理学分子生物学英文10.pptx
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1、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 regula
2、ted 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 miRNAs
3、2.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
4、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
5、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 Den
6、nis 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
7、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
8、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-
9、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
10、 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-7Pasquinel
11、li 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(680
12、8):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,bi
13、rds,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 mechan
14、ism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-interferenc
15、e 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 fluoresce
16、nce 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 mRNAlevel
17、s 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 dis
18、covery 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
19、 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
20、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 silenc
21、ing1.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 th
22、at 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 dire
23、ct 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 domainth
24、e 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 its
25、elf 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 characteriz
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