RNA和RNA組學(xué)RNomics 研究進(jìn)展 - 上海中醫(yī)藥大學(xué)

RNA和RNA組學(xué)（RNomics)

研究進(jìn)展1Reference:AnexpandinguniverseofnoncodingRNA.2002.

Science.296:1260-1263()GlimpsesofatinyRNAworld.2001.

Science.294:797-799.

Non-codingRNAs:thearchitectsofeukaryoticcomplexity.2001.EMBOReports.2(11):986-991.()2DNARNA蛋白質(zhì)基因組學(xué)RNA組學(xué)蛋白質(zhì)組學(xué)3蛋白質(zhì)編碼基因數(shù)量人：30,000果蠅和線蟲：12,000-14,000釀酒酵母：6,200假單孢菌：5,500人和鼠的蛋白質(zhì)編碼基因99%是共同的。人個體間單倍體基因組的堿基差異300萬個，其中1萬個（0.3%）出現(xiàn)在蛋白質(zhì)編碼基因中，且絕大多數(shù)存在于第3字母。98%轉(zhuǎn)錄物是非編碼蛋白RNA。2倍2倍4RNA的種類及主要功能RibosomalRNAs(rRNAs):translationTransferRNAs(tRNAs):translationMessengerRNAs(mRNAs):proteintemplateNoncodingRNAs(ncRNAs):varioustypesandfunctions5mRNA、tRNA、rRNA的功能6多聚核糖體的電鏡和示意圖7原核生物核糖體的大亞基和小亞基8原核和真核生物的核糖體9原核和真核生物核糖體的裝配10原核和真核生物的轉(zhuǎn)錄和翻譯過程11核糖體的解聚與重新裝配12蛋白質(zhì)的翻譯過程13翻譯過程中的轉(zhuǎn)位14翻譯過程的終止15基因的三聯(lián)密碼表16三聯(lián)密碼子按簡并性排列17tRNA中參與氨酰tRNA合成酶的識別因子18酵母丙氨酸t(yī)RNA的一級結(jié)構(gòu)19tRNA的氨?；磻?yīng)20tRNA的氨酰化反應(yīng)21TheCompleteAtomicStructureoftheLargeRibosomalSubunitat2.4

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ResolutionScience.2000.289:905-919.NenadBan,1*PoulNissen,1*JeffreyHansen,1PeterB.Moore,1,2ThomasA.Steitz1,2,3

1DepartmentofMolecularBiophysics&

Biochemistry,and

2DepartmentofChemistry,YaleUniversity,and

3HowardHughesMedicalInstitute,NewHaven,CT06520-8114,USA.

*

Thesetwoauthorscontributedequallytothiswork.

22核糖體的電子密度圖23核糖體大亞基旋轉(zhuǎn)花冠圖2423SrRNA的二級結(jié)構(gòu)25核糖體大亞基RNA的四級和二級結(jié)構(gòu)26核糖體大亞基RNA的四級和二級結(jié)構(gòu)（續(xù)）2723SrRNA的保守和擴(kuò)展序列28大亞基核糖體蛋白質(zhì)骨架結(jié)構(gòu)29核糖體大亞基表面的蛋白質(zhì)30蛋白質(zhì)延伸入RNA和多域的相互作用31TheStructuralBasisofRibosomeActivityinPeptideBondSynthesis

Science.2000.289:920-930.PoulNissen,1*JeffreyHansen,1*NenadBan,1*PeterB.Moore,1,2ThomasA.Steitz1,2,3

1DepartmentofMolecularBiophysicsandBiochemistryand

2DepartmentofChemistry,YaleUniversity,and

3HowardHughesMedicalInstitute,NewHaven,CT06520-8114,USA.

*

Theseauthorscontributedequallytothiswork.32肽基轉(zhuǎn)移酶底物和類似物的化學(xué)結(jié)構(gòu)33底物類似物的相位電子密度圖氧：紅磷：黃氮：蘭碳：綠底物：灰34A-和P-位點結(jié)合的組合模型3523S\5SrRNA、蛋白質(zhì)和組合CCA的填空模型36域5活性位點區(qū)的立體圖37TheclosestapproachofpolypeptidestothepeptidyltransferaseactivesitemarkedbytheYarusinhibitor,CCdA-p-Puro38ConservednucleotidesinthepeptidyltransferaseregionwithboundCCdA-p-Puro39肽基轉(zhuǎn)移酶活性位點的催化裝置40多肽的出口通道41CrystalStructureoftheRibosomeat5.5

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ResolutionScience.2001.292:883-896.MaratM.Yusupov,1*GulnaraZh.Yusupova,1*AlbionBaucom,1KateLieberman,1ThomasN.Earnest,2J.H.

D.Cate,3HarryF.Noller1

1CenterforMolecularBiologyofRNA,SinsheimerLaboratories,UniversityofCaliforniaatSantaCruz,SantaCruz,CA95064,

USA.

2BerkeleyCenterforStructuralBiology,PhysicalBiosciencesDivision,LawrenceBerkeleyNationalLaboratory,Berkeley,CA94720,

USA.

3WhiteheadInstitute,Cambridge,MA01242,

USA.

42ElectrondensityoftRNAMetfboundtothePsiteofthe70Sribosome,at5.5

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resolution4370S核糖體的結(jié)構(gòu)44Secondaryandtertiarystructuresof16S,23S,and5SrRNAs45ConformationaldifferencesbetweenrRNAsin70Sribosomesand30Sand50Ssubunits46亞基間橋47亞基間橋（續(xù)）48tRNA-ribosomeinteractions49TwoviewsoftheA-tRNAanticodonstemloopboundtoitscodoninthe30SsubunitAsite50InteractionofE-tRNAwiththeribosome.Secondarystructuresof16Sand23SrRNA,showingmolecularcontactswithA-tRNA(gold),P-tRNA(orange),andE-tRNA(red)51StructuralBasisofTranscription:RNAPolymeraseIIat2.8

?ngstromResolutionScience.2001.292:1863-1876.PatrickCramer,*DavidA.Bushnell,RogerD.Kornberg

DepartmentofStructuralBiology,StanfordUniversitySchoolofMedicine,Stanford,CA94305-5126,USA.

52RefinedPolIIstructure53StructureofRpb154(AtoD)StructureofRpb255StructureandlocationoftheRpb3/10/11/12subassembly56StructureandlocationofRpb5,Rpb6,Rpb8,andRpb957Surfacechargedistributionandfactorbindingsites58FourmobilemodulesofthePolIIstructure59Activecenter.StereoviewfromtheRpb2sidetowardtheclamp60RNAexitandRpb1COOH-terminalrepeatdomain(CTD)61ProposedpathforstraightDNAinaninitiationcomplex62SequenceidentitybetweenRNApolymerases63AconservedRNApolymerasecorestructure64非編碼RNA的結(jié)構(gòu)與功能65NoncodingRNAs(ncRNAs)ineukaryotesSmallRNA(sRNA)inbacteriaSmallnuclearRNAs(snRNAs)SmallnucleolarRNAs(snoRNAs)MicroRNA(miRNA)SmalltemporalRNAs(stRNAs)SmallinterferingRNAs(siRNAs)RNAinterference(RNAi)GuideRNAs(gRNAs)tRNA/mRNA:tmRNA非編碼RNA的名稱與縮寫符號66克隆ncRNA的方法計算機(jī)分析法基因克隆法蛋白質(zhì)-RNA分離法67計算機(jī)分析法大致過程：利用計算機(jī)軟件從已知基因組序列中尋找基因間區(qū)中的snoRNA序列，然后模擬其高級結(jié)構(gòu)，設(shè)計引物擴(kuò)增其序列和克隆，或人工合成其序列，測定其功能。存在的問題：未分析mRNA、rRNA和蛋白質(zhì)編碼基因中的反義鏈。68小RNA的克隆和鑒定小鼠腦組織勻漿總RNA8%PAGE回收50-110nt(fractionI)和110-500nt(fractionII)Poly(A)聚合酶加C尾cDNApSPORT1PCR高密度陣列雜交除去高豐度、已知的小RNA未雜交的cDNA測序詳見：存在問題：基因的特異時空和瞬時表達(dá)69蛋白質(zhì)-RNA復(fù)合物分離法分離蛋白質(zhì)-RNA復(fù)合物，除去蛋白質(zhì)，純化RNA分子，反轉(zhuǎn)錄成cDNA，克隆，測序，結(jié)果分析。所得RNA的cDNA克隆必須進(jìn)行Northern雜交，以確認(rèn)其轉(zhuǎn)錄物大小是否相符。70NoncodingRNAfunctionsandinvolvinginprocesses71ncRNA(red)的各種作用方式直接配對模擬其他核酸結(jié)構(gòu)形成RNA-蛋白質(zhì)復(fù)合物72Comparisonoftheprokaryoticandproposedeukaryoticgeneticoperatingsystems73eRNA在真核生物網(wǎng)狀系統(tǒng)中的作用74RNomics:anexperimentalapproachthatidentifies201candidatesfornovel,small,non-messengerRNAsinmouse.TheEMBOJ.200120(11):2943-2953AlexanderHüttenhofer1,MartinKiefmann,SebastianMeier-Ewert2,3,JohnO’Brien2,4,HansLehrach2,Jean-PierreBachellerie1,5andJürgenBrosius1

InstituteofExperimentalPathology/MolecularNeurobiology,ZMBE,48149Münster,2Max-Planck-InstituteofMolecularGenetics,14195Berlin-Dahlem,Germanyand5LaboratoiredeBiologieMoléculaireEucaryoteduCNRS,UniversitéPaul-Sabatier,31062Toulouse,France3Presentaddress:GPCBiotechAG,82152Plannegg-Martinsried,Germany4Presentaddress:DepartmentofClinicalPharmacology,RCSI,Dublin2,Ireland1Correspondingauthorse-mail:,or75測序與結(jié)果分析先從2個cDNA文庫中隨機(jī)測序400個克隆用BLASTN進(jìn)行數(shù)據(jù)分析和歸類從富集后的cDNA文庫中再測序2500個克隆76隨機(jī)測序的cDNA類別和比例77snoRNA的類別和功能C/D盒snoRNAs:引導(dǎo)rRNA甲基化C/D盒snoRNAs:引導(dǎo)snRNA甲基化C/D盒snoRNAs:未鑒定目標(biāo)H/ACA盒snoRNAs:引導(dǎo)rRNA假尿嘧啶化H/ACA盒snoRNAs:引導(dǎo)snRNA假尿嘧啶化H/ACA盒snoRNAs:未鑒定目標(biāo)H/ACA盒和C/D盒snoRNAs:腦特異性，未鑒定目標(biāo)位于mRNA編碼區(qū)內(nèi)的RNAs位于mRNA的5’-或3’-端非編碼區(qū)的RNAs類似重復(fù)因子的RNAs未知序列和結(jié)構(gòu)的snmRNAs78snoRNAs的引導(dǎo)(A)和產(chǎn)生(B)79鼠H/ACAsnoRNA與鼠rRNA(A)或snRNA間的堿基配對80IdentificationofNovelGenesCodingforSmallExpressedRNAs

Science2001294:853-858MarianaLagos-Quintana,ReinhardRauhut,WinfriedLendeckel,ThomasTuschl*

DepartmentofCellularBiochemistry,MaxPlanckInstituteforBiophysicalChemistry,AmFassberg11,

D-37077G?ttingen,Germany.81SlicinganddicingmiRNAs82ExpressionofmiRNAsRepresentativeexamplesofNorthernblotanalysisaredepicted83GenomicorganizationofmiRNAgeneclusters84PredictedprecursorstructuresofD.

melanogaster

miRNAs85PredictedprecursorstructuresofhumanmiRNAs86miRNAsE,embryo;L,larvalstage;P,pupa;A,adult;S2,Schneider2cells87HumanmiRNAs88AnAbundantClassofTinyRNAswithProbableRegulatoryRolesinCaenorhabditiselegans

Science2001294:858-862NelsonC.Lau,LeeP.Lim,EarlG.Weinstein,DavidP.Bartel*WhiteheadInstituteforBiomedicalResearch,andDepartmentofBiology,MassachusettsInstituteofTechnology,9

CambridgeCenter,Cambridge,MA02142,

USA.

89Fold-backsecondarystructuresinvolvingmiRNAs(red)andtheirflankingsequences(black)90UniquesequencefeaturesofthemiRNAs91miRNAclonedFrom92ExpressionofnewlyfoundmiRNAsandlet-7RNAduringC.

elegansdevelopment93AnExte