【US10504519B1】Transcriptionofcommunications【专利】
international journal of acarology投稿须知
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translational oncology杂志的 decision in process -回复
translational oncology杂志的decision inprocess -回复decision in process，即审稿流程的决策过程。
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分子生物学英文文献6
Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryfindings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantification of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsflanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-specific primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-specific primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Purification 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-specific primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into afilter cartridgewithin a collection bel thefilter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the samefilter.17.Centrifuge at10,000rpm for15s at RT.18.Collect thefiltrate(theflow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theflow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newfilterbel thefilter as small RNA.When you have >700μL of thefiltrate mixture,apply it in successive appli-cation to the samefilter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theflow-through and repeat until all of thefiltratemixture is passed through thefilter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to thefilter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theflow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to thefilter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theflow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer thefilter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of thefilter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave thefilter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube briefly.3.Incubate for1h at37◦C.3.3.Purification 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a purificationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard thefiltrate(theflow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of thefilter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let thefilter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube briefly.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-specific primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube briefly.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-specific primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which thefluorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method amplifies over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-specific primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefficiency.Good efficiencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially amplifies maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). AcknowledgmentsThe authors would like to thank Jonathan Cho for reading andediting the text.This work was supported by grants from theNational Institute of Health(HD048855and HD050281)toW.Y.References1.Ambros,V.(2004)The functions of animalmicroRNAs.Nature,431,350–355.2.Bartel,D.P.(2004)MicroRNAs:genomics,biogenesis,mechanism,and function.Cell, 116,281–297.3.Chang,T.C.and Mendell,J.T.(2007)Theroles of microRNAs in vertebrate physiol-ogy and human disease.Annu Rev Genomics Hum Genet.4.Kim,V.N.(2005)MicroRNA biogenesis:coordinated cropping and dicing.Nat Rev Mol Cell Biol,6,376–385.5.Kim,V.N.(2006)Small RNAs just gotbigger:Piwi-interacting RNAs(piRNAs) in mammalian testes.Genes Dev,20, 1993–1997.6.Kotaja,N.,Bhattacharyya,S.N.,Jaskiewicz,L.,Kimmins,S.,Parvinen,M.,Filipowicz, W.,and Sassone-Corsi,P.(2006)The chro-matoid body of male germ cells:similarity with processing bodies and presence of Dicer and microRNA pathway components.Proc Natl Acad Sci U S A,103,2647–2652.7.Aravin,A.A.,Lagos-Quintana,M.,Yalcin,A.,Zavolan,M.,Marks,D.,Snyder,B.,Gaaster-land,T.,Meyer,J.,and Tuschl,T.(2003) The small RNA profile during Drosophilamelanogaster development.Dev Cell,5, 337–350.8.Lee,R.C.and Ambros,V.(2001)An exten-sive class of small RNAs in Caenorhabditis ele-gans.Science,294,862–864.u,N.C.,Lim,L.P.,Weinstein, E.G.,and Bartel,D.P.(2001)An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.Science,294, 858–862.gos-Quintana,M.,Rauhut,R.,Lendeckel,W.,and Tuschl,T.(2001)Identification of novel genes coding for small expressed RNAs.Science,294,853–858.u,N.C.,Seto,A.G.,Kim,J.,Kuramochi-Miyagawa,S.,Nakano,T.,Bartel,D.P.,and Kingston,R.E.(2006)Characterization of the piRNA complex from rat testes.Science, 313,363–367.12.Grivna,S.T.,Beyret,E.,Wang,Z.,and Lin,H.(2006)A novel class of small RNAs inmouse spermatogenic cells.Genes Dev,20, 1709–1714.13.Girard, A.,Sachidanandam,R.,Hannon,G.J.,and Carmell,M.A.(2006)A germline-specific class of small RNAs binds mammalian Piwi proteins.Nature,442,199–202.304Ro and Yan14.Aravin,A.,Gaidatzis,D.,Pfeffer,S.,Lagos-Quintana,M.,Landgraf,P.,Iovino,N., Morris,P.,Brownstein,M.J.,Kuramochi-Miyagawa,S.,Nakano,T.,Chien,M.,Russo, J.J.,Ju,J.,Sheridan,R.,Sander,C.,Zavolan, M.,and Tuschl,T.(2006)A novel class of small RNAs bind to MILI protein in mouse testes.Nature,442,203–207.15.Watanabe,T.,Takeda, A.,Tsukiyama,T.,Mise,K.,Okuno,T.,Sasaki,H.,Minami, N.,and Imai,H.(2006)Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes.Genes Dev,20,1732–1743.16.Vagin,V.V.,Sigova,A.,Li,C.,Seitz,H.,Gvozdev,V.,and Zamore,P.D.(2006)A distinct small RNA pathway silences selfish genetic elements in the germline.Science, 313,320–324.17.Saito,K.,Nishida,K.M.,Mori,T.,Kawa-mura,Y.,Miyoshi,K.,Nagami,T.,Siomi,H.,and Siomi,M.C.(2006)Specific asso-ciation of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome.Genes Dev,20, 2214–2222.18.Ro,S.,Song,R.,Park, C.,Zheng,H.,Sanders,K.M.,and Yan,W.(2007)Cloning and expression profiling of small RNAs expressed in the mouse ovary.RNA,13, 2366–2380.19.Ruby,J.G.,Jan,C.,Player,C.,Axtell,M.J.,Lee,W.,Nusbaum,C.,Ge,H.,and Bartel,D.P.(2006)Large-scale sequencing reveals21U-RNAs and additional microRNAs and endogenous siRNAs in C.elegans.Cell,127, 1193–1207.20.Terns,M.P.and Terns,R.M.(2002)Small nucleolar RNAs:versatile trans-acting molecules of ancient evolutionary origin.Gene Expr,10,17–39.21.Ouellet,D.L.,Perron,M.P.,Gobeil,L.A.,Plante,P.,and Provost,P.(2006)MicroR-NAs in gene regulation:when the smallest governs it all.J Biomed Biotechnol,2006, 69616.22.Maatouk,D.and Harfe,B.(2006)MicroR-NAs in development.ScientificWorldJournal, 6,1828–1840.23.Kim,V.N.and Nam,J.W.(2006)Genomics of microRNA.Trends Genet,22, 165–173.24.Bohnsack,M.T.,Kos,M.,and Tollervey,D.(2008)Quantitative analysis of snoRNAassociation with pre-ribosomes and release of snR30by Rok1helicase.EMBO Rep,9, 1230–1236.25.Hertel,J.,de Jong, D.,Marz,M.,Rose,D.,Tafer,H.,Tanzer, A.,Schierwater,B.,and Stadler,P.F.(2009)Non-codingRNA annotation of the genome of Tri-choplax adhaerens.Nucleic Acids Res,37, 1602–1615.26.Kim,M.,Patel,B.,Schroeder,K.E.,Raza,A.,and Dejong,J.(2008)Organization andtranscriptional output of a novel mRNA-like piRNA gene(mpiR)located on mouse chro-mosome10.RNA,14,1005–1011.27.Mishima,T.,Takizawa,T.,Luo,S.S.,Ishibashi,O.,Kawahigashi,Y.,Mizuguchi, Y.,Ishikawa,T.,Mori,M.,Kanda,T., and Goto,T.(2008)MicroRNA(miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary.Reproduction,136, 811–822.28.Papaioannou,M.D.,Pitetti,J.L.,Ro,S.,Park, C.,Aubry, F.,Schaad,O.,Vejnar,C.E.,Kuhne, F.,Descombes,P.,Zdob-nov, E.M.,McManus,M.T.,Guillou, F., Harfe,B.D.,Yan,W.,Jegou,B.,and Nef, S.(2009)Sertoli cell Dicer is essential for spermatogenesis in mice.Dev Biol,326, 250–259.29.Ro,S.,Park,C.,Sanders,K.M.,McCarrey,J.R.,and Yan,W.(2007)Cloning and expres-sion profiling of testis-expressed microRNAs.Dev Biol,311,592–602.30.Ro,S.,Park,C.,Song,R.,Nguyen,D.,Jin,J.,Sanders,K.M.,McCarrey,J.R.,and Yan, W.(2007)Cloning and expression profiling of testis-expressed piRNA-like RNAs.RNA, 13,1693–1702.31.Ro,S.,Park,C.,Young,D.,Sanders,K.M.,and Yan,W.(2007)Tissue-dependent paired expression of miRNAs.Nucleic Acids Res, 35,5944–5953.32.Siebolts,U.,Varnholt,H.,Drebber,U.,Dienes,H.P.,Wickenhauser,C.,and Oden-thal,M.(2009)Tissues from routine pathol-ogy archives are suitable for microRNA anal-yses by quantitative PCR.J Clin Pathol,62, 84–88.33.Smits,G.,Mungall,A.J.,Griffiths-Jones,S.,Smith,P.,Beury,D.,Matthews,L.,Rogers, J.,Pask, A.J.,Shaw,G.,VandeBerg,J.L., McCarrey,J.R.,Renfree,M.B.,Reik,W.,and Dunham,I.(2008)Conservation of the H19 noncoding RNA and H19-IGF2imprint-ing mechanism in therians.Nat Genet,40, 971–976.34.Song,R.,Ro,S.,Michaels,J.D.,Park,C.,McCarrey,J.R.,and Yan,W.(2009)Many X-linked microRNAs escape meiotic sex chromosome inactivation.Nat Genet,41, 488–493.Quantitative Analysis of Small RNAs30535.Wang,W.X.,Wilfred,B.R.,Baldwin,D.A.,Isett,R.B.,Ren,N.,Stromberg, A.,and Nelson,P.T.(2008)Focus on RNA iso-lation:obtaining RNA for microRNA (miRNA)expression profiling analyses of neural tissue.Biochim Biophys Acta,1779, 749–757.36.Wu,F.,Zikusoka,M.,Trindade,A.,Das-sopoulos,T.,Harris,M.L.,Bayless,T.M., Brant,S.R.,Chakravarti,S.,and Kwon, J.H.(2008)MicroRNAs are differen-tially expressed in ulcerative colitis and alter expression of macrophage inflam-matory peptide-2alpha.Gastroenterology, 135(1624–1635),e24.37.Wu,H.,Neilson,J.R.,Kumar,P.,Manocha,M.,Shankar,P.,Sharp,P.A.,and Manjunath, N.(2007)miRNA profiling of naive,effec-tor and memory CD8T cells.PLoS ONE,2, e1020.38.Yan,W.,Morozumi,K.,Zhang,J.,Ro,S.,Park, C.,and Yanagimachi,R.(2008) Birth of mice after intracytoplasmic injec-tion of single purified sperm nuclei and detection of messenger RNAs and microR-NAs in the sperm nuclei.Biol Reprod,78, 896–902.39.Guryev,V.and Cuppen,E.(2009)Next-generation sequencing approaches in genetic rodent model systems to study func-tional effects of human genetic variation.FEBS Lett.40.Li,W.and Ruan,K.(2009)MicroRNAdetection by microarray.Anal Bioanal Chem.41.Ro,S.,Park,C.,Jin,JL.,Sanders,KM.,andYan,W.(2006)A PCR-based method for detection and quantification of small RNAs.Biochem and Biophys Res Commun,351, 756–763.。
Transcriptomeprofiling
OWS 2013Transcriptome profiling--Past, Present and FutureWei ChenBerlin Institute for Medical Systems BiologyMax-Delbrueck-Center for Molecular MedicineWhy RNA?DNA (m)RNA Protein TranslationTranscriptionSplicingLocalization…snRNARNA editingdegradationRNAi (yeast) LincRNA Promoter associated RNA enhancer associated RNA … tRNA rRNA miRNA … 5’ capping3’ poly ATranscriptome profiling• Past– Pre-genome era– Genome era• Present• Ongoing and further developmentPre-genome era (1960s)DNAProtein Fractionation technique (Count Concurrent Distribution for tRNAisolation )During his (R.Holley) 3 years of work on the structure of the alanine tRNA, Holley used a total of only 1 g of highly purified material, which he isolated from approximately 200 g of bulk yeast tRNA, which in turn was obtained by phenol extraction of approximately 140 kg of commercial bakers' yeast.RNA sequencingSpecifically, Holley, George A. Everett, James T. Madison, and Ada Zamir first used pancreatic ribonuclease to cleave the RNA chain next to pyrimidine nucleotides and then used takadiastase ribonuclease T1 to cleave the RNA chain at guanylic acid residues. They isolated the resulting fragments by ion-exchange chromatography. The components of dinucleotide fragments were then identified by chromatographic and electrophoretic properties and spectra…rRNA tRNA mRNAPre-genome era (1970s and 1980s)• Reverse transcriptase (Temin and Baltimore,1970, Nobel prize 1975)• PCR (Mullis, 1983, Nobel Prize 1993)• Sanger Sequencing (Sanger, 1977, Nobel Prize 1980)• Northern Blot (Alwine, Kemp, and Stark, 1977 )– one-shot sequencing of a clone cDNA/mRNA– Several hundred bps, 3’, 5’ or random• D iscovery of expressed (m)RNAs from different tissues• P hysical mapping of genes into chromosome• D esign of expression microarrayGenome era (1990s, 2000s)• Series Analysis of Gene expression (SAGE)Genome era (1990s, 2000s)• MicroarrayLimitationsa. Available annotationb. Cross hybridizationc. Limited dynamicrange/sensitivityMassive parallel RNA sequencing (2005-present)• S mall RNA sequencing• m iRNA, piRNA, siRNA…• R NA-seq• >200ntSmall RNA library prep (miRNA, PiRNA...)• L igation: 5’ phosphate and 3’ OH, ligation bias• R T-PCR: strong bias due to 2nd structureSmall RNA sequencing result10-40nt 40-90ntLi et.al, NAR 41(6) 3619-3634UNG treatmentRNA-seq vs ArrayWang et.al, Nature Review Genetics (10) 57-63Findings• Novel miRNAs• Novel PiRNAs• Endo-siRNAs• Novel isoforms (5’/3’ end, alternative splicing) • Promoter associated RNAs• Enhancer RNAs• LincRNAs• Circular RNAsLincRNAs• Negative definition• Not protein coding• Not overlapping with other defined transcripts• PolII transcripts– Cap, polyA, often splicing• A heterogeneous group with diverse properties and functionsLincRNA detection• FANTOM project (cDNAclone and Sanger seq)– >34000 in differentmouse tissues• Tiling array– define transcribed regionw/o transcript model• RNA-seq & de novoassembly• Chromatin map• Other supporting data– CAGE, 3-PIgor Ulitsky and David P. Bartel Cell (154) 26-46Non-coding vs codingIgor Ulitsky and David P. Bartel Cell (154) 26-46LincRNA association with RibosomeGuttman et.al. Cell (154) 240-251LincRNA genomics• Preferentially surrounding developmental TFs– Regulate gene is cis (e.g. HOTTIP)– Act in concert and benefit from co-regulation (e.g. Six3 and Six3os)– Accommodating environment for the emergence of newlincRNAs• Low expression and tissue specific (brain and testis) – median 1/10 protein-coding• Subcellular localization– both nuclear and cytoplasmeDiverse functions of lincRNAsCis-regulation• Association with PRC2, CTCF…• Direct chromatin modifying complex to DNA via nascent transcript or triplex interaction• Paring ofAlu-repeat induces STAU1 action.• miRNA sponge• Malat1 binds multiple proteins in paraspeckles• Gadd7 & TDP-43Igor Ulitsky and David P. Bartel Cell (154) 26-46Circular RNAsJeremy E. Wilusz and Phillip A. Sharp Science (340) 4401. Cocquerelle, C., et al, Mis-splicing yields circular RNA molecules. FASEB J. 7, 155–160 (1993).2. Capel, B. et al. Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73, 1019–1030 (1993).3. Chao, C. W., et al., The mouse formin (Fmn) gene: abundant circular RNA transcripts and gene-targeted deletion analysis. Mol. Med. (1998).4. Suzuki, H. et al. Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res. (2006).5. Burd, C. E. et al. Expression of linear and novel circular forms of an INK4/ARF- associated non-coding RNA correlates with atherosclerosis risk. PLoS Genet. (2010).6. Hansen, T. B. et al. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. EMBO J. 30, 4414–4422 (2011).7. Salzman,J.et al. , Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS ONE 7, e30733 (2012).8. Jeck, W. R. et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19, 1–17 (2013).Detection of circular RNAMemczak et.al Nature. (7441):333-8Memczak et.al Nature. (7441):333-8Memczak et.al Nature. (7441):333-8Memczak et.al Nature. (7441):333-8Possible functions of circular RNAsMatthias W Hentze and Thomas Preiss Embo J (32) 923–925Ongoing and further development• Full length RNA sequencing• Single cell transcriptome profiling• Direct RNA sequencing• Discovery and Profiling of RNA modification • In situ RNA sequencingTranscriptome assembly- state-of-art State-of-Art transcriptome assembly using short readsFull length cDNA sequencingFull length cDNA sequencing—a hybridapproachYou et.al. UnpublishedSingle cell RNA-seqSingle cell RNA-seq (Fluidigm)Direct RNA-seq (Helicos)Fatih Ozsolak and Patrice M. Milos, Nature Review Genetics (12) 87-98Direct RNA-seq (Helicos)—mapping 3’ endOzsolak et.al. Nature. (461) 814-8Direct RNA-seq (PacBio)Vilfan et.al. Journal of Nanobiotechnology 11:8PacBio RNA-seq—RNA modificationVilfan et.al. Journal of Nanobiotechnology 11:8In situ RNA-seqKe et.al. Nature Methods (2013) doi:10.1038/nmeth.2563In situ RNA-seq (2)Ke et.al. Nature Methods (2013) doi:10.1038/nmeth.2563。
transcript名词解释
transcript名词解释transcript，英语单词，主要用作为名词，译为“成绩单；抄本，副本；文字记录”。
transcript 双语例句1. The name of the minor will be listed in the graduate list, transcript and degree certificate of students if they have completed all required courses and received the required credits of the minor upon graduation.修满辅系规定之科目与学分成绩及格者，其毕业生名册、历年成绩表及学位证书应加注辅系名称。
2. Finally, our data provide estimates of absolute transcript abundance, and suggest there is significant transcriptional heterogeneity within a clonal, synchronized bacterial population.研究人员还发现，RNA测序本质上只是一种高通量的计数技术，它提供了一种方法来决定细胞中的转录有多丰富。
3. Transcript structure, operon linkages, and absolute abundance information all provide valuable insights into gene function and regulation, but none has ever been determined on a genome-wide scale for any bacterium.为了解决这个问题，乔治亚理工学院和生命技术公司的研究人员开发了一套可让RNA测序适用于任何细菌的程序。
转录子
Bacterial RNA Polymerases
How does RNA polymerase work?
A single type of RNA polymerase is responsible for almost all synthesis of mRNA, rRNA and tRNA in a eubacterium.
Insensitive Low Conc. (1-10 nM) High conc.
Major Products
rRNA hnRNA tRNA, 5S RNA and small RNAs
All have in common 2 large subunits and a number of smaller subunits,
Intrinsic terminatorsρ-independent
An inverted repeat that allows a hairpin to form at the end of the transcripts A string of T’s in the nontemplate strand that results in a string of weak rU-dA base pairs holding the transcript to the template strand
Similarity between replication and transcription
• Both processes use DNA as the template.
• Phosphodiester bonds are formed in both cases.
• Both synthesis directions are from 5´ to 3´.
通信原理英文
(MF)
direction-finding, distress calling, coast guard
17
Division of frequency band
Frequency band (MHz)
3 – 30
30 – 300
300 – 3000
Name
Typical application
High frequency (HF)
# Ex: “Rainfall will be 1 mm tomorrow” – information content small
“Rainfall will be 1 m tomorrow” – information content large
“The sun will rise in the east tomorrow morning” – information content equals zero # Information content I = I [ P(x) ]，P(x) – Occurrence probability # Definition：I = loga [1/P(x)] = -logaP(x) # Usually, set a = 2, the unit of the information content will be called a bit.
5
1.3 Digital Communication
1.3.1 Basic concept
Two categories of signals • Analog signal：Its voltage or current
can be expressed by a continuous function of time. For example, speech signal.
Transcript-Assisted Transcriptional Proofreading.
DOI: 10.1126/science.1127422, 518 (2006);313 Science et al.Nikolay Zenkin Transcript-Assisted Transcriptional ProofreadingThis copy is for your personal, non-commercial use only.clicking here.colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to othershere.following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles): October 7, 2014 (this information is current as of The following resources related to this article are available online at/content/313/5786/518.full.html version of this article at:including high-resolution figures, can be found in the online Updated information and services, /content/suppl/2006/07/25/313.5786.518.DC1.htmlcan be found at:Supporting Online Material /content/313/5786/518.full.html#related found at:can be related to this article A list of selected additional articles on the Science Web sites /content/313/5786/518.full.html#ref-list-1, 12 of which can be accessed free:cites 24 articles This article 23 article(s) on the ISI Web of Science cited by This article has been /content/313/5786/518.full.html#related-urls 30 articles hosted by HighWire Press; see:cited by This article has been/cgi/collection/molec_biol Molecular Biologysubject collections:This article appears in the following registered trademark of AAAS.is a Science 2006 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n O c t o b e r 7, 2014w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mTranscript-AssistedTranscriptional ProofreadingNikolay Zenkin,1*Yulia Yuzenkova,1Konstantin Severinov 1,2,3*Fidelity of template-dependent nucleic acid synthesis is the main determinant of stable heredity and error-free gene expression.The mechanism (or mechanisms)ensuring fidelity of transcription by DNA-dependent RNA polymerases (RNAPs)is not fully understood.Here,we show that the 3¶end–proximal nucleotide of the nascent transcript stimulates hydrolysis of the penultimatephosphodiester bond by providing active groups and coordination bonds to the RNAP active center.This stimulation is much higher in the case of misincorporated nucleotide.We show that during transcription elongation,the hydrolytic reaction stimulated by misincorporated nucleotides proofreads most of the misincorporation events and thus serves as an intrinsic mechanism of transcription fidelity.The mechanism of transcription is high-ly conserved in all living organisms.In the RNAP elongation complex,the 3¶end of the nascent RNA can occupy a post-translocated,a pretranslocated,or a back-tracked state (Fig.1A).In each of these states,the RNAP active center performs different reactions,i.e.,the forward reaction of nucleoside triphosphate (NTP)addition or hydrolytic cleavage of the nascent RNA (Fig.1A).Catalysis by the RNAP active center depends on two Mg 2þions (1–7)that activate reacting groups and stabilize leav-ing groups during nucleophilic attack on the phosphorus (4,8).In cellular RNAPs,only one Mg 2þion (MgI)is bound tightly in the ac-tive center.The other Mg 2þion,MgII (2,4–7),is bound weakly,but its binding is stabilized by the triphosphate moiety of the incoming NTP (5).The hydrolytic transcript cleavage reaction,characteristic of pretranslocated and backtracked elongation complexes (9)(Fig.1A),is slow compared to the forward RNA polymerization reaction,presumably because of poor binding of MgII (4).Although the rate of misincorporation of nucleotides by RNAP is much slower than the rate of incorporation of correct nucleo-side 5¶-monophosphates (NMPs)(10,11),the relatively low selectivity of RNAP (12)makes misincorporation unavoidable,suggesting the existence of a proofreading mechanism.To define such a mechanism,12complexes with mismatched NMP at RNA 3¶end (misincorpo-rated elongation complexes,MECs)modelingall possible misincorporation events and 4correct complexes (correct elongation com-plexes,CECs)were assembled by means of 4DNA templates that differed from each other only by a base pair at the þ1register (corresponding to the transcript 3¶end)and 45¶end–labeled RNAs that were identical except for the 3¶-terminal base (13)(fig.S1).Complexes were assembled in the absence of Mg 2þand were therefore inactive.Upon addition of Mg 2þto MECs,RNAP efficiently cleaved the penultimate (P2)phos-phodiester bond.No P1(ultimate phospho-diester bond)(Fig.1A)cleavage was observed (Fig.1,B and C),suggesting that MECs are backtracked by 1base pair relative to the pretranslocated state (Fig.1A).Cleavage of P2was much slower in CECs than in the cor-responding MECs (Table 1),as expected for active,not backtracked,complexes.However,because no P1cleavage was observed in CECs (Fig.1,B and C),stabilization of the backtracked state in MECs cannot explain preferential P2cleavage,which was also observed with eukaryal and archeal RNAPs (10,14–17)and appears to be a general phenomenon.Noncomplementary NTPs bind in the RNAP E-site close to the active center and stimulate P1cleavage (4).Addition of nonhydrolyzable E to prevent any possibility of (mis)incorporation in the nascent RNA ^NTP analog APcPP A adenosine-5¶-E (a ,b )-methyleno ^triphosphate Z (Fig.1C),noncomplementary to the DNA template guanosine in register þ2,led to stimulation of P1cleavage in CECs.No such stimulation was observed in MECs,indicating that base-pairing of the RNA _s 3¶end is required for NTP-assisted cleavage.Noncomplementary NTPs also inhibited P21Waksman Institute,2Department of Molecular Biology and Biochemistry,Rutgers University,Piscataway,NJ 08854,USA.3Institute of Molecular Genetics,Russian Academy of Sciences,Moscow,123182Russia.*To whom correspondence should be addressed at Waksman Institute,190Frelinghuysen Road,Piscataway,NJ 08854,USA.E-mail:nicserzen@mail.ru (N.Z.);severik@(K.S.)Fig.1.Cleavage in misincorporated (MECs)and correct (CECs)elongation complexes.(A )Schematic representation of catalytic reactions character-istic of transcription elongation complexes in different states.The red circle represents the active center that contains two Mg 2þions.(B )MECs (lanes 1to 6,13to 24)and a corresponding CEC (lanes 7to 12)(with CMP at the RNA 3¶end as an example)were supplied with 10mM Mg 2þand incubated for various times at pH 7.9(40-C).For each MEC,the first letter indicates misincorporated 3¶NMP,and the correct nucleotide that it replaces is indicated in parentheses (fig.S1).(C )CECs and MECs [A-CEC and U(A)MEC are shown as examples]were supplied with 15mM Mg 2þand incubated for various times with or without 1mM noncomplementary nonhydrolyzable NTP (APcPP).REPORTS28JULY 2006VOL 313SCIENCE518cleavage in both CECs and MECs in a dose-dependent manner (Fig.1C).Noncomplemen-tary NTPs are known to bind in the so-called E-site of the RNAP active center,the same site where initial interaction of correct NTPs with RNAP occurs.To explain the inhibitory effect,we postulate that in backtracked com-plexes,the 3¶-terminal NMP also occupies the E-site (or an overlapping site,Fig.2)and activates P2cleavage in a way that is simi-lar to P1cleavage activation by noncomple-mentary NTP.Binding of NTP in the E-site displaces the transcript _s 3¶end and destabi-lizes the backtracked state,thus inhibiting P2cleavage.Noncomplementary NTP activates P1cleavage by stabilizing MgII through interac-tion with the b and g phosphates (4,5).Because these phosphates are absent in the 3¶-terminal NMP,MgII coordination and/or P2cleavage may be stimulated by the terminal NMP itself.This hypothesis predicts that Mg 2þdependence of P2cleavage,which reflects the complex affinity for MgII (4),should have a lower dissociation constant (K d )than the intrinsic (unassisted by non-complementary NTP)K d of P1hydrolysis (9100mM)(4).This expectation was fulfilled for both MECs and CECs (Table 1).The lowest apparent K d observed (8mM)is close to the K d of P1hydrolysis stimulated by noncomplementary NTP (4).Thus,the 3¶-terminal NMP,either matched or mismatched,increases the P2cleavage velocity by increasing affinity for MgII.A high rate of P2cleavage could not be solely due to a decreased K d for MgII,be-cause cleavage velocity at saturating Mg 2þconcentrations (k cat )differed depending on the nature of 3¶-terminal NMP (Table 1).For example,comparisons of complexes con-taining A,G,or U instead of correct C at the 3¶end (Table 1rows 2,7,and 15,cor-respondingly)reveal that the cleavage re-action k cat values in different complexes differ significantly (0.14,0.028,and 0.015s j 1,respectively).This suggests that some groups of the transcript _s 3¶-end NMPs par-ticipate,directly or indirectly,in cleavage.Whereas the k cat of P2cleavage in MECs is determined by the properties of the reaction itself,in CECs it is strongly influenced by base-pairing of the 3¶end with the template strand,which affects the probability of back-tracked state occupancy.To avoid this compli-cation,we focused on MECs only (supporting online text).High pH deprotonates the active water molecule stimulating phosphodiester hydroly-sis by the RNAP active center.The stimula-tion depends on the reaction mechanism and should plateau at a pH equal to the system p K value.Therefore,if mismatched nucleotides were involved in cleavage,different profiles of cleavage reaction dependence on pH are expected for different MECs.This expecta-tion was fulfilled (fig.S2).The shapes of pH curves were different from that of the previously reported P1cleavage curve (4)(dotted line in fig.S2),indicating that the mechanism of P2cleavage was distinct from intrinsic RNAP-catalyzed P1hydrolysis.Whereas most P2cleavage profiles pla-teaued at about pH 9.5,some had a different E U(C)MECs ^plateau or even double (A-MECs)plateaus.Thus,different acid/base systems provided by the transcript 3¶-terminal nucle-otide participate in P2cleavage in different MECs.The dependence of cleavage reaction properties for complexes containing misin-corporated cytidine 5¶-monophosphate (CMP)and uridine 5¶-monophosphate (UMP)on the þ1DNA template-strand base may be ex-plained by effects of local sequence-dependent deviations of nucleic acids structure near the active center on the reaction pathway (support-ing online text).To check which chemical groups of 3¶-terminal NMP participate in MgII stabilization and P2hydrolysis,we determined the cleavage reaction K d and k cat in MECs with RNAs containing chemical modifications in the phos-phate,sugar,and base of the 3¶-terminal nucleo-tide (Fig.2).The results (Table 1and table S1),discussed in detail in the supporting online text,are summarized below (see also fig.S3).With misincorporated adenosine 5¶-mono-phosphate,one of the P1oxygens interacts with the 3¶-hydroxyl,which in turn coordinates MgII.Another P1oxygen orients the active water molecule.The 2¶-hydroxyl does not participate in the reaction.N-7of the purine ring co-ordinates MgII;the amino group in position 6participates in water-molecule orientation or,alternatively,acts,together with nitrogen in position 1,as a general acid-base system.For misincorporated guanosine 5¶-monophosphate (GMP),one of the P1oxygens orients active water.The 2¶and 3¶hydroxyls do not participate in the reaction.N-7of the base coordinates MgII.The amino group in position 2fixes the GMP moiety,probably through interactions with the protein,making the reaction insensitive to local variations in nucleic acid structure.Table 1.K d for Mg 2þand k cat of P2cleavage for all possible CECs and MECs.All experiments were carried out in pH 7.9(40-C).K d and k cat values were calculated with the Michaelis-Menten equation.Complex 3¶-endNMP ofthe RNA Incorporated instead of K d (Mg 2þ)(mM)k cat(s j 1)CEC AA 100.004MECC 90.14G 80.12U 90.11CEC GG 90.001MECA 110.024C 150.028U 140.027CEC CC 570.001MECA 490.026G 150.029U 460.026CEC UU 370.001MECA 230.054C 80.015G300.043Fig. 2.Modifications of misincorporated 3¶-terminal nucleotides used in this study.A schematic representation of the active center of RNAP in MEC that is consistent with our findings is shown on the left.Structures of modified bases,phosphate groups,and sugars are shown.REPORTS SCIENCEVOL 31328JULY 2006519With misincorporated CMP,sequence de-pendence of the cleavage reaction in C-MECs is due to differences in the P1bond orientation,which appears to be sensitive to local variations of nucleic acids structure.In one type of complex,P1interacts with the 3¶-hydroxyl,which coordinates MgII.In other complexes,this interaction is absent,and the 3¶-hydroxyl does not chelate MgII.P1also participates in a network of hydrogen bonding that positions the active water molecule.The 2¶-hydroxyl is dispensable.Nitrogen in position 3of the base chelates MgII.Finally,with misincorporated UMP,P1interacts with the 3¶-hydroxyl,po-sitioning it to coordinate MgII or to orient the active water molecule.P1also participates in coordination of the active water molecule.The 2¶-hydroxyl is dispensable.The keto group in position 4of the base either positions the water molecule or acts in concert with N-3as a general base/acid.Taken together,the results indicate that nucleotides that are misincorporated at the transcript 3¶end participate in their own ex-cision.In contrast to the previously described stimulation of transcript cleavage by noncom-plementary NTP (4),which can be regarded as B substrate-assisted catalysis [(18,19),the reaction described here represents B product-assisted catalysis [and,therefore,can directly affect transcription fidelity.To show that excision of misincorporated NMP via P2cleavage can prevent transcription past misincorporated NMP,we supplied MECs with NTP specified by the þ2register of the template and monitored transcript extension (Fig.3).As noted for RNAPs from eukaryotes and archaea (10,17,20,21),the rate of incorporation of NTPs by MECs was much lower than by CECs (7,22)and was compara-ble (k obs ,0.03s j 1in the presence of 1mM NTP)to the rate of P2cleavage (Table 1).Presumably,slow elongation of misincorpo-rated transcripts is due to stabilization of MECs in a backtracked state and to the occupancy of the primary NTP binding site,the E-site,by misincorporated NMP.At 100m M NTP,only 5to 13%of MECs (30%for G-MEC)extended the RNA,whereas the rest of RNA was cleaved and,therefore,the misincorporated NMP was removed (Fig.3B).In the presence of 1mM NTP (a physiological concentration),È30%of complexes (50%for G-MEC)extended past incorrect NMP,whereas the remainder underwent cleavage (Fig.3B).When NTP was added together with transcript cleavage factor GreA,very low (except 20%for G-MEC)incorporation was detected,and mismatched NMP was removed (Fig.3B).Thus,cleavage stimulated by misincorporated nucleotides is sufficient to proofread most misincorpora-tion events.This activity is stimulated by transcript cleavage factors that were previ-ously suggested to contribute to transcrip-tional fidelity (10,12,17,21)and that act by direct stabilization of MgII in the RNAP ac-tive site (23).The importance of transcriptional proof-reading for error-free gene expression was suggested (24).In addition,complexes con-taining misincorporated nucleotides elongate RNA slowly,which should impede expression of actively transcribed genes and may interfere with DNA replication.Cleavage factors cannot be solely responsible for removal of misin-corporated nucleotides,because they are not essential for cells.Our results reveal a proof-reading mechanism that may be sufficient to control transcription misincorporation in theabsence of cleavage factors.The mechanism,which is likely evolutionarily conserved,also allows the removal of 2¶-deoxy NMPs erro-neously incorporated in RNA,because ribo and 2¶-deoxy NMPs cleaved out with the same efficiency.In the RNA-protein world,when RNAP was likely replicating RNA genomes (25),the rela-tively low fidelity of RNAP-catalyzed synthesis could not have been sufficient for stable maintenance of large RNA genomes in the absence of cleavage factors (24).A proofreading and repair mechanism similar to the one de-scribed here could have allowed a large RNA genome of the last common universal ancestor to exist.References and Notes1.T.A.Steitz,Nature 391,231(1998).2.P.Cramer,D.A.Bushnell,R.D.Kornberg,Science 292,1863(2001).3. D.G.Vassylyev et al.,Nature 417,712(2002).4.V.Sosunov et al.,EMBO J.22,2234(2003).5.K.D.Westover,D.A.Bushnell,R.D.Kornberg,Cell 119,481(2004).6.H.Kettenberger,K.J.Armache,P.Cramer,Mol.Cell 16,955(2004).7. D.Temiakov et al.,Mol.Cell 19,655(2005).8.T.A.Steitz,J.A.Steitz,Proc.Natl.Acad.Sci.U.S.A.90,6498(1993).9.M.Orlova,J.Newlands,A.Das,A.Goldfarb,S.Borukhov,Proc.Natl.Acad.Sci.U.S.A.92,4596(1995).10.M.J.Thomas,A.A.Platas,D.K.Hawley,Cell 93,627(1998).11.G.Bar-Nahum et al.,Cell 120,183(2005).12. D.A.Erie,O.Hajiseyedjavadi,M.C.Young,P.H.vonHippel,Science 262,867(1993).13.Materials and methods are available as supportingmaterial on Science Online.14.H.Guo,D.H.Price,J.Biol.Chem.268,18762(1993).15.M.G.Izban,D.S.Luse,J.Biol.Chem.268,12864(1993).16.S.K.Whitehall,C.Bardeleben,G.A.Kassavetis,J.Biol.Chem.269,2299(1994).nge,W.Hausner,Mol.Microbiol.52,1133(2004).18.P.Carter,J.A.Wells,Science 237,394(1987).19.W.Dall’Acqua,P.Carter,Protein Sci.9,1(2000).20.H.Matsuzaki,G.A.Kassavetis,E.P.Geiduschek,J.Mol.Biol.235,1173(1994).21. C.Jeon,K.Agarwal,Proc.Natl.Acad.Sci.U.S.A.93,13677(1996).22.J.E.Foster,S.F.Holmes,D.A.Erie,Cell 106,243(2001).ptenko,J.Lee,I.Lomakin,S.Borukhov,EMBO J.22,6322(2003).24. A.M.Poole,D.T.Logan,Mol.Biol.Evol.22,1444(2005).25. zcano,J.Fastag,P.Gariglio,C.Ramirez,J.Oro,J.Mol.Evol.27,365(1988).26.This work is dedicated to the memory of Dmitry Salonin.We thank E.P.Geiduschek for fruitful discussions.This work was supported by NIH grant RO1GM64530and a Burroughs Wellcome Career Award (to K.S.).Supporting Online Material/cgi/content/full/313/5786/518/DC1Materials and Methods SOM Text Figs.S1to S3Table S1References14March 2006;accepted 8June 200610.1126/science.1127422Fig.3.P2cleavage and transcriptional proofread-ing.(A )CECs and MECs [C-CEC (lanes 1and 2)and C(G)MEC (lanes 3to 12)are shown as exam-ples]were supplied with 15mM Mg 2þat pH 7.9(40-C)and incubated for various times with or without different concen-trations of CTP ,specified by the þ2register of template DNA and 1m M GreA.(B )Plots represent the relative amounts of MECs [A(U)MEC,C(G)MEC,G(C)MEC,and U(A)MEC are shown as examples]that incorporated NMP specified by the þ2reg-ister (white bars)and those that underwent P2cleavage (black bars)in an experiment similar to that shown in(A).REPORTS28JULY 2006VOL 313SCIENCE 520。