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1、三、RNA加工水平的调控,RNA加工:剪切(cleavage)修剪(trimming)剪接(splicing)修饰(modification)戴帽”(capping)m7Gppp“接尾”(tailing)PolyA.编辑(editing)RNAi降解,(一)切除多余序列,1.基因间序列的除去2.内元的除去 可变剪接(alternative splicing)调控基因的表达,以适应生理的需求。可变剪切的结果,一个单基因转录得到的mRNA前体,可以产生多个蛋白质,即蛋白质同源体(isoform)。,(二)碱基修饰,差不多所有RNA都是含有不同数量的修饰成分,尤以tRNA和snRNA为最多。迄今RN
2、A中的修饰成分发现已近百种,但对其功能的认识还不多。,(三)RNA编辑,1986年Berme等人从锥体虫中发现的一种新的RNA加工方式。当时发现锥体虫线粒体细胞色素C氧化酶亚基(Co)基因与酵母或人的相应基因相比,在编码蛋白的170位氨基酸附近有一移码突变。比较锥体虫Co基因与其转录物的序列,发现转录序列在相应上述移码突变位点附近有四个不被基因DNA所编码的额外的尿苷酸,这四个尿苷酸正好校正了基因的移码突变。,480 490 500 510 mRNA:UUA GGU AUA AAA GUA GAU UGU AUA CCU GGU AGG UGU AAU蛋白序列:L G I K V D C I
3、P G R C N DNA:TTA GGT ATA AAA GTA GA*G*A*A CCTGGT AGG TGT AAT核酸序号以mRNA起始密码AUG的A开始编号。下划线标出与人及酵母相应蛋白相同的氨基酸。,锥体虫T.buucei亚种mRNA的编辑(不是全部),mRNA 起始密码 编辑区域 5区 编码区 3及PolyA 区Co+40Co?39418 321Cyb+340 140 MURF2+264 斜线坐标时插右边表示删去U。斜线右表示基因中有无起始密码。右边表示编辑中有无构建起始密码。,Mechanism of edting,RNA editing is catalyzed by a c
4、omplex with seven major polypeptides,two of which correspond to distinct ligases.U deletion and U insertion involve gRNA-directed endonuclease cleavage of the pre-mRNA(shown by an arrowhead),either 3-U-exo or TUTase acting on the upstream fragment,and RNA ligase rejoining the mRNA.,R,purine(s);Y,pyr
5、imidine(s).These two forms of editing use distinct cleavage activities and distinct ligase enzymes,and the 3-U-exo is not a reverse TUTase reaction,which is indicated by the unequal sign.gRNAs have three main portions:a 5 region to anchor the gRNA to the mRNA just downstream of the editing site and
6、direct the cleavage,a central region to guide the U deletions and insertions at mismatches in base pairing with the pre-mRNA,and a 3 oligo(U)that may tether the very purine-rich upstream pre-mRNA.Many additional conserved gRNA features have also been noted.Dotted lines indicate potential base pairs
7、that could serve in a ligation bridge.,Model of the 19S and 3540S RNA editing complexes revisited.Polycistronic minicircle transcripts assemble into 19S gRNA processing complexes,which process the 5 most gRNAs into monocistrons(gray boxes).These complexes further associate with pre-edited mRNA and o
8、ther proteins to form 3540S RNA editing complexes that produce edited mRNAs.Red,endonuclease;yellow,TUTase;green,RNA ligase;closed arrowhead,editing site.,Current understanding of U deletion and U insertion.,编辑的生物学意义,1.校正移码突变:Co2.控制转译:产生起始密码,MURF2 除去起始密码,锥体虫(C.fasciculate)MURF-3 产生终止密码,T.brucel Co.3
9、.扩充遗传信息。T.burceil Cyb基因:在其固有起始密码上游构造新的起始密码,从而扩展了21个核苷酸的转译序列。另外两种锥体虫C.fasclculata和L.tarentolae的Co基因也用这种方法增加了18个密码子。,T.brucel的Co最突出,在158个位点插入394个U,在9个位点删去18个U,实际增加376个U,增加数占成熟的Co转录物编码区总长度的55。RNA编辑是对中心法则的重要补充。,(四)反义RNA,基本原理是通过碱基配对与mRNA结合,形成二聚体阻断其表达功能。1.反义RNA在胞质内与mRNA形成RNA:RNA二聚体使其不能与核糖体结合。2.反义RNA在核内与新生
10、mRNA结合,二聚体不能运输出核外。3.反义RNA:mRNA二聚体易被酶降解。4.碱基修饰作用。mRNA中A转变为 I,A I,I-U对不稳定,可使双链拆开。,(五)RNA干涉(RNA interference,RNAi)与microRNAs(miRNAs),RNA干涉是1998年首次在线虫(C.elegans)中发现并证明的转录后水平的基因沉默机制,利用双链RNA 的介导可以特异性地降解相应的mRNA,阻断相应基因的表达,随后发现这种现象广范存在于从真菌到植物、从无脊椎动物到哺乳动物的各种生物中。,RNAi和miRNA的特征,转录后水平的基因沉默机制具有很高的特异性能够非常特异地降解与之相应
11、的单个内源基因的mRNA,RNAi抑制基因的表达具有很高的效率,远远少于内源mRNA的数量的双链RNA能完全抑制相应基因的表达。这表明很可能存在干涉效应分子的扩增机制。,RNAi抑制基因表达的效应可以在不同细胞间长距离传递和维持,在线虫中干涉效应甚至可以传到后带中去,这说明RNAi干涉机制中可能存在维持mRNA降解序列特异性的信号小分子。,RNAi的意义及应用前景,生物的watchdog 抵抗病毒入侵,抑制转座子活动,防止自私基因序列的过度增殖,对生物体的发育和基因的调控可能也有重要作用。,产生抗病毒的植物和动物研究新基因的功能人类疾病的基因治疗,国外医学分子生物学分册()P267。RNAi的
12、作用机制及其应用。,RNAi的负向调控网络,在基因的转录产物中,只要有这一段很短的同源序列,表达就会被抑制。,(六)mRNA的降解,mRNA的结构,国外医学分子生物学分册()P。真核生物中降解机制研究进展。,影响mRNA稳定的因素,5端帽子结构5非编码区(5UTR)开放阅读框的降解序列3非编码区(3UTR)IRE(铁离子相关区域),AREBP在一般的细胞RNA代谢中起着重要作用,其结合力受细胞内铁离子的调节,保护mRNA的稳定性。ARE(A-U富集区),含50150碱基,有多个AUUUA重复序列,U的百分比含量对稳定有重要意义。polyA和PABP Human PABP MW 72000KD
13、形成2527核苷酸结合区。,Formation of circular eukaryotic mRNA by protein-protein interactions of eIF4E and eIF4G(binding to the m7G cap),poly(A)-binding protein I(PABP),Force-fieldelectron micrograph,1.脱腺苷酸依赖型降解,真核细胞中最主要的降解方式,其降解过程如下:polyA核酸水解酶水解polyA尾巴,polyA缩短。polyA能结合一种称为polyA结合蛋白(PABP)的特异蛋白,只有当polyA缩短至1015
14、个残基时,PABP便不能与之结合,脱帽过程被启动。脱帽时由脱帽酶Dcp1切除mRNA5端鸟苷酸形成的帽子结构。脱帽后的mRNA 很容易被5 3核酸外切酶识别水解,另外真核生物的mRNA也可以在脱腺苷酸后以3 5方向被降解,但这种作用很小。,脱polyA尾巴的调节 Poly(A)核酸酶的活性受Ccr4和Caf1的正调节脱帽过程的调节 在翻译过程中,mRNA被翻译起始因子eIF4E,eIF4G及PABP包围。eIF4E蛋白与5端帽子连接,而且eIF4G可加强他们之间的连接,eIF4G是一种大分子蛋白质,同时与eIF4E和PABP发生关联。这种结构保护mRNA的5和3端不受脱帽酶和脱腺苷酶的降解。一
15、旦上述的稳定环状结构被破坏,polyA水解,PABP脱离,紧接着发生脱帽反应。,线粒体中的mRNA的降解,与真核生物的细胞质中的情形相反 polyA促进降解,是降解的信号。,2.无义密码介导的mRNA的降解,Nonsense mediated mRNA decay N M D,突变和剪接错误,突变和剪接错误,突变和剪接错误,突变和剪接错误,突变和剪接错误,突变和剪接错误,发生无义突变和移码突变的mRNA和从核内逃逸的未经剪接还有内含子的mRNA,Is it important?,Absolutely!为害极大!NMD probably evolved to eliminate abnormal
16、 transcripts due to routine errors in gene expression.For example,inefficient or inaccurate pre-mRNA splicing can generate an intron-derived in frame nonsense codon,or a shift in the reading frame and an exon-derived nonsense codon downstream of the shift.,NMD降解过程:,当mRNA转运进入细胞质后,立即同核糖体结合并开始翻译,如果存在无义
17、突变密码子则会使翻译提前终止。随着翻译的终止,一组由核糖体部分组分和相关因子组成的物质会继续向下游移动与一段下游元件(DSE)相遇,形成一个异常的核蛋白结构,引起mRNA的5端水解一个或二个核苷酸残基,完成脱帽反应,脱帽后的mRNA则迅速被5核酸酶水解。,Nonsense mediated decay of mRNAs,DAHLBERG J.E.et.al.RNA 2003;9:1-8,Copyright 2003 by RNA Society,Nonsense mediated decay of mRNAs,DAHLBERG J.E.et.al.RNA 2003;9:1-8,Copyrigh
18、t 2003 by RNA Society,exon junction complex(EJC),Nonsense-mediated decay(NMD).Following splicing in the nucleus,the exon junction complex(EJC),which contains UPF3(a core protein of the NMD pathway),is associated with the transcript,and the resulting messenger ribonucleoprotein is exported to the cyt
19、oplasm.In the cytoplasm,a second NMD core protein,UPF2,binds to UPF3.Ribosomes associate and translate the mRNA,but are stalled on encountering a premature termination codon(PTC).This results in binding of the SURF complex(comprising SMG1,UPF1 and the peptide-release factors eRF1 and eRF3)to the rib
20、osome.UPF1 also binds UPF2,thereby linking the EJC to the PTC.Phosphorylation of UPF1 by SMG1 leads to dissociation of eRF1 and eRF3 and binding of the SMG7 adaptor protein.Subsequent steps that are still being elucidated lead to mRNA decay by various pathways.b|Non-stop decay.Translation of an mRNA
21、 that lacks a stop codon results in ribosomes traversing the poly(A)tail,displacing poly(A)-binding protein(PABP)and stalling at the 3 end of the mRNA.One model proposes that,in yeast and mammalian cells,Ski7,an adaptor protein that functions as a molecular mimic of tRNA,binds to the A site on the s
22、talled ribosome to release the transcript,and then recruits the exosome.The exosome degrades the poly(A)tail and mRNA body.In another pathway described in Saccharomyces cerevisiae,in the absence of Ski7,the displacement of PABP by the translating ribosome renders the mRNA susceptible to decapping an
23、d 5 3 decay by the 5 3 exoribonuclease Xrn1.c|No-go decay.Ribosomes can stall within the open reading frame(ORF),for example,on encountering a strong secondary RNA structure.The Dom34 and Hbs1 proteins bind the transcript near the stalled ribosome and initiate an endonucleolytic cleavage event near
24、the stall site.This releases the ribosome and generates two mRNA fragments,each with a free end exposed for exonucleolytic decay by the exosome and Xrn1,respectively.,mRNA编码区3端附近的一个下游元件(DSE)是无义mRNA降解必需的无义密码子与最靠近3端的外显子之间的相对位置决定了无义mRNA是否被降解大于5055个核苷酸,降解;小于50个核苷酸则不被降解。,Garneau et al.Nature Reviews Mole
25、cular Cell Biology 8,113126(February 2007),Nonsense-mediated mRNA decay:from vacuum cleaner to Swiss army knifeGenome Biology 2004,5:218,Vacuum cleaner,Swiss army knife,1.国外医学分子生物学分册():。真核生物细胞中的无义介导mRNA降解机制。2.遗传学报Acta Genetica Sinica,November 2004,31(11):13211326 NMD 作用的顺式调控元件3.Nonsense-Mediated mRN
26、A Decay:Splicing,Translation And mRNP DynamicsLynne E.MaquatNature Reviews Molecular Cell Biology 5,89-99(February 2004),参考文献,3.无终止密码子mRNA降解途径,These data suggest that nonstop decay is initiated when the ribosome reaches the 3 terminus of the message RNA,Science,2002;295:2258An mRNA Surveillance Mech
27、anism That Eliminates Transcripts Lacking Termination Codons.Science,2002;295:2262Exosome-Mediated Recognition and Degradation of mRNAs Lacking a Termination Codon.,4.no-go decay,So what happens when RNA accumulates a mutation that hampers the ribosomes ability to translate it?For example if a large
28、 stem loop of other RNA fold impeded the ribosomes path along the mRNA.,5.核酸内切酶降解mRNA,直接降解核酸内切酶活性受到调节 如哺乳动物的RNaseL通常不具活性,但其与2,5磷酸二酯键相连的寡腺苷酸结合时才具有活性,而后者在双链RNA存在时才产生。,6.马方综合症(Marfan syndrome),马方综合征,是一种常染色体显性遗传的全身性结缔组织疾病,主要累及眼、骨骼和心血管系统,95%的患者死于心血管并发症-主动脉夹层、破裂和充血性心力衰竭。未治患者的平均寿命,男性30岁左右,女性约40岁。婴幼儿患者多因心脏瓣
29、膜病变所致的充血性心力衰竭而夭折。,马方综合症是一种结缔组织疾病,所以影响许多结构,包括骨胳、肺、心脏、眼睛和血管。该疾病通常以细长肢为特征。,流行病学和发病率,1990年我国的一组流行病学统计报告发病率约为1.72/10,000人,1995年在苏格兰进行的一项人口调查报告MS的最低流行病率为1/14,217(7.03/100,000人),发病率达1/9802,最近估计的发病率约为1/3-5,000人。,Figure 2:Pedigrees representing inheritance of Marfan Syndrome in the Summers family.,1896年Antoi
30、ne Bernard Marfan第一次描述了一个叫Gabrielle P.的5岁半女孩,并称之为瘦长肢体症(dolichostenomelia)。1972年Hecht和Beals认为其实该女孩患的是先天性挛缩性蜘蛛指症(Congenital Contractural Arachnodactyly,CCA,也称作Beals综合征)。,马方综合症相关历史名人和报道,三国志中对天生异相的蜀主刘备的描述是:“身长七尺五寸,垂手下膝,顾自见其耳。”美国总统林肯 篮球球星克里斯帕顿(Chris Patton)奥运会排球队队员海曼(Flo Hyman),死于马方综合征中的主动脉瘤破裂小提琴家帕格尼尼四川排
31、球运动员朱刚、杨怀清喀麦隆球星维维安福“恐怖大亨”拉登?,Health note:Does Obama have Marfan Syndrome?Posted on July 1st,2008 by Nancy Reyes,分子遗传学表明符合诊断标准的MFS患者有7090发生了致病性FBN1基因突变。,FBN1是一种分子量为320kd的糖蛋白,由2871个氨基酸组成,是构成细胞外微纤丝的主要蛋白之一。,B.Loeys等通过筛选95个诊断为马方综合征病人FBN1基因的所有65个外显子,发现在93个马方综合征病人中发现了85个FBN1基因突变,频率为91。鉴定出的突变主要由三类组成:.41个错义突
32、变;.13个无义突变和20个移码突变引起的提前终止密码;.11个突变影响剪接或引起框缺失。如果NMD起作用,则这些突变基因的mRNA降解,不发生马方综合症;否则产生大量的异常蛋白质。,7.玉米CMS-S育性恢复机理研究,CMS产生的原因,CMS的遗传特性,Fertile,Sterile,Fertile,Fertile,Gametophytic CMS,Sporophytic CMS,Now that 12 mtDNA regions associated with CMS have been identified,it is striking how often the events invo
33、lve ATP synthase subunit gene promoter regions and portions of coding regions(Hanson and Bentolila,2004,Plant Cell,preview).,与CMS相关的线粒体片断,实验材料,S-Mo17rf3rf3(S),S-Mo17Rf3Rf3(F),N-Mo17rf3rf3(N),orf355-orf77的不同拷贝,orf355/BamHI,2.8kb和1.6kb转录本的末端位点,1583bp,2741bp,1583bp,1160bp,5末端,3末端,引物延伸法分析5末端位点,tttctatggt,ORF17,Transcripts degraded by the exo-and endo-ribonucleases,Collapsed pollen,Restroed pollen,玉米CMS-S不育机理与育性恢复机理,Degradation,Degradation,