現(xiàn)代分子生物學(xué)之轉(zhuǎn)錄剪切培訓(xùn)資料

現(xiàn)代分子生物學(xué)之轉(zhuǎn)錄剪切Intronnumberindifferentorganisms2SequenceswithintheRNAdeterminewheresplicingoccursTHECHEMISTRYOFRNASPLICINGTheconsensussequencesforhuman5’

splicesite:

theexon-intronboundaryatthe5’endoftheintron3’splicesite:

theexon-intronboundaryatthe3’endoftheintronBranchpointsite:

anAclosetothe3’endoftheintron,followedbyapolypyrimidinetract(Pytract).3Theintronisremovedasa“Lariat”Twosuccessive

transesterification:Step1:TheOHoftheAatthebranchsiteattacksthephosphorylgroupoftheGinthe5’splicesite.--the5’exonisreleasedandthe5’-endoftheintronformsathree-wayjunctionstructure.Three-wayjunction

4Step2:TheOHofthe5’exonattacksthephosphorylgroupatthe3’splicesite.---------the5’and3’exonsarejoinedandtheintronisliberatedina“l(fā)ariat”.CanbeotherexonsTrans-splicing:twoexonscarriedondifferentRNAmoleculescanbesplicedtogether5Pre-mRNAsplicingiscarriedoutbyalargecomplex--spliceosomeSmallnuclearribonuclearproteins(snRNPs,

“snurps”).ThespliceosomeisthelargestsnRNP,andtheexactmakeupdiffersatdifferentstagesofthesplicingreactionThreerolesofsnRNPsinsplicing1.Recognizingthe5’splicesiteandthebranchsite.2.Bringingthosesitestogether.3.Catalyzing(orhelpingtocatalyze)theRNAcleavage.Mechanisms:RNA-RNA,RNA-protein

andprotein-proteininteractionsComplexesofRNAs(snRNA:smallnuclearRNA:U1,U2,U4-6,100-300nt,byRNAPOLIII)andproteins(~150proteins)6Functionsofinitiatorprotein:bindstoreplicator,distorts/unwindsaregionofDNAinteractswithandrecruitsadditionalreplicationfactorsNucleotideExcisionRepair:1.Recognitionofdamagedbase(s)2.Assemblyofmultiproteincomplexatthesite3.Cuttingofthedamagedstrandseveralnucleotidesupstreamanddownstreamofthedamagesiteandremovalofthenucleotides(~30)betweenthecuts4.UseoftheundamagedstrandasatemplateforDNApolymerasefollowedbystrandligationHowdotheymove:CommonfeaturesaboutCSSRandTransposition:Recombinasesrecognizeandbringspecificsitestogethertoformaprotein-DNAcomplexbridgingtheDNAsites:thesynapticcomplex.Withinthesynapticcomplex,therecombinasecatalyzesthecleavageandrejoiningoftheDNAmoleculeseithertoinvertaDNAsegmentortomoveasegmenttoanewsite.transpososome:Astableprotein-DNAcomplexpre-initiationcomplexintranscription:

Corepromoter+regulatoryelements+GTFs+

Mediatorcomplex+Transcriptionalregulatory

proteins

+

Nucleosome-modifyingenzymes+RNAPAreviewaboutDNA/RNA-proteininteractionSplicingpathway:

Assembly,rearrangement,and

catalysis

withinthespliceosome2.OnesubunitofU2AFbindstoPytract,andtheothertothe3’splicesite.TheformersubunitsinteractswithBBPandhelpsitbindtothebranchpoint.Assemblystep1:Early(E)complex

formsU1recognizes5’splicesite.

8Assemblystep2U2bindstothebranchsite,andAcomplexisformed,withbranchsiteAis

extruded.9Assemblystep31.

U4,U5andU6formthetri-snRNPParticle.2.Withtheentryofthetri-snRNP,theAcomplexisconvertedintotheBcomplex.10Assemblystep4U4leaves,allowingU6tointeractwithU2.Ccomplexforms.U6replacesU1atthe5’splicesiteCatalysisStep1:

U2andU6RNAsbeingbroughttogethertoproducestheactivesiteTransesterficationCatalysisStep2:U5helpstobringthetwoexonstogether,andaidsthesecondtransesterificationreaction11Asmallgroupofintronaresplicedbyminorspliceosome:AT-ACspliceosomeTheintronscontainAUat5’ss,andACatthe3’ss.Thechemicalpathwayisthesameasthemajorspliceosome,butU11andU12areusedinplacesofU1andU2,respectively.12GroupIintronsuseafreeG,toattackthe5’splicesiteSelf-splicingintronscanremovethemselvesfrompre-RNAsinabsenceofanyproteinsorotherRNAsSelf-splicingintrons

foldintoaspecificconformationwithintheprecursorRNA,andcatalyzethechemistryoftheirownreleaseandtheexonligation.13GroupIIintronsandU2-U6snRNAcomplexaresimilarinstructures,forprocessingthefirsttransesterification14SmallerthangroupIIintronsShareaconservedsecondarystructure,withan“internalguidesequence”

base-pairingwiththe5’splicesitesequenceintheupstreamexon.Theirtertiarystructurecontainsabindingpocketthatwillaccommodatetheguaninenucleotideornucleosidecofactor.FeaturesofgroupIintrons1516Howtomakethesplice-siterecognitionspecific?Challengeforexonidentification:Theaverageexonis150nt,andtheaverageintronisabout3,000nt(upto800,000nt).Thesplicesiteconsensus

sequenceareratherloose17WaystoavoidtheerrorsCo-transcriptionalloading

ofsplicingproteins

toRNAPII

C-terminaltailensuresallsplicesitesemergingarereadily

recognized,preventingexonskipping18SR(serinearginine-rich)

proteinsrecruitsplicingmachineryto

nearbysplicesites-----

the

sitesclosetoexonsarerecognizedpreferentiallySomeSRproteinsareexpressedpreferentiallyincertaincelltypesESEs:ExonicsplicingenhancersControlsplicingincell-typespecificpatterns19Alternativesplicingisachievedbyusingactivatorsandrepressors

undervariousconditionsMostrepressorsarehnRNP(heterogeneousnuclearribonucleoprotein).TheybindRNA,butlacktheRSdomainsActivatorsareSRproteins:withRNA-recognitionmotifandRSdomain(forinteractionswithsplicingmachinery)20DifferentwaysofalternativesplicingAlternativesplicing:somepre-mRNAscanbesplicedinmorethanoneway,generatingalternativemRNAs.75%ofthehumangenesaresplicedalternatively.21DrosophilaDSCAMgenecanbesplicedin38,000alternativewaysDownsyndromecelladhesionmolecule:Animmunoglobulinsuperfamilymember,encodesanaxonguidancereceptor

---OverexpressedinthebrainsofDownsyndromepatients,implicatedinmentalretardation.22JExpBiol.2011May1;214(Pt9):1523-32.

Bodyweight-dependenttroponinTalternativesplicingisevolutionarilyconservedfrominsectstomammalsandispartiallyimpairedinskeletalmuscleofobeseratsAmechanismthroughwhichskeletalmusclesmatchtheirperformanceandexperiencedloadAmJPhysiolCell

Physiol.2012Aug1;303(3):C298-307.

Cell-autonomousregulationoffasttroponinTpre-mRNAalternativesplicinginresponsetomechanicalstretch23AlternativesplicingcanbeeitherconstitutiveorregulatedConstitutive:morethanoneproductisalwaysmadefromapre-mRNARegulative:differentformsofmRNAatdifferenttime,underdifferentconditions,orindifferentcellortissuetypesAnexampleofCAS:

SplicingoftheSV40TantigenRNAsst:splicesiteforT/tmRNA24Theoutcomeofalternativesplicing

1.Producingmultipleproteinproducts---isoforms,withsimilar,distinctorantagonisticfunctions--------[Onegeneencodesmultiplefunctions]

2.Switchingonandofftheexpressionofagivengenethatencodesonlyonefunction.[Whentheexoncontainingastopisincludedtoproducenonfunctionalprotein,ortheintronisincludedtopreventmRNAtransport]

25RNAediting-----anotherwayofchangingthesequenceofanmRNAattheRNAlevelThehumanapolipoproteingene:ApoBApoB100ApoB4826SitespecificdeaminationTheprocessoccursonlyincertaintissuesorcelltypes,inaregulatedmanner.Adenosinedeamination:Theenzyme

ADAR(adenosinedeaminaseactingonRNA)convertAintoInosine.Insonecanbase-pairwithC,andthischangecanalterthesequenceoftheprotein27gRNAshavethreeregions:anchor