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1、Post-Transcriptional Regulation of Eukaryotic Genes(真核基因的转录后调控)RNA silencing(siRNA and miRNA)Protein degradation,郭红卫 2008.11.24 PKU,Reference：Genes IX(Benjamin Lewin)现代分子生物学(朱玉贤),Transcriptional Regulation of Eukaryotic Genes(真核基因的转录调控)Transcriptional initiationHistone modificationDNA methylation,Ac
2、etylation of histones activates chromatin,and methylation of DNA and histones inactivates chromatin.Methylation of DNA and of histones is associated with heterochromatin.The two types of methylation event may be connected.,DNA甲基化的位点DNA甲基化主要形成5甲基胞嘧啶（5-mC）和少量的N6-甲基嘌呤（N6-mA）及7甲基鸟嘌呤（7-mG）,C,5-mC,1、原核生物中
3、，DNA甲基化是为了抵抗噬菌体侵害而发生碱基C和A上的化学修饰。如大肠杆菌的限制修饰系统中，自身DNA特定位点的甲基化可以避免限制性内切酶的切割。2、真核生物中，甲基化被分为对称性甲基化（canotical/symmetric methylation），包括CpG和CpNpG），以及非对称甲基化（asymmetric methylation），包括CpHpH。多数细胞 5甲基胞嘧啶主要出现在CpG中。DNA甲基化能引起染色质结构、DNA构象、组蛋白修饰及DNA与蛋白质相互作用方式的改变，从而控制基因表达。,多个CpG序列集合成簇形成了富含甲基化位点的CpG岛（CpG island），具有很高的
4、序列保守性。真核生物约一半的存在于所有组成型表达的管家基因中，但这些CpG岛处于组成型非甲基化状态；另外一半出现在部分（40%）组织特异性调控基因的启动子中。,由于5甲基胞嘧啶脱氨后生成胸腺嘧啶（T），不易被识别校正，因此DNA甲基化提高了该位点的突变频率。,CpG island作为甲基化调控基因转录的单位,CpG islands are regulatory targetsCpG island is a stretch of 1-2 kb genomic sequence that is rich in unmethylated CpG doublets.Key Concepts CpG i
5、slands surround the promoters of constitutively expressed genes where they are unmethylated(why?).They are also found at the promoters of some tissue-regulated genes.There are 29,000 CpG islands in the human genome.Methylation of a CpG island prevents activation of a promoter within it.Repression is
6、 caused by proteins that bind to methylated CpG doublets.,A fully methylated site is a palindromic sequence that is methylated on both strands of DNA.Most DNA methylations are found on cytosine on both strands of the CpG doublet.A hemi-methylated site is a palindromic sequence that is methylated on
7、only one strand of DNA.Replication converts a fully methylated site to a hemi-methylated site.A demethylase is a casual name for an enzyme that removes a methyl group,typically from DNA,RNA,or protein.A methyltransferase(Methylase)is an enzyme that adds a methyl group to a substrate,which can be a s
8、mall molecule,a protein,or a nucleic acid.A de novo methylase adds a methyl group to an unmethylated target sequence on DNA.A maintenance methylase adds a methyl group to a target site that is already hemimethylated.,terms,DNA methylation is perpetuated by a maintenance methylase,De novo and perpetu
9、ation methylases are known,but demethylases have not been identified.,DNA methylase甲基化转移酶：包括日常性甲基化转移酶和从头合成型甲基化转移酶日常性甲基化转移酶是遗传DNA甲基化状态最重要的酶类，它可以在甲基化母链模板的指导下甲基化新合成链的相应位点，使DNA迅速由半甲基化状态转变为完全甲基化状态，即参与甲基化的维持（maintenance）。,从头合成型甲基化转移酶可以催化为甲基化的CpG成为mCpG，此过程不需母链指导，但速度很慢。但这一类甲基化酶是特异基因受甲基化调控的主要因子，在基因表达的表观遗传学调控
10、中起十分重要的作用。,真核生物中甲基化转移酶的种类：DRM1/2(plants)/Dnmt3a/b(mammal):establish all de novo DM and maintain partial CpNpG and nearly all CpHpH MET class(plants)/Dnmt1(mammal):maintenance of CpGCMT3(plant specific):maintain the rest CpNpG and CpHpH两类甲基化酶协同作用：,DNA去甲基化酶-目前最具争议的领域,MBD2 in mammal,Nature 1999,ROS1 in
11、 Arabidopsis,PNAS 2006,DNA甲基化可以使特定的阻遏物结合到DNA上两种结合到甲基化CpG序列上的蛋白可以阻遏转录：MeCP1:结合到DNA上时需要有几个甲基化同时存在，多结合与CpG岛上MeCP2及其相关家族蛋白:能够结合到单个甲基化的CpG碱基上，这也使得转录起始时需要一块无甲基化区域。,甲基化调控基因转录的两种机制：用于结合某些因子的位点被甲基化后不能再结合蛋白质。这些例子发生在调节位点中而不是在启动子上。,MeCP2通过结合到启动子上的复合体相互作用来直接抑制转录。MeCP2通过与具有组蛋白去乙酰化酶活性的Sin3阻抑物复合体结合，从而协同组蛋白的乙酰化调节基因的
12、转录活性。DNA的甲基化与组蛋白的乙酰化可以互相引发：,histone methylation and DNA methylation are connected,SUVAR39H:histone methyltransferase(methylation on H3-K9)HP1:heterochromatin-associated protein 1,研究DNA甲基化的方法：,Genomic DNA fractionated by methylation-sensitive restriction enzyme digestion(individual sites)PCR amplific
13、ation products from bisulfite-treated DNA(hundreds of sequences by direct sequencing or genome-wide sequences by microarray analysis/pyrosequencing)Direct sequencing of methylated DNA fragments isolated by affinity purification of MeCP1/2 protein(genome-wide),The restriction enzyme MspI cleaves all
14、CCGG sequences whether or not they are methylated at the second C,but HpaII cleaves only nonmethylated CCGG tetramers.,Methylation-sensitive restriction enzyme digestion,Bisulfite sequencing,Bisulfite(HSO3)can switch unmethylated C into U,(or microarray analysis),DNA甲基化的分布染色体水平上，DNA甲基化在着丝粒附近水平最高,基因水
15、平上，DNA甲基化高水平区域涵盖了多数转座子，假基因和小RNA编码区,在最新的研究发现，甲基化似乎对长度较短的基因有较强的转录调控能力，而对长基因的调控能力十分微弱。,Two examples of DNA methylation on gene regulation,X chromosome inactivationimprinting,X chromosome inactivation:In mammals,one of the two female X chromosomes is inactivated completely.The active X chromosome of fem
16、ales and the single X chromosome of males are expressed at the same level.,The Xic(X inactivation center)is a cis-acting region(450 kb)on the X chromosome(128 Mb)that is necessary and sufficient to ensure that only one X chromosome remains active.Xic includes the Xist gene which codes for an RNA(17
17、kb)that is found only on inactive X chromosomes.The mechanism that is responsible for preventing Xist RNA from accumulating on the active chromosome is unknown.,X-inactivation involves stabilization of XistRNA,which coats the inactive chromosome.,Active X chromosome:Xist:methylated,offOther sites:un
18、methylated,onInactive X chromosome:Xist:unmethylated,onOther sites:methylated,off,The typical pattern for imprinting is that a methylated locus is inactive.If this is the maternal allele,only the paternal allele is active,and will be essential for viability.The methylation pattern is reset when game
19、tes are formed,so that all sperm have the paternal type,and all oocytes have the maternal type.Imprinted genes(17 genes in mouse genome)occur in clusters and may depend on a local control site where de novo methylation occurs.,DNA methylation is responsible for imprinting,Gene Silencing,Heterochroma
20、tinDNA methylation and histone modificationRNA interference(siRNA and miRNA),DNA,Transcription,Protein,Translation,mRNA,Splicing,exon,intron,AAAAAAAAA,Polyadenylation,Protein Coding Gene,Folding,Modification,Transport,Complex Assembly,Protein Complex,Transcription,Processing(nucleus),Processing(cyto
21、plasm),Modification/Complex Assembly(cyt.or nuc.),ncRNA Gene,RNP,primary transcript/pre-mRNA,Export,Coding gene and non-coding gene expression,Terminology,RNA SilencingRNA interference(RNAi),PTGS post-transcriptional gene silencingTGS transcriptional gene silencingCo-suppressionHomology-dependent ge
22、ne silencingQuellingsiRNA/miRNA silencing,1.RNAi的发现,Nature 333:866(1988),Napoli,Lemieux,Jorgensen Plant Cell 2:279(1990),Inhibition by injected anti-sense RNA,Sense RNA had similar effects!Double-stranded RNA not tested-that was how to lose a Nobel prize!,Double-stranded RNA is the trick!,Effect of
23、mex-3 RNA interference on levels of endogenous mRNANegative control Normal pattern of mex3 expression embryo from a parent-uninjectedc)Embryo from a parent injected with antisense RNAd)Embryo from a parent injected with dsRNA,Fire,Xu,Montgomery,Kostas,Driver,Mello(1998),Nature 391:806-811,小RNA的发现,Ha
24、milton,Baulcombe，Science 286:950(1999),长度约为25 nt 正义（sense）和反义（anti-sense）转基因植株皆存在,Zamore P,Tuschl T et al(2000)Cell,David Baulcombe,For the discovery of RNA interference gene silencing by dsRNA and small RNA,RNA interference(RNAi),2.小干扰RNA(Small interfering RNA,siRNA)的特点,P,P,19-nt 双链2-nt 3末端突出5-磷酸基3
25、-OH:3 被阻碍后便失去活性植物siRNAs 3甲基化分为引导链与乘客链（Guide vs passenger strands）具有高度保守的种子序列（Seed region，nt 2-8)具有双链的不对称性,Tuschl T Gene Dev 15:188(2001);Nature 411:494(2001),是引发RNAi的充分条件，并且为Dicer的产物,3.siRNAs 是Dicer作用的产物,Dicer的结构,A model for dsRNA processing by Dicer,The PAZ domain of Dicer,a module that binds the e
26、nd of dsRNA,is separated from the two catalytic ribonuclease III(RNase III)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 c
27、leaves a specified distance from the helical end.,The PAZ domain binds the 2nt 3 overhang of a dsRNA terminus.The RNaseIII domains form a pseudo-dimer.Each domain hydrolyzes one strand of the substrate.The binding site of the dsRBD is not defined.Hammond(2005)FEBS Lett 579:5822-5829,Model for Dicer
28、catalysis,4.siRNA 的装载与激活（Loading&activation of siRNA）,siRNA 的装载需要一个双链RNA结合蛋白R2D2。R2D2包含两个一前一后的双链RNA结合结构域Dicer-2 与R2D2形成了一个异源二聚体R2D2 将与siRNA热稳定性更高的一端结合（3端）siRNA 的不对称性（siRNA asymmetry）:引导链的5端稳定性较差,siRNA asymmetry,FEBS Letters 579:5850(2005),The guide strand of an effective siRNA will begin an A or U,o
29、r have a mismatched nucleotide at the 5end.(top strand is the guide strand),Argonaute&slicing,Hammond(2005)FEBS Lett 579:5822,RNAi essential:rde1 in C.elegans(1999)qde-2 in Neurospora(2000)ago-1 in A.thaliana(2000)A core component of RISC in Drosophila(2001):AGO-24 AGOs 2 AGOs&3 PIWIs in flieshAGO2
30、is sufficient for the slicer activity,The PAZ domain PIWI is an RNase H domain,110 aa domain found in Piwi,Ago,Zwille&Dicer proteinsA binding pocket in PAZ accommodates the 2 nt overhangNo interactions found between the 2 nts with the pocket,suggesting that the pocket accommodates all nucleotide com
31、binations,Ma et al.Nature 429,318-322(2004),The similarity was not obviousat the primary sequence level!,Song et al.Science 305,1434-1437.,The siRNA guide strand is bound at the 5 end by the PIWI domain and at the 3 end by the PAZ domain.The 5 phosphate is coordinated by conserved basic residues.mRN
32、A targets are initially bound by the seed region of the siRNA and pairing is extended to the 3 end.The RNaseH fold hydrolyzes the target in a cation dependent manner.Slicer cleavage is measured from the 5 end of the siRNA.,Model for Slicer catalysis,5.RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase，RDRP)&
33、transitive RNAi,最早克隆自被类病毒侵染的番茄中(1998,Plant Cell)与RNA病毒编码的RdRP 基本没有同源性在果蝇和哺乳动物基因组中不存在是产生次级小干扰RNA的放大效应的主要因子RdRP 在RNAi过程中的研究主要集中在以下物种中Neurospora 1st RNAi pathway gene identified(1999,Nature)Arabidopsis(2000,Cell)C.elegans(2000,Curr Biol)&for TGS in fission yeast(Science 2002),RNA-dependent RNA polymera
34、se(RDR)&transitive RNAi,Required for transitive RNAi in C.elegans(2001)&Arabidopsis(2002)Synthesis of new dsRNA,leading to production of secondary siRNAsPossibly primer-independentSpreading 5 3 from the antisense strand of target mRNA in C.elegansMost of siRNAs detected in worms are secondary&antise
35、nse.But spreading occurs both directions in plantsAlso play a role in non-cell autonomous RNAi,Probe for2nd siRNAs,Probe for2nd siRNAs,6.非细胞自主(non-cell autonomous)的RNA沉默与系统性(systemic)RNA沉默,RNAi 作用可以向其它细胞传递，因此受到其他细胞或组织传来的小RNA的抑制成为非细胞自主的RNA沉默，可以分为两类：细胞与细胞间的传递：包括近程与远程两种系统性传递：通过传输器官（如韧皮部）在不同组织间广泛传播。,7.非
36、细胞自主的RNA沉默的抑制子,First panel:These results suggest that production and transmission of the systemic silencing signal are largely unaffected by HC-Pro,implying that HC-Pro suppression of silencing occurs downstream of the signal.Second panel(additional grafting experiment):showing denitively that HC-Pr
37、o works downstream of the systemic silencing signal:The rootstock is known to send a signal;therefore,in the presence of HC-Pro,the scion either fails to perceive the signal or fails to respond to it.,可以抑制细胞与细胞间的传递的抑制子,microRNA(miRNA),miRNA的发现：在线虫（C.elegans）当中,通过功能缺失突变体的筛选，找到了let-7/lin-41基因,在lin-14基
38、因的3UTR区域发现了lin4的互补区,不同物种中的let-7基因具有序列保守性,在lin-41基因的3UTR区域发现了let-7的互补区,lin-4 is another miRNA,lin-4/let-7 miRNA调控发育过程,miRNA biogenesis,Du and Zamore,Development 132,4645-4652.,animals,plants,Transcription of miRNA genes by RNA Pol II,Transcribed by pol II to pri-miRNA(primary precursor)Pri-miRNA cont
39、ains the 7-methylguanosine cap and a poly(A)tailPol II is physically associated with miRNA gene promotersmiRNA gene transcription is sensitive to-amanitinPol II dependent transcription enables temporal and spatial regulation of miRNA production.Some viral miRNAs are transcribed by pol III.,基因组上的分布和基
40、因结构,植物 miRNA 基因似乎具有它们独立于蛋白编码基因的自身的转录单位,Altuvia et al.发现人类已知miRNAs中 37%都以两个或多个聚集的形式存在于配对的染色体上3kb的区域.以前的研究表明聚集的 miRNAs 作为 polycistronic messages被转录.植物的 miRNAs 不聚集(exception:moss).,基因组上的分布和基因结构,Mirtrons 果蝇中miRNA合成的新途径,microRNA靶作用位点在植物当中的预测和验证,植物 miRNAs 与 mRNAs具有很高的序列互补性。.植物 miRNAs 更趋向于在蛋白编码的基因家族中设置靶位点靶
41、位点预测的原则是基于实验验证的靶位点的特征错配/G:U pairs/bulges的数量和位置双链的最小自由能Allen et al.Cell 121,207-221.,Rhoades et al.Cell 110,513-520.,植物miRNAs 可以利用类似于siRNA介导的剪切机制去剪切靶mRNAs如何研究miRNA的功能？,miRNA 基因敲除突变体miRNAs基因过表达突变体在内源启动子作用下表达miRNA-resistant targets 约半数预测的保守miRNA的靶位点都存在于转录因子的mRNAs中(转录因子只占基因的 6%),动物 miRNAs 只与它们的靶位点保持很低的互
42、补性：仅为miRNAs中 2-7个核苷酸(成为种子区seed sequence)，且决定了miRNA的功能。Within miRNA target sites of invertebrate miRNAs,residues that pair with nucleotides 2-7 of the miRNAs are conserved in orthologous mRNAs of other species.Nucleotide 2-7 of the miRNA are the most conserved among homologous metazoan miRNAs.Experim
43、ental evidence also indicates that nucleotides 2-7 in siRNAs are more important than others in guiding cleavage,microRNA靶作用位点在动物当中的预测和验证,Pairing to the seed is necessaryAdditional pairing at nt 12-17 enhances miRNA targetingBinding site location preference：Local AU rich region,microRNA的作用机理,Inhibiti
44、on of mRNA translationlin-4 mediated regulation of lin-14;let-7 mediated regulation of lin-41Other animal miRNAs cause reduced target protein levels without affecting target mRNA levels in cell cultureAt least three examples of plant miRNAs affecting target protein but not mRNA levelsReduction of mR
45、NA stabilityPlant miRNAs guide cleavage of target mRNAs(however only a few miRNA targets have been examined at the protein level)Animal miRNAs also reduce stability of target mRNAs,Translation inhibition-a role for the anti-association factor eIF6,Human RISC(Dicer,TRBP,and Ago2)is found in a larger
46、complex containing nearly all subunits of the 60S(but none of the 30S subunits)and eIF6.eIF6 is a 60S subunit associated factor that prevents the assembly of a translationally competent(80S)ribosome.Depletion of eIF6 in human cells abolishes let7-mediated repression of reporter gene expression.Deple
47、tion of eIF6 in C.elegans diminishes lin-4-mediated repression of the endogenous lin-14 and lin-28 target mRNA and protein levels.,miRNA的生物学功能,Plant miRNAs Many act in cell differentiation and developmental patterning by targeting transcription factor mRNAsOther miRNAs target non-transcription facto
48、r mRNAs and may play a role in physiological processes or stress responsesEssential functions of miRNAs illustrated by the embryo lethal phenotype of dcl1 null mutantsAnimal miRNAsDevelopmental patterningES cells lacking Dicer are viable but cannot differentiate in vitro and in vivo.Dicer knockout z
49、ebrafish lacking both maternal and zygotic Dicer have intact patterning in the first 24 h but fail to continue with morphogenesisPhysiological functionsCancer,siRNA途径与表观遗传学调控,tasiRNA:trans-acting siRNAAllen et al.(2005)Cell 121:207-221,miRNA-guided tasiRNA biogenesis,nat-siRNAs:siRNAs derived from n
50、atural antisense transcripts produced by convergent overlapping genes,nat-siRNAs,rasiRNA and piRNA,rasiRNAs:repeat associated siRNA 24-27 nt long enriched in the testes and early in developmentderived from retrotransposons and other repetitive elements3 ends are methylated(Saito et al.,2006;Vagin et
