RNA和RNA组学RNomics 研究进展 - 上海中医药大学

RNA和RNA组学（RNomics)

研究进展1Reference:AnexpandinguniverseofnoncodingRNA.2002.

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

Science.294:797-799.

Non-codingRNAs:thearchitectsofeukaryoticcomplexity.2001.EMBOReports.2(11):986-991.()2DNARNA蛋白质基因组学RNA组学蛋白质组学3蛋白质编码基因数量人：30,000果蝇和线虫：12,000-14,000酿酒酵母：6,200假单孢菌：5,500人和鼠的蛋白质编码基因99%是共同的。人个体间单倍体基因组的碱基差异300万个，其中1万个（0.3%）出现在蛋白质编码基因中，且绝大多数存在于第3字母。98%转录物是非编码蛋白RNA。2倍2倍4RNA的种类及主要功能RibosomalRNAs(rRNAs):translationTransferRNAs(tRNAs):translationMessengerRNAs(mRNAs):proteintemplateNoncodingRNAs(ncRNAs):varioustypesandfunctions5mRNA、tRNA、rRNA的功能6多聚核糖体的电镜和示意图7原核生物核糖体的大亚基和小亚基8原核和真核生物的核糖体9原核和真核生物核糖体的装配10原核和真核生物的转录和翻译过程11核糖体的解聚与重新装配12蛋白质的翻译过程13翻译过程中的转位14翻译过程的终止15基因的三联密码表16三联密码子按简并性排列17tRNA中参与氨酰tRNA合成酶的识别因子18酵母丙氨酸tRNA的一级结构19tRNA的氨酰化反应20tRNA的氨酰化反应21TheCompleteAtomicStructureoftheLargeRibosomalSubunitat2.4

Å

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核糖体的电子密度图23核糖体大亚基旋转花冠图2423SrRNA的二级结构25核糖体大亚基RNA的四级和二级结构26核糖体大亚基RNA的四级和二级结构（续）2723SrRNA的保守和扩展序列28大亚基核糖体蛋白质骨架结构29核糖体大亚基表面的蛋白质30蛋白质延伸入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肽基转移酶底物和类似物的化学结构33底物类似物的相位电子密度图氧：红磷：黄氮：兰碳：绿底物：灰34A-和P-位点结合的组合模型3523S\5SrRNA、蛋白质和组合CCA的填空模型36域5活性位点区的立体图37TheclosestapproachofpolypeptidestothepeptidyltransferaseactivesitemarkedbytheYarusinhibitor,CCdA-p-Puro38ConservednucleotidesinthepeptidyltransferaseregionwithboundCCdA-p-Puro39肽基转移酶活性位点的催化装置40多肽的出口通道41CrystalStructureoftheRibosomeat5.5

Å

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

Å

resolution4370S核糖体的结构44Secondaryandtertiarystructuresof16S,23S,and5SrRNAs45ConformationaldifferencesbetweenrRNAsin70Sribosomesand30Sand50Ssubunits46亚基间桥47亚基间桥（续）48tRNA-ribosomeinteractions49TwoviewsoftheA-tRNAanticodonstemloopboundtoitscodoninthe30SsubunitAsite50InteractionofE-tRNAwiththeribosome.Secondarystructuresof16Sand23SrRNA,showingmolecularcontactswithA-tRNA(gold),P-tRNA(orange),andE-tRNA(red)51StructuralBasisofTranscription:RNAPolymeraseIIat2.8

ÅngstromResolutionScience.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非编码RNA的结构与功能65NoncodingRNAs(ncRNAs)ineukaryotesSmallRNA(sRNA)inbacteriaSmallnuclearRNAs(snRNAs)SmallnucleolarRNAs(snoRNAs)MicroRNA(miRNA)SmalltemporalRNAs(stRNAs)SmallinterferingRNAs(siRNAs)RNAinterference(RNAi)GuideRNAs(gRNAs)tRNA/mRNA:tmRNA非编码RNA的名称与缩写符号66克隆ncRNA的方法计算机分析法基因克隆法蛋白质-RNA分离法67计算机分析法大致过程：利用计算机软件从已知基因组序列中寻找基因间区中的snoRNA序列，然后模拟其高级结构，设计引物扩增其序列和克隆，或人工合成其序列，测定其功能。存在的问题：未分析mRNA、rRNA和蛋白质编码基因中的反义链。68小RNA的克隆和鉴定小鼠脑组织匀浆总RNA8%PAGE回收50-110nt(fractionI)和110-500nt(fractionII)Poly(A)聚合酶加C尾cDNApSPORT1PCR高密度阵列杂交除去高丰度、已知的小RNA未杂交的cDNA测序详见：存在问题：基因的特异时空和瞬时表达69蛋白质-RNA复合物分离法分离蛋白质-RNA复合物，除去蛋白质，纯化RNA分子，反转录成cDNA，克隆，测序，结果分析。所得RNA的cDNA克隆必须进行Northern杂交，以确认其转录物大小是否相符。70NoncodingRNAfunctionsandinvolvinginprocesses71ncRNA(red)的各种作用方式直接配对模拟其他核酸结构形成RNA-蛋白质复合物72Comparisonoftheprokaryoticandproposedeukaryoticgeneticoperatingsystems73eRNA在真核生物网状系统中的作用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测序与结果分析先从2个cDNA文库中随机测序400个克隆用BLASTN进行数据分析和归类从富集后的cDNA文库中再测序2500个克隆76随机测序的cDNA类别和比例77snoRNA的类别和功能C/D盒snoRNAs:引导rRNA甲基化C/D盒snoRNAs:引导snRNA甲基化C/D盒snoRNAs:未鉴定目标H/ACA盒snoRNAs:引导rRNA假尿嘧啶化H/ACA盒snoRNAs:引导snRNA假尿嘧啶化H/ACA盒snoRNAs:未鉴定目标H/ACA盒和C/D盒snoRNAs:脑特异性，未鉴定目标位于mRNA编码区内的RNAs位于mRNA的5’-或3’-端非编码区的RNAs类似重复因子的RNAs未知序列和结构的snmRNAs78snoRNAs的引导(A)和产生(B)79鼠H/ACAsnoRNA与鼠rRNA(A)或snRNA间的碱基配对80IdentificationofNovelGenesCodingforSmallExpressedRNAs

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

DepartmentofCellularBiochemistry,MaxPlanckInstituteforBiophysicalChemistry,AmFassberg11,

D-37077Göttingen,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