分子生物学Chapter 9 RNA Processing.docx

分子生物学Chapter 9 RNA Processing.docx

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分子生物学Chapter9RNAProcessing

Chapter9RNAProcessing

Introduction

VeryfewRNAmoleculesaretranscribeddirectlyintothefinalmatureRNA.

vMostnewlytranscribedRNAmolecules（primarytranscripts）undergovariousalterationstoyieldthematureproduct

vRNAprocessingisthecollectivetermusedtodescribethemoleculareventsallowingtheprimarytranscriptstobecomethematureRNA.

1.BasicFeaturesofRNAProcessing

vProkaryoticRNA

2-5'halflife;polycistronic;seldommodified;usedimmediately

Coupledtranscriptionandtranslation;manyribosomesbindsameoperon,andsynthesizepolypeptidesimultaneously...calledpolyribosomesorpolysomesforshort.

PolycistronicmRNAs:

coordinateexpressionof>1gene:

operon

MultipletranslationalstartsitespermRNA

 EukaryoticRNA:

vlonghalflife;monocistronic;alwaysmodified;transporttocytoplasm

vMostpost-transcriptionmodificationsoftheprimarytranscriptoccurinnucleus,beforetransporttocytoplasm.

vthesemodificationsconvertthetranscript（HeterogeneousNuclearRNAhnRNA）intomRNA

1.1MajorEukaryoticModificationsevents

vGcappingof5'endofpre-mRNA

CleavageandPoly（A）additionto3'end

Splicingout（removalof）Introns

Somemethylationofbases

1.1.1.Poly（A）on3'end

vTranscriptsarecleavedat3'endpastahighlyconservedAAUAAAsequence.

SuchCleavageinvolvessomeproteincofactors

vThePoly（A）polymerase（PAP）adds~200AresiduestomostmRNAs

vPoly（A）-BindingProtein（PABP）bindstothepoly（A）stoichiometrically,oneproteinevery10-20bases...increasesstabilityandenhancestranslation

vHistonemRNAsarepoly（A）-free.

1.1.2MethylatedGCapat5'end:

vGuanylyltransferasecatalyzesadditionofGTPin5'->5'direction,withreleaseofPPifromtheGTP,andreleaseofPifromtheterminaltriphosphateoftheRNAtranscript.

Thenoneof3methylCappingreactionsoccurs

vA）Cap0:

amethylgroupisaddedtotheG-7posn:

Guanine-7-MethylTransferase

PresentinalleukaryoticmRNA...nofurthercappinginunicelluareukaryotes

vB）Cap1:

amethylgroupisthenaddedtothe2'-OHoftheriboseofthe1stnucleotideintheoriginaltranscript:

2'-O-MethylTransferase

PresentinmosteukaryoticmRNA...ifbaseisA,ittoocanbemethylated

vC）Cap2:

asimilarmethylationofthe2ndnucleotideoftheoriginaltranscript

Presentinabout10-15%oftotalcappedpopulation.

Capat5'end

1.1.3.InternalMethylation:

vN-6methylationofAdenines,atfrequencyofabout0.1%

1.2.EukaryoticRNASplicing:

IntronsandExons

vEukaryoticgenesareofteninterruptedgenes:

Codingsequenceisinterruptedbynoncodingsequences.

vRemovaloftheIntronsinRNAtranscriptmodificationiscalledRNAsplicing

vExonsareExpressedsequences:

thesesequencesarethosepresentinmaturemRNA

vIntronsareInterVeningSequences（IVS）,sequencesfoundbetweenExonsequencesinthegenomicDNA,i.e.inthegene

Chapter9RNAProcessing

2.NuclearRNAsplicing

vRNAsplicingoccursinthenucleus,ontheinitialtranscripts（pre-mRNAorhnRNA）complexedwiththenuclearproteins:

hnRNPparticles.

2.1.GU-AGrule

vGUalwaysat5'endofintron:

Donorsite:

AG|GUAAGU

AGalwaysat3'endofintron:

Acceptorsite:

（Py...Py）12NCAG|N

2.2.Spliceosomes

vSpliceosomesarecomposedofSplicingFactorproteins（~40）plussmallnuclearRNAs（snRNA）foundinnucleoprotein（~10proteins）complexes...50-60Ssize...

TheseribonucleoproteincomplexesarecalledsnRNPparticles,orsnurps

snRNAs.

vThesnRNPsareU1,U2,U5,andU4/U6,containingU1,U2,U4,U5,U6snRNAs.

FunctionalrolesforeachsnRNP:

snRNPU1:

v

vStructure:

4stem-loopDomains（A,B,C,D）

Sm-bindingsite:

non-pairedregionbetweenDomainsCandB-bindingofsnRNPproteins

5'non-pairedend:

H-bondstoDonorsiteofpre-mRNA...

Thispairingisessentialforsplicing;somepairingsitesmoreimportantthanothers

vU1-initiallyboundto5’-splicesite

v-isreleaseduponrecruitmentofU4/U5/U6

v（determineswhich5’-splicesiteisused?

）

U1SnRNA

vMutationsthatabolishfunctionofthe5’splicingsitecanbesuppressedbycompensatingmutationsinsnRNAthatrestorebasepairing

U2snRNA

v-initiallybindsthebranchpointrecognitionsequence

v-formstwoduplexeswithU6,bringingtheintron5’splicesiteclosetothebranchpoin

U4RNA

v-initiallycomplexedwithU5andU6

v-keepsU6inanunfoldedconformation

v-isreleasedafterdeliveringU6tothe5’-splicesite

v（U6chaperone?

）

U5RNA

v-initiallycomplexedwithU4andU6

v-bindsexonsequencesupstreamofthe5’-splicesite

vanddownstreamofthe3’-splicesite

v（guidesequenceforholdingexonstogether?

）

U6RNA

v-initiallycomplexedwithU4andU5

v-displacesU1from5’-splicesite,formingduplexwithintronsequences

v-complexeswithU2,bringingtheintron5’-splicesiteclosetothebranchpoint.

v（catalyticactivity?

）

vU6-U4pairingisincompatiblewithU6-U2pairing.WhenU6joinsthespliceosomeitispairedwithU4.ReleaseofU4allowsaconformationalchangeinU6;onepartofthereleasedsequenceformsahairpin,andtheotherpartpairswithU2.BecauseanadjacentregionofU2isalreadypairedwiththebranchsite,thisbringsU6intojuxtapositionwiththebranch.

2.3SplicingReaction:

vStage1:

DonorSiteiscut,5'endiscovalentlyattachedtoaBranchsiteintheIntronina5'-2'phosphodiesterbondformingaLariatIntermediate.

vStage2:

AcceptorSiteiscut,exonsarejoined,intronisreleasedastheLariat

vStage3:

TheLariatis"debranched"anddegraded.

Splicingofexonsinpre-mRNAoccursviatwotransesterificationreactions

vOH-nucleophilicattackon5'-Pofintron:

1sttransesterificationreaction

vExonA3'-OHnucleophilicattackon3'-Pofintron:

2ndtransesterificationreaction

vExonsjoined;Intronexcised,eitheraslinearRNAorasbranchedlariatstructure

2.4RoleofsnRNPparticlesinthe3Stages

（1）

v1）.U1snRNPbindstoDonorsite...

v2）U2snRNPH-bondstoBranchsiteofIntron,usingATPhydrolysisandinvolvingasnRNP"auxilliaryfactor"（U2AF）:

CommitmenttoSplicing.

v3）U1snRNPinteractswithU2snRNP,bringingDonorsiteandBranchsitetogether...

RoleofsnRNPparticlesinthe3Stages

（2）

v4）U5/U4/U6trimercomplexnowbindstoU1andU2,withATPhydrolysis;U5bindstheintronDonorSite;U6bindsU2:

ThisisthecompleteSpliceosome.

ATPprovidesenergyineachofthesereactionsforconformationalchanges...

U4interactswithU6viaH-bonds.

2.4RoleofsnRNPparticlesinthe3Stages（3）

v5）U5shiftspositionfromtheintronparttotheexonpartofDonorsite,andU2H-bondswithU6,displacingU4fromitsinteractionwithU6

1stTransesterificationreactionnowoccurs;enzymemaybetheU2-U6complex

v6）U5snRNPnowinteractswiththeintronAcceptorsite,aswellaswithU2,catalyzingcuttingattheintron-exonjunctionandjoiningofthetwoExons

2ndTransesterificationReaction

2.4RoleofsnRNPparticlesinthe3Stages（4）

v7）Spliceosomeisnowdisassembled,withreleaseofU1,U2,U4,U5,U6,joinedexons,andintronLariat

v8）IntronLariatisDebranchedanddegraded

TheRNAsplicingmechanism.

vThesplicingreactionproceedsthroughdiscretestagesinwhichspliceosomeformationinvolvestheinteractionofcomponentsthatrecognizetheconsensussequences.

Chapter9RNAProcessing

3.GroupIIIntrons

vfungalmitochondrialintrons...fairlyuncommon.

vSimilartonuclearintrons:

GU-AGrule;splicingviaaLariatintermediate;thetwotransesterificationreactionsaresimilar:

...BUT...IntronisSelf-Splicing.

v

vThreeclassesofsplicingreactionsproceedbytwotransesterifications.

SchematicdiagramscomparingthesecondarystructuresofgroupIIself-splicingintrons（a）andUsnRNAspresentinthespliceosome（b）.

vBranchsiteA-OH->DonorsiteG;DonorsiteG-OH->AcceptorsiteA

vDomains5and6inRNA2·structurearesimilartoU2-U6andU2-introninteractions;thisprovidestheactivesitefortheRNAcatalyticactivity.

v

ＷＨＹ：

vNuclearsplicingandgroupIIsplicinginvolvetheformationofsimilarsecondarystructures.Thesequencesaremorespecificinnuclearsplicing;groupIIsplicingusespositionsthatmaybeoccupiedbyeitherpurine（R）oreitherpyrimidine（Y）.

Chapter9RNAProcessing

4.rRNAProcessing

vGroupIIntrons

vFoundinrRNAgenesoflowereukaryotesandinfungalmitochondrialgenes

AlsofoundinthreegenesinE.coliphageT4!

!

!

...theonlyexampleofprokaryoticintrons.

vTwocommonproperties:

1.Self-Splicing:

abilityoftheRNAtospliceitself...intramolecular...RNAcatalyst

2.CharacteristicSecondaryStructureof9stem-loops

SplicingmechanismsingroupIandgroupIIself-splicingintronsandspliceosome-catalyzedsplicingofpre-mRNA

4.1.TetrahymenarRNA

vPrecursor:

35Sprocessed,asabove,into->18S,5.8S,28SrRNA

28Shasone400baseIntroninsomeTetrahymenastrains:

ThisIntroncanSelf-Splice;requiresionsandaG-nuc（GTP,GDP,GMP,GR）

4.2.Self-SplicingReaction

1）G3'-OHattacks5'endofIntron

2）3'-OHofExonAattacks5'endofExonB:

exonsjoin

3）LinearIntron,withG（414）at5'end,nowcircularizes

2）

4.3CircularizationoftheLinearIntron:

v1）3'-OHofIntron,onG（414）,DIFFERENTfromthethefreeGabove,attackspA（16）orpU（20）,yieldingcyclizedIntronandsplittingoff5'end15or19nucsfromtheIntron

v2）WaterhydrolyzesthecyclizedIntronatSAMEposition

v3）IfFirstcyclizationwereatpA（16）,then3-OHofIntronattackspU（20）,yieldingcyclizedIntronandsp