环状RNAPPT课件

基因组 暗物质 环状RNA 2014 10 08Group1 1953年 克里克和沃森提出中心法则 唯一以法则命名的生物学理论 核酶 1989年诺贝尔化学奖美国科学家切赫加拿大科学家奥尔特曼 RNAi 2006年诺贝尔医学奖安德鲁 法尔克雷格 梅洛 几乎所有的RNA都是线性的 为数不多的关于植物和动物中的环状RNAs的记述 遗传意外 实验人为因素 环形RNA分子 20世纪70年拟病毒也称为类类病毒 它是一种环状单链RNA 它的侵染对象是植物病毒 绒毛烟 苜蓿 茛菪 地下三叶草 1 1979年 Hsu和Coca Prados利用电子显微镜第一次观察到RNA可以以环状的形式存在于真核细胞的细胞质中 2 Arnberg等在酵母的线粒体中又发现了circRNA3 1993年 人们在人体细胞的转录本中也发现了一些由外显子构成的circRNA 外显子转录本发生错误剪接而形成的低丰度RNA分子 Jeck等在人类成纤维细胞中检测出了高达25000多种的circRNA Memczak等通过RNA seq鉴定出1950种人类circRNA 1903种小鼠circRNA 其中81种与人类circRNA相同 和724种线虫circRNA 线性RNAs的优势有可能一直是假象 经典的RNA测序方法 具有特征性分子 尾巴 的那些分子 环状RNAs的末端连接在一起 缺乏这些尾巴 因此被普遍忽略掉 2012年 斯坦福大学医学院的分子生物学家JuliaSalzman和同事们他们报道称在寻找用传统方法可能忽视的RNA过程中 发现了过量的环状人类RNAs 并进行了研究 2013年 Rajewsky研究小组环状RNA充当分子 海绵 结合并封闭了称作microRNAs的微小基因调控子 并且在斑马鱼中表达这一环状RNA或敲除miR 7可以改变大脑发育 2013年 丹麦奥胡斯大学的ThomasHansen和JorgenKjems1500个核苷酸构成的一个环状大RNA上包含了70个miR 7的结合位点 MicroRNAs是一些通过结合和阻止mRNA翻译阻断基因表达的短片段RNA 2014年 WilliamRJeck1和NormanESharpless系统检测 识别circRNA的方法 全新的对环形RNA进行研究的方法与认识 DetectingandcharacterizingcircularRNAs circRNAs与正常RNAs竞争 机体还以牺牲正常RNA为代价来生成它们 在大脑中环状RNA分子以高水平生成 在许多情况下来自具有非常重要功能的一些基因 circRNAs在脑功能以及有可能在脑疾病中发挥重要作用 与阿尔兹海默发生相关 muscleblind可以促进和调控一组环状RNAs的生成 表明circRNAs有可能参与了营养不良性肌强直的形成 27February2013NaturalRNAcirclesfunctionasefficientmicroRNAspongesThomasB Hansen TrineI Jensen BettinaH Clausen JesperB Bramsen BenteFinsen etal Nature495 384 388doi 10 1038 nature1199327February2013CircularRNAsarealargeclassofanimalRNAswithregulatorypotencySebastianMemczak MarvinJens AntigoniElefsinioti FrancescaTorti JannaKrueger etal Nature495 333 338doi 10 1038 nature1192827February2013Molecularbiology CirclesreshapetheRNAworldKennethS KosikNature495 322 324doi 10 1038 nature11956 Rajewsky研究小组在斑马鱼中表达这一环状RNA或敲除miR 7可以改变大脑发育 环状RNA充当分子 海绵 结合并封闭了称作microRNAs的微小基因调控子 ToidentifycircRNAs designedanalgorithm ToidentifycircRNAs designedanalgorithm IdentifythatcircRNAsarenotjustrareandspecificallyexpressed SequenceconservationwithincircRNAs qPCRtestcircRNA DigestwithRNaseR 24hafterblockingtranscriptioncircRNAswerehighlystable exceedingthestabilityofthehousekeepinggeneGAPDH circRNAswereidentifiedanditscharacterization NotrareSpecifically spatialandtemporal CDSexonsConservationRNaseRresistantstable CancircularRNAbeamiRNAsponge ThecircRNACDR1asisboundbythemiRNAproteinAGO ThecircRNACDR1asiscytoplasmicandhighlyexpressed 每个细胞中CDR1as可能最多结合20 000个miR 7分子 蓝色 种子匹配 暗红 AGOPAR CLIP阅读 siRNAdepletionofCDR1asinducesrepressionofmiR 7targetgenes CDR1asandmiR 7haveoverlappingandspecificexpressioninneuronaltissues KnockdownofmiR 7expressionofCDR1ascausesmidbraindefects ExpressionofCDR1ascausesmidbraindefects KnockdownofmiR 7orexpressionofCDR1ascausesmidbraindefects ABOUTAUTHORS SebastianMemczakNikolausRajewsky computationalanalysisofanimalsmallRNAdeepsequencingdatafocusingontheidentificationofmiRNAsandtheirtargetgenescomputationallydetectfunctionalsmallpeptides socalled micropeptides inflies ScientificHeadofthe BerlinInstituteforMedicalSystemsBiology SystemsBiologyofGeneRegulatoryElements Max Delbru ck CenterforMolecularMedicine Robert Ro ssle Strasse10 13125Berlin GermanyTheRajewskylabcombinestheoretical computationalandexperimentalmethodstounderstandmoreaboutgeneregulationinanimalsAmajorfocusisonpost transcriptionalgeneregulationbysmallRNAs forexamplemicroRNAs andRNAbindingproteins DISCUSSION CDR1ascanactasapost transcriptionalregulatorbybindingmiR 7inbraintissues CDR1asisdenselyboundbymiRNAeffectormoleculesCDR1asharbours74miR 7seedmatches oftendeeplyconservedCDR1asisexpressedhighly stablyandmostlycytoplasmicCDR1asandmiR 7sharespecificexpressiondomainsinmouseembryonicbrainhuman mouseCDR1asiscircularizedinvivoandisnotdetectableasalinearmoleculehuman mouseCDR1assequences wheninjectedintozebrafish andmiR 7knockdownhavesimilarphenotypesinbrain FuturestudiesshouldelucidatehowCDR1ascanbeconvertedintoalinearmoleculeandtargetedfordegradationCDR1asmiR 671miR 7PAK1 FAK1 ThephenotypeinducedbyCDR1asexpressioninzebrafishwasonlypartiallyrescuedbyexpressingmiR 7 indicatingthatCDR1ascouldhavefunctionsbeyondsequesteringmiR 7bindintrans3 UTRsoftargetmRNAsmiR 7bindsCDR1asassemblyoflargercomplexesofRNAorprotein HowmanyothercircRNAsexist certainlymuchlargerafewtissues developmentalstageswithstingentcutoffscircularRNAsinfibroblastsweredescribed circRNAsprobablycompetewithotherRNAsformiRNAbindingcircRNAfromtheSRYlocushasseedsitesformurinemiRNAs Itisappealingtospeculatethatoccasionalcircu larizationofexonsiseasytoevolveandmayprovideamechanismforrapidevolutionofstablyandwellexpressedregulatoryRNAs ThereisnoreasontothinkthatcircRNAsfunctionpredominantlytobindingmiRNAsseedmatchesforviralmiRNAswithinhumancircRNAsRBPsmiRNAspongesinbacteria SupplementaryReading High throughputsequencing RNA seq librariespreparedfromribosome depletedRNAIdentified 25 000distinctRNAspeciesinhumanfibroblaststhatcontainednoncolinearexons a backsplice reproduciblyenrichedbyexonucleasedegradationoflinearRNA circularRNA ecircRNA ratherthanlinearRNACircularRNA ecircRNA morestablethanassociatedlinearmRNAsinvivo theabundanceofcircularmoleculesexceededthatofassociatedlinearmRNAby 10 foldecircRNAswerenotboundtoribosomesdegradedbysiRNAs mayactascompetingendogenousRNAs ApplicationofthismethodtomurinetestisRNAidentified69ecircRNAsinpreciselyorthologouslocationstohumancircularRNAs paralogouskinasesHIPK2andHIPK3produceabundantecircRNAfromtheirsecondexoninbothhumansandmice ThatecircRNAsareabundant stable conservedandnonrandomproductsofRNAsplicingthatcouldbeinvolvedincontrolofgeneexpression Abstract Introduction Purpose BiochemicalenrichmentofnonlinearRNAs detectionofmorerareanddiversecircularRNAforms Hs68cells RNaseR Preference ContinuousmappingSplicedmappingsFusionmappings TreatmentwithRNaseRDecreasedcoverageoflinearproducts Enrichmentofreadsfromexonsincludedincircularproducts increasedreadsmappingasbacksplicejunctions UnbiasedidentificationofRNAcircles MappingartifactsNonsequentialexonsharboredinlinearproducts resultingfromeitherRNAtrans splicingorcleavageofecircRNAs EnrichmentofcircularRNAsbyCircleSeq SingleexonecircRNAs TheinterveningexonsnotdirectlypartofthebacksplicealsoshowedenrichmentbyRNaseRIntronsaresplicedfrommostcircularforms Thatbacksplice containingtranscriptsidentifiedbythismethodarediverse generallyRNaseR resistantandincludemostpreviouslydescribedcircularRNAs CircularRNAscontainpredominantlyexonicsequence Abundence Therelativeraterangingfrom3200 Intronsaresplicedfrommostcircularforms Indicate TheformationofcircularRNAsisconsiderable RareecircRNAsarisingfrompervasivebackgroundlevelsofRNAcircularization occasionalerrorinsplicing NovelbackspliceeventsANRIL14 5 ASXL1 FOXN2 HIPK3 KIAAO182 LPRAR1 MYO9B ZFY EnrichmentLinearRNASTBP GAPDH 18S Decreased circularRNA asubsetofexonsTrans splicedproducts repeatedexons longerthanfull length Characters 1 containbacksplices 2 areenrichedbyRNaseRtreatment 3 arenotpolyadenylated and 4 areofsmallersizethanlinearmRNAsemanatingfromthesamelocusEcircRNAsresultingfromcis ratherthantrans splicing CircularRNAvalidation SingleexonecircRNAs TheinterveningexonsnotdirectlypartofthebacksplicealsoshowedenrichmentbyRNaseRIntronsaresplicedfrommostcircularforms Thatbacksplice containingtranscriptsidentifiedbythismethodarediverse generallyRNaseR resistantandincludemostpreviouslydescribedcircularRNAs Lariatdetectionandbranchpointmapping Indicate CircleSeqapproachcanalsoidentifylariatbranchpointsasevidencedby3 taildegradationandbranchpointspanningreads Lariats CreatedbyRNAsplicingContainsignificantintronicsequenceInvolvea2 5 phosphodiesterlinkageatabranchpoint Don thaveenhancement Inefficienttraversal2 5 junctionsbyreversetranscriptase Abundence Therelativeraterangingfrom3200 Intronsaresplicedfrommostcircularforms Indicate TheformationofcircularRNAsisconsiderable ConservationofcircularRNAproductioninparalogousgenesorthologousgenesIndicatesEvolutionarypreservationofcircularRNAformation Conservationofabundantcircularizedtranscripts RepresentativeecircRNAsUntranslatedTargetedbysiRNA EcircRNAscanbetargetedbyRNAinterference CircularRNAsmightregulatetranscriptionthroughaneffectonmicroRNAbinding RepresentativeecircRNAsareuntranslatedbutcanbetargetedbysiRNA Method TreatedwithactinomycinDMeasurement Half life Datasets thecircularRNAisoformswerehighlystable withtranscripthalf livesexceeding48h Associatedlineartranscriptsexhibitedhalf livesof 20hCircularformswerepreferentiallylocalizedinthecytoplasm ResultsEcircRNAseitherundergonuclearexport releasedtothecytoplasmExtraordinarystability resistancetodebranchingenzymesandRNAexonucleases ecircRNAsarepredominantlycytop