)for several reasons.First,RRL presents a robust source of highly active extract for in vitro analysis.RRL demonstrates very low lot-to-lot var-iability,as consistency between preparations is critical dur-ing in vitro assay development.Second,RRL demonstrates high specific translation activity.In other words,even small amounts of input RNA yield high levels of encoded pro-tein.Indeed,one major insight towards the development of in vitro miRNA-dependent translation repression was reducing input mRNA levels far below the generally rec-ommended levels used in general application of RRL to produce proteins.Third,RRL demonstrates mRNA cap-independent translation.Because the cap-dependency of miRNA-dependent translational repression was not known and RRL permits both cap-dependent and cap-indepen-dent translation,RRL permitted the greatest chance of identifying miRNA activity in extracts.The essential properties of this miRNA-directed transla-tional silencing in vitro are that(1)miRNA-directed trans-lational gene silencing occurs during translation,not by simply reducing steady-state mRNA levels;(2)transla-tional gene silencing requires50-seed region complementar-ity between miRNAs and target mRNAs[9,19,48,49];(3) miRNAs require50-phosphates[63]for translational gene silencing activity;and(4)miRNA-targeted mRNAs require both a7-methyl G cap[20,36]and a poly(A)tail[20]for translational gene silencing,whereas siRNA-targeted mRNAs may be uncapped and unpolyadenylated and still be cleaved.While the use of unphosphorylated siRNAs is common in transfection of cells,our studies indicate that the kinase activity that phosphorylates siRNAs in cells is either absent or weak in RRL and thus phosphorylated small RNAs should be used.The sequences of the CXCR4(test)siRNA are50-P-GUUUUCACUCCAGCUAACACA-30(sense strand)and50-P-UGUUAGCUGGAGUGAAAACUU-30 (antisense strand).The sequences of the GFP(control) siRNA are50P-GGCUACGUCCAGGAGCGCACC-30 (sense strand)and50-P-UGCGCUCCUGGACGUAGC CUU-30(antisense strand).The20O-methylated RNAs used to inhibit small RNAs have identical base sequence to the antisense strand of the CXCR4siRNA with a20car-bon O-methyl substituent on every nucleotide,though other modifications to the inhibitory RNA backbones are possible.One important consideration for robust repression is the amount of starting mRNA that is added and,related to this,the stoichiometry of small RNA to mRNA.Another important consideration is the order of addition ofreagents.The observation that small RNAs and targetRNAs should be mixed and heated togetherfirst was anindication that a RISC loading complex(RLC)activityand a miRNP loading activity(miRLC)was absent fromthe RRL[23].One consequence of the lack of RLC andmiRLC activities is that the two strands must be separatedprior to beginning the gene silencing reactions.In the caseof the translation repression assay,the best repressionoccurred when the miRNA was pre-incubated with thereporter mRNA[23].miRNA function in vitro is optimalwhen the stoichiometry of the small RNAs matches thestoichiometry of the miRNA-binding sites.The fact thatthe miRNA appears to bind the target before the miRNPloads may be the explanation for the strict stoichiometricrequirements for miRNA function in vitro.However,theexact reason for this stoichiometric relationship is stillunknown.Another important consideration is the time atwhich the reaction is terminated and repression is mea-sured.Our studies indicate that repression is mostpotent at early time points,indicating that the miRNPor other trans-acting factors have a limited half-lifein vitro.Further underscoring this idea,we have foundthat the number of freeze-thaw cycles for the lysate iscritical,as each additional cycle dramatically reducesactivity.There are two general methods to visualize the resultsof miRNA-dependent gene silencing reactions,either gelelectrophoresis followed by transfer and autoradiographyor dual luciferase assay.Whereas autoradiography istime-consuming and more accurate,luciferase assays aremore rapid and cost effective.The method of autoradiog-raphy is more accurate because it permits direct visualiza-tion of full-length products offirefly and Renillaluciferases.Non-specific and/or prematurely terminatedproducts offirefly and Renilla luciferases during transla-tion can be excluded from quantitation.In addition,amore precise calculation can be obtained if the back-ground is subtracted from mature bands scanned fromphosphorimager analysis.Nonetheless,measuring genesilencing by luciferase assay has the advantage of immedi-ate results and is suitable for screening large numbers ofreactions.Importantly,luciferase assays require very littlereporter RNAs.Thus,luciferase assays are more compat-ible to test gene silencing of reporter mRNAs in parallelwith testing other assays such as ribosome binding assay(described below).In addition,autoradiography of 35S-incorporated protein production is not possible in assays using50-end-labeled reporter mRNAs(see below).In these cases,luciferase assays are the preferred methodof measuring gene silencing.5.1.Gene silencing assays1.Obtain nuclease-treated RRL from Promega(Cat.#L4960).B.Wang et al./Methods43(2007)91–104952.Mix0.025pmol test or control reporter mRNAs with0.15pmol test or control siRNAs in a total reaction vol-ume of2.2l L.In this example using an mRNA reporter with six binding sites,the ratio of siRNA to mRNA is 6:1.3.Heat the reaction mixture to75°C for3min and cooledto room temperature for5min.4.Place the reaction mixtures on ice.5.Prepare translation master mixes by assembling7l LRRL,0.2l L of4–8U RNasin(Promega),0.2l L 20l M amino acid mixture minus methionine and cys-teine(Promega),and0.4l L(5.7l Ci)Promix L-[35S] in vitro cell labeling mix(Amersham Biosciences).6.Add7.8l L of master mix to each siRNA–mRNAreaction mixture for a total reaction volume of 10l L.7.Incubate the reaction at30°C for10to60min.8.Stop the reactions by transferring the reaction mixturesto ice.5.2.Gel electrophoresis1.Resolve reaction products by12%SDS–PAGE in1·SDS–PAGE buffer(25mM Tris base,200mM glycine,and0.1%SDS)at120V at room temperature for1–2h.2.Transfer proteins from SDS–PAGE to PVDF mem-branes(Bio-Rad)in chilled1·Western buffer composed of20mM Tris base and200mM glycine at80V for1h.3.Quantitate gene silencing by PhosphorImager(Molecu-lar Dynamics).e ImageQuant software.Measure the intensity of eachvisible band and the background.To obtain the absolute volume of each band,subtract the background from each band intensity.5.3.Dual luciferase activity assay1.Perform dual luciferase activity assay according tomanufacturer’s protocol(Promega)using mRNA labeled with[a-32P]ATP in the poly(A)tail in transla-tional gene silencing reactions.2.Measurefirefly luciferase activity in96-well reactiontrays.This method keeps luciferase reaction volumes low and minimizes reaction volume losses.a.Add2l L of each reaction to one well of a96-wellplate.b.Immediately add20l L LAR II(Promega)to eachwell.c.Countfirefly luciferase activity in a luminometer(Victor3V,Perkin–Elmer)for5s.3.Measure Renilla luciferase activity.a.Add20l L Stop&Glo(Promega)to each well andmix.b.Count Renilla luciferase activity for another5s.5.4.Calculate gene silencing1.Normalize gene silencing by measuring the ratio offire-fly luciferase activity to Renilla luciferase activity in each reaction.2.Calculate gene silencing using the following formula:1-[(+siRNA FL6X/RL0)/(ÀsiRNA FL6X/RL0)].5.5.Northern blotting analysisOne method to assess the effect of gene silencing on reporter mRNAs is to perform Northern blotting reac-tions.Even though the reactions are labeled with35S-methionine and35S-cysteine,the degree of radioactivity emitted form this source is minimal relative to the b emis-sion from32P used in traditional Northern blotting.More-over,most of the35S should be extracted away from the RNAs in thefirst step of this procedure.Thus,reactions assessing amino acid incorporation do not substantially affect the signal measured in Northern blotting.This method has the advantage of directly assessing the effect of gene silencing on mRNAs directly without enzymatic amplification required by real-time,reverse transcription PCR.1.Extract RNAs from gene silencing reactions by phe-nol:choloroform:isoamyl alcohol(25:24:1,pH6.0,Ambion).2.Precipitate RNA in2.5volumes of100%ethanoland1/10volumes of3M NaOAc(pH5.5)at–80°C for1h.3.Resuspend the precipitate in10l L of DEPC-trea-ted H2O and quantitate RNAs by UVspectrophotometry.4.Resolve RNAs on a1%agarose–formaldehyde gel.5.Transfer resolved RNAs to Hybond N+mem-branes(Amersham Biosciences).6.Hybond N+membranes are auto UV-crosslinkedat1200l F.7.Generate the probe tofirefly luciferase mRNA bydouble digesting the pGL3vector with ScaI andSphI(New England Biolabs).8.Gel-purify the498bp band.9.Generate the probe to Renilla luciferase RNA byamplifying a200-bp PCR product using thepRL-TK vector as template.Primer sequencesare50-GCTTATCTACGTGCAAGTGATGATTTACC-30and50-TTGAGAACTCGCTCAACGAACG-30.10.Random-label probes using the DECA-Prime II kit(Ambion)in the presence of[a-32P]ATP accordingto manufacturer’s protocols.11.Remove unincorporated isotopes by MicroSpinG-50columns(Amersham Biosciences).12.Hybridize membranes with the labeled probes at42°C overnight.96 B.Wang et al./Methods43(2007)91–104。
miRNA、lncRNA、circRNA的基础知识详解
miRNA、lncRNA、circRNA的基础知识详解miRNA1、背景介绍小分子DNA(miRNA)是一类存在于动植物体内、大小为2l一25 nt的内源性非编码单链小分子RNA，对生物体转录后的基因表达调控起关键作用。
1993年，首次在秀丽隐杆线虫中发现miRNA zBt_4；7年后，在果蝇中发现第2个IIliRNA 如t一7。
在进化中的保守性分析使科学家惊异地发现miRNA如卜7的形成至少需要有Dmsha，DGCR8(Pasha)、Dicer等2种RNA酶(RNaseⅢ)的参与。
Dmsha，DGCR8定位于细胞核内，它能剪切miRNA前体转录物(研一miRNA)，从而释放出具有发夹结构、大小为70 nt左右的pre—miRNA，后者在转运受体Exportin一5(Exp5)的作用下被转运至细胞质，然后被胞质中的另一种RNaseⅢ蛋白Dicer剪切，最终被船工成成熟的miRNA。
动物的miRNA位于前体mRNA的内含子中，这种安排将使mRNA基因和内含子中miRNA共同转录。
近年来，发现和鉴定的miRNAs越来越多，但植物miRNAs仅占很小一部分，且主要集中于拟南芥和水稻等少数模式植物中，植物miRNA的靶基因大多编码转录因子，与植物的生长、发育密切相关；而在动物和人中发现大量miR—NA，已证实在动物的生长、发育和疾病发生等过程中起重要作用。
2、miRNA的生物功能真核生物miRNA在调节植物对环境胁迫如干旱、盐害和养分的胁迫反应等方面起着重要的作用。
成熟的miRNA先与一种称为RNA诱导沉默复合体(RNA—indlIced silencing complex，R1SC)的复合物结合，再特异性地与目标mRNA结合，引起靶mRNA的降解。
由于植物miRNA与其靶mRNA具有很高的碱基互补性，因而植物miRNA的作用方式可能更像小分子RNA干涉(smallinte如而ng RNA，siRNA).与植物相反，在动物细胞中大多数miRNA与其靶mRNA并不完全互补，miRNA则通过与对应mRNA的3’端非翻译区(3’UTR)结合阻止转录后的翻译，从而起到调节基因表达的作用。