理学分子生物学英文10.pptx

Welcome Each of You to My Molecular Biology Class2Molecular Biology of the Gene,5/E -Watson et al.(2004)Part I:Chemistry and Genetics Part II:Maintenance of the Genome Part III:Expression of the GenomePart IV:RegulationPart V:Methods2005-5-103Chapter 16 Regulation principles and How genes are regulated in bacteriaChapter 17 Basic mechanism of gene expression in eukaryotesChapter 18 The mechanism of RNAi and the role of miRNA in development and cancer4Chapter 18:Chapter 18:RNAi and miRNA regulationRNAi and miRNA regulationMolecular Biology Course5Outlines1.The background and discovery of miRNAs2.RNAi discovery and mechanism3.miRNA biogenesis and regulation4.miRNA roles in development,cell differentiation and virus5.miRNA in cancer6.siRNA application6Topic 1:The background Topic 1:The background and discovery of miRNAsand discovery of miRNAsCHAPTER 18 RNAi and miRNA regulation一、一、miRNA发现的背景和发现的背景和miRNA发现发现7后基因组时代的分子生物学后基因组时代的分子生物学Maintenance of the Genome(基因组保持基因组保持)Expression of the Genome(基因组表达基因组表达)Regulation(调控调控)8DNA Double Helix(1953)Francis H.Crick James D.Watson 生命科学生命科学20世纪的里程碑世纪的里程碑中心法则中心法则The Central Dogma(1953-1956)是分子生物学的基本框架是分子生物学的基本框架DNARNAProteinReplicationReplication复制复制复制复制TranscriptionTranscription转录转录转录转录TranslationTranslation翻译翻译翻译翻译10后基因组时代的中心法则后基因组时代的中心法则RNA processingRNA processingGene regulationDNA repair and recombination基基因因组组的的保保持持基基因因组组的的表表达达11后基因组时代的基因调控：后基因组时代的基因调控：RNA 调控调控Most of the RNA transcribed from your genome doesnt make protein.Carina Dennis talks to the revolutionaries who believe that it functions in gene-regulatory networks that underlie the complexity of higher organisms.121.含有含有30亿对碱基的人类基因组仅含有亿对碱基的人类基因组仅含有23万万个蛋白质基因，是果蝇的两倍，啤酒酵母的个蛋白质基因，是果蝇的两倍，啤酒酵母的4倍。显而易见，倍。显而易见，生物的复杂性不由编码蛋白质生物的复杂性不由编码蛋白质生物的复杂性不由编码蛋白质生物的复杂性不由编码蛋白质的数目决定的数目决定的数目决定的数目决定。2.人类基因组的蛋白质编码区的总和占总基因组人类基因组的蛋白质编码区的总和占总基因组长度为长度为12，那么其他，那么其他9898的基因组有什的基因组有什的基因组有什的基因组有什么功能么功能么功能么功能呢？当然，在这呢？当然，在这98的非蛋白质编码的非蛋白质编码基因组序列里，约基因组序列里，约24为插入编码序列的内为插入编码序列的内含子序列；人类基因平均每个基因有含子序列；人类基因平均每个基因有7个内含个内含子。但这么冗长的内含子序列有什么生物学功子。但这么冗长的内含子序列有什么生物学功能呢？能呢？人类基因组草图带给科学家们的困惑人类基因组草图带给科学家们的困惑13人类基因组绝大部分都被转录成人类基因组绝大部分都被转录成RNA，细胞内非编码细胞内非编码RNA的的数量是编码数量是编码RNA的上百倍。这促使许多科学家认为生物体复的上百倍。这促使许多科学家认为生物体复杂性被隐藏在它们所输出的非编码杂性被隐藏在它们所输出的非编码RNA内，而非编码序列内。内，而非编码序列内。14The discovery of miRNAs miRNA was first discovered in 1993 by Victor Ambros at miRNA was first discovered in 1993 by Victor Ambros at Harvard(Harvard(lin-4lin-4)The second miRNA The second miRNA Let-7Let-7 was discovered in 2000 by Frank was discovered in 2000 by Frank Slack as a postdoc at HarvardSlack as a postdoc at Harvard (Ruvkun lab)(Ruvkun lab)Victor AmbrosVictor AmbrosGary Ruvkun15The first discoered miRNA:lin-4Ruvkun G,Wightman B,Ha I.The 20 years it took to recognize the importance of tiny RNAs.Cell.2004 Jan 23;116(2 Suppl):S93-6.Lee R,Feinbaum R,Ambros V.A short history of a short RNA.Cell.2004 Jan 23;116(2 Suppl):S89-92Thought to be an oddity not a general phenomenon16Breakthrough with BlastN of the second miRNA(stRNA)let-7Pasquinelli AE,Reinhart BJ,Slack F,Martindale MQ,Kuroda MI,Maller B,Hayward DC,Ball EE,Degnan B,Muller P,Spring J,Srinivasan A,Fishman M,Finnerty J,Corbo J,Levine M,Leahy P,Davidson E,Ruvkun G.Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.Nature.2000 Nov 2;408(6808):86-9.17microRNAs had been neglected for so many years because of their small size.OOPs!1819The number of the identified miRNAs is growing rapidly in recent years.Over 5000 miRNAs have been found until the August of this year(The miRBase Sequence Database).These miRNAs are from primates,rodents,birds,fish,worms,flies,plants and viruses.The data are freely available to all through the web interface at http:/microrna.sanger.ac.uk/sequences/and in flatfile form from ftp:/ftp.sanger.ac.uk/pub/mirbase/sequences/.2021Topic 2:RNA interference Topic 2:RNA interference and its mechanismand its mechanism二、二、RNA干扰及其机制干扰及其机制CHAPTER 18 RNAi and miRNA regulation221 Double-stranded RNA inhibits expression of genes homologous to that RNA.phenomena-现象现象双链双链RNA抑制抑制含其同源序列含其同源序列基因的表达基因的表达232006年的诺贝尔生理学奖获得者：年的诺贝尔生理学奖获得者：Andrew Z.FireAndrew Z.FireCraig C.MelloCraig C.Mello24Fig 2.Analysis of RNA-interference effects in Fig 2.Analysis of RNA-interference effects in individual cells.individual cells.Fluorescence micrographs show progeny of injected animals from GFP-reporter strain GFP-reporter strain PD4251(a C.elegans strain expressing GFP fluorescence PD4251(a C.elegans strain expressing GFP fluorescence protein)(protein)(使用外源导入的报告基因使用外源导入的报告基因使用外源导入的报告基因使用外源导入的报告基因).Young larva(幼虫幼虫)Adult(成虫成虫)adult body wall at high magnification(高放大倍数的高放大倍数的成虫体壁成虫体壁)Control dsRNAds-gfp RNA25Fig 3.Effects of mex-3 RNA interference on Fig 3.Effects of mex-3 RNA interference on levels of the endogenous mRNAlevels of the endogenous mRNA(in situ hybridization in embryos)(胚胎的原位杂交胚胎的原位杂交).adult body wall at high magnification(高放大倍数的高放大倍数的成虫体壁成虫体壁)No hybridization and staining+hybridization(endogenous mex-3 RNA)+antisense+hybridization+ds mex-3 RNA+hybridization26The discovery of RNAi explains the virus-The discovery of RNAi explains the virus-induced gene silencing in plants(induced gene silencing in plants(植物病毒引起的植物病毒引起的植物病毒引起的植物病毒引起的基因沉默基因沉默基因沉默基因沉默).).Most plant viruses have single-stranded RNA genomes,which are released from the protein coat of their virus particles as they enter a cell.Their genomic RNA is then replicated by the virus encoded RNA-dependent RNA polymerase to produce sense and antisense RNA,which can hybridize to form dsRNA and trigger an RNAi response against their own sequences.272.Short interfering RNA(siRNAs)are produced from dsRNA and direct machinery that switch off genes in various way.Mechanism-机制机制从双链从双链RNA产生的小干扰产生的小干扰RNA可以指可以指导用不同机制关闭基因的细胞机器导用不同机制关闭基因的细胞机器28The question to be addressed is“Why exogenous dsRNA can inhibit expression of genes homologous to that RNA?”29Figure 17-30 RNAi silencingExogenous dsRNA外源双链外源双链RNA30The targets of the RNAi-directed gene silencing1.Degradation of the target mRNA(引引起靶标起靶标mRNA的降解的降解),2.Inhibition of translation of the target mRNA(抑制靶标抑制靶标mRNA的翻译的翻译),3.Silencing the gene transcription from the target promoter(引起靶标启引起靶标启动子的转录沉默动子的转录沉默).31The heart of the RNAi mechanism1.Dicer:Dicer:an RNaseIII-like multidomain ribonuclease that first processes input dsRNA into small fragments called short interfering RNAs(siRNAs)or microRNAs(miRNA).Dicer then helps load its small RNA products into RISC.2.RISCRISC(RNA induced silencing complexes)(RNA induced silencing complexes)(RNA诱导的沉默复合体诱导的沉默复合体):a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression.32Dicer:Structural organization:-A PAZ domain,binds the end of the dsRNA-Two RNase III domains-Other non-conserved domains.贾第鞭毛虫贾第鞭毛虫33The crystal structure of the Giardia intact Dicer enzyme shows that the PAZ domainthe PAZ domain,a module that binds the end of dsRNA,is separated from the two catalytic RNase two catalytic RNase III domainsIII domains by a flat,positively charged surface.The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA.Thus,Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.34RISC:the key component is Argonaute(AGO)the key component is Argonaute(AGO)Argonaute(AGO)Argonaute(AGO):A large protein family that constitutes key components of RISCs.-AGO proteins are characterized by two unique two unique domains,PAZ and PIWI,domains,PAZ and PIWI,whose functions are not fully understood.Current evidence suggests that the PAZPAZdomain binds the 3-end two-nucleotide overhangs of the siRNA duplex,whereas the PIWI PIWI domain of some AGO proteins confers slicer activity.PAZ and PIWIdomains are both essential to guide the interaction between the siRNA and the target mRNA for cleavage or translational repression.-Distinct AGO members have distinct functionsDistinct AGO members have distinct functions.For example,human AGO2 programs RISCs to cleave themRNA target,whereas AGO1 and AGO3 do not.3536A model for siRNA-guidedmRNA cleavage by Argonaute37The multiple functions of RNAi38Topic 3:miRNA biogenesis Topic 3:miRNA biogenesis and regulationand regulation三、三、miRNA生成和调控生成和调控CHAPTER 18 RNAi and miRNA regulation391.MicroRNA(miRNA)&its processing微小微小RNA及其加工及其加工40MicroRNA(miRNA):A type of non-coding small RNA(2123 nucleotides)produced by Dicer from a stem-loop structured RNA precursor(70-90 nts ong)(结构和来源结构和来源).miRNAs are widely expressed in animal and plant cells and functions in the form of RNAprotein complexes,termed miRISCs.miRNAs have been implicated in the control of development because they lead to the destruction or translational suppression of target mRNAs with homology to the miRNA(生物学功能和机制生物学功能和机制).41The miRNA genes and Structure of pri-miRNAsPri-miRNAs bear the 5 cap and 3 poly(A)tails42miRNA processingPri-miRNA(miRNA初级转初级转录产物录产物)DroshaDrosha(1)(1)pre-miRNA(miRNA前体前体)DicerDicer(2)(2)miRNAExportin 5(Exp5)transports pre-miRNA to the cytoplasm431.A typical metazoan pri-miRNA consists of a stem of approximately 33 bp,with a terminal loop and flanking segments.2.The terminal loop is unessential,whereas the flanking ssRNA segments are critical for processing.3.The cleavage site is determined mainly by the distance(approximately 11 bp)from the stem-ssRNA junction.44Han et al.,Cell 125,887901,June 2,200645Human Drosha and Dicer share the same RNase III domains and dsRNA binding domain.462.MicroRNA(miRNA)targets and regulation.47A comparison between miRNA and siRNA48RNA silencing in different organisms49Three strategies of miRNA and target recognition(targets are locating in 3 UTRs).50Topic 4:miRNA roles in Topic 4:miRNA roles in development,cell development,cell differentiation and virusdifferentiation and virusCHAPTER 18 RNAi and miRNA regulation四、四、MicroRNA在发育中的调控作用，在发育中的调控作用，及其他作用及其他作用51Victor R.Ambros秀丽线虫秀丽线虫 C.elegans1.miRNA in 1.miRNA in C.elegansC.elegans development development52lin-4 and let-7 miRNAs control the developmental time of C.elegans.53Expression of lin-4 allows C.elegans to proceed to the late developmental stage54lin-4 binds its target mRNAs by imperfect base pairing.552.miRNAs in vertebrate development:There are a lot unknown because the the lack of efficient methods to uncover the targets of miRNAs.56Figure 2.Expression of Expression of miR-124amiR-124a and and miR-1miR-1 in in Zebrafish,Medaka,Mouse,and Fly.Zebrafish,Medaka,Mouse,and Fly.miR-124a is restrictedly expressed in the brain and the spinal cord in fish and mouse or to the ventral nerve cord in the fly.The expression of miR-1 is restricted to the muscles and the heart in the mouse.青鳉青鳉斑马鱼斑马鱼小鼠小鼠果蝇果蝇Learning the miRNA function from its expression pattern573.miRNA controls plant phenotypes(控控制植物表型特征制植物表型特征)Jaw-miRNA 控制拟南芥叶形变化(Nature,2003)58(Science 2004)3 3种种miRNAmiRNA控制造血干细胞向淋巴细胞的分化过程控制造血干细胞向淋巴细胞的分化过程4.miRNA controls the differentiation of the hematopoietic stem cell(调控造血干细调控造血干细胞的分化胞的分化)595.Some viruses encode miRNAs(有些病毒有些病毒编码编码miRNAs)6061Topic 5:miRNA in cancerTopic 5:miRNA in cancer五、微小五、微小RNA在癌症发生中的作用在癌症发生中的作用CHAPTER 18 RNAi and miRNA regulation62miRNAs in human:There are about 500 miRNAs from human have been found and annotated.They are named as has-miRhas-miRx.63miRNA expression pattern changes during oncogenesis,and is unique for each cancer.微小微小RNARNA在癌症发生中表达谱的变化在癌症发生中表达谱的变化6465Figure 3,Comparison between normal and tumor samples reveals global changes in miRNA expression.66One mechanism of miRNA controlling oncogene expression 微小微小RNARNA调控癌基因表达的一种机制。调控癌基因表达的一种机制。671.c-Mycc-Myc is a helixloophelix leucine zipper transcription factor that regulates an estimated 1015%of genes in the human and Drosophila genomes.2.c-Mycc-Myc activates expression of a cluster of six miRNAs on human chromosome 13.(Figure 1)3.E2F1E2F1 is the transcription factor,which is a target of c-Myc that promotes cell cycle progression.4.Expression of E2F1 is negatively regulated Expression of E2F1 is negatively regulated by two miRNAs in this cluster,miR-17-5p by two miRNAs in this cluster,miR-17-5p and miR-20a.and miR-20a.(Figure 1)6869Used 2-O-methyl Antisense oligonucleotides to downregulate the level of miR-17-5p and miR-20a,and then analyzed the protein(B-Western)and mRNA levels(C-Northen)of E2F1.70Some microRNAs are potential oncogenes 有些微小有些微小RNARNA可能是致癌基因。可能是致癌基因。71B-B-细胞淋巴瘤细胞淋巴瘤72Figure 1.The mir-1792 cluster shows increased Figure 1.The mir-1792 cluster shows increased expression in B-cell lymphoma samples and cell lines.expression in B-cell lymphoma samples and cell lines.The level of mir-1792 pri-miRNA was determined by real-time quantitative RT-PCR in 46 lymphomas and 47 colorectal carcinomas,and compared to levels found in corresponding normal tissues from five individuals.73Figure 2.Overexpression of the mir-1719b cluster Figure 2.Overexpression of the mir-1719b cluster accelerates c-myc-induced lymphomagenesis in mice.accelerates c-myc-induced lymphomagenesis in mice.74Topic 6:siRNA applicationTopic 6:siRNA applicationCHAPTER 18 RNAi and miRNA regulation六、六、siRNA的应用的应用75siRNA application in mammalianTransfect exogenous siRNA into cellsTransfect exogenous siRNA into cells(transient expression)Chemical synthesis:expensiveIn vitro transcription of pre-miRNA with T7 promoter.In vitro transcription of long dsRNA by that are then cleaved by E.coli RNase III or RNase III-like DICER.Expression of siRNA in cultured cells or in Expression of siRNA in cultured cells or in animal modelsanimal modelssiRNA produced with pol III promoter from the transfected DNA plasmids.761.Transcription from RNAP III promoters of U6 and H1 are well characterized.RNAP III transcription uses a well-defined termination signal(TTTTT)and the products have no extra sequence.2.Transcription from these promoters is very efficient in various tissues.Expression of hairpin RNA(shRNA)using Pol III promoters7778A mammalian expression vector designed to direct the intracellular synthesis of siRNAs.791.Create induced phenotypes that can be observed over long time spans 2.Create a stably engineered cells can be assayed either in vitro or in vivo,perhaps testing the angiogenic(血管生血管生成成)or metastatic(转移转移)potentials of tumor cells in xenograft models(异种移异种移植模型植模型)。3.Create hypomorphic alleles(亚等位基因亚等位基因)rapidly in transgenic mice.804.Combine shRNAs with existing high-efficiency gene delivery vehicles to create bona fide RNAi-based therapeutics.For example,ultimately,to silence a disease-causing mutant allele specifically.81Research Applications of RNAi:A new strategy of reverse genetics&a novel way of gene knock-outnIt can be used in reverse genetics(反反向遗传学向遗传学)to identify the cellular or biological function of a gene.nIt can be combined with genomics to perform large-scale genetic screens aimed at gene discovery.82Therapeutic Applications of RNAi:A new strategy to invitation of new drugs and gene therapynsiRNAs