Human triplet repeat RNA-binding proteins: involvement in mRNA biogenesis and genetic disease

194 Pages·1998·6.2 MB·English

by  Miller, Jill Watkins

HUMANTRIPLETREPEATRNA-BINDINGPROTEINS:
INVOLVEMENTINmRNABIOGENESISANDGENETICDISEASE
By
JILLWATKINSMILLER
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1998
Copyright1998
by
JillWatkinsMiller
Thisworkisdedicatedtomyparents,JohnandRobbieWatkinsandtomyhusband,
DavidMiller,wholovemenomatterwhat.
ACKNOWLEGMENTS
Iwouldliketofirstthankmyhighschoolscienceteacher,Mr.Lavinghausez,for
encouragingmetopursuemyideasandforsettingintomotionalife-longloveofscience.
IthankPeterStacpoole,MaryJoKoroly,andNancyDenslowforseeingmypotentialand
givingmeaplacetobegin. Iwouldespeciallyliketothankmycommitteemembers,
JohnAris,HenryBaker,BertFlanegan,GerryShaw,andmyoutsideexaminer,Jack
Griffith,fortheirinterest,help,andguidance. TheadministrativestaffoftheDepartment
ofMolecularGeneticsandMicrobiologyhavebeentremendouslyhelpful. Iwouldlike
toespeciallythankBradleyMooreandJoyceConnersformakingsurethatthingswere
doneright. IwouldliketothankallofthemembersoftheSwansonlabfortheirhelpand
adviceandforbeingsuchafungroupwithwhichtowork. Iwouldespeciallyliketo
thankDrueDeVoreforhistremendouscontributiontotheproject. Mostofall,Iwould
liketothankMaurySwansonforhishelp,enthusiasm,mentorship,andforactingasa
fabulousrolemodel. Hiswisdom,skill,anddedicationaresomethingtowhichIaspire.
iv
TABLEOFCONTENTS
ACKNOWLEDGMENTS iv
ABSTRACT vii
INTRODUCTION 1
TripletRepeatExpansionDisorders 1
MechanismofExpansion 4
MyotonicDystrophy 8
ClinicalManifestations 8
Genetics 10
ModelsforDMPathogenesis 13
HaploinsufficiencyModel 13
ChromatinStructureModel 14
RNADominantMutationModel 16
DM2andPR0MM 17
NucleicAcidTripletRepeatStructures 18
NuclearPre-mRNAMetabolism. 20
Protein/RNARecognition 20
HeterogenousNuclearRibonucleoproteins:
StructureandFunction 23
hnRNPsinSaccharomycescerevisiae 25
Capping 26
Pre-mRNASplicing 27
AlternativePre-mRNASplicing 29
3'EndFormation 32
NuclearmRNAExport 39
MATERIALSANDMETHODS 46
MediaandCellCulture 46
^^S-LabelingofCells 47
PreparationofTotalCellularProteinsand
NuclearExtracts 48
CellTransformationandPlasmidRescue 49
IsolationofPINs 51
Quantitativep-GalactosidaseLiquidAssay 51
cDNALibraryScreening 52
PurificationofCUG-BPandBandshiftAnalysis 54
MonoclonalAntibodyPreparation 54
SDS-PAGEandImmunoblotAnalysis 55
V
IndirectCellularImmunofluorescence 55
CellFractionationandImmunoprecipitation
ofhnRNPComplexes 56
NucleicAcidMethods 57
RNABlotAnalysis 60
PlasmidConstructs 61
PhotocrosslinkingandImmunopurification 62
RESULTS 65
ProteinsthatInteractwithNab2pareInvolvedin
mRNAExportandPolyadenylation 65
CharacterizationofhNabSO:ANovelHumanhnRNP 72
SubcellularLocalizationofhNabSO 78
hNabSOisaPoly(A)"^RNA-BindingProtein 81
hNabSOisImmunologicallyConserved 85
-VariableSequencesFoundinhNab503'-UTR 88
hNab50istheCUGRepeatRNA-BindingProtein 91
CUG-BP/hNab50:Disease-AssociatedChangesinDM 93
hNab50isaDMPKTranscript-BindingProtein 96
AccumulationofMutantTranscriptsinDMCellsLines 99
Another(CUG)Repeat-ContainingmRNAisNotBound
byhNabSO 105
hNabSOShowsIncreasedBindingto
MutantDMPKTranscripts 108
IdentificationofTripletRepeatExpansion(EXP)
RNA-BindingProteins 113
StrategiesforIdentifyingtheEXPProteins 130
CandidateProteins 130
ExpressionScreening 134
RNAAffinityChromatography 135
PreparationofAnti-EXPAntibodies 136
DISCUSSION 139
RNADominantMutationModel 139
IsolationoftheFirstEukaryoticTriplet-Repeat
RNA-BindingProtein 141
TheExpansion(EXP)BindingProteins 153
ConclusionsandFutureStudies 157
REFERENCES 160
BIOGRAPHICALSKETCH 183
SIGNATUREPAGE 184
vi
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
HUMANTRIPLETREPEATRNA-BINDINGPROTEINS:INVOLVEMENTIN
mRNABIOGENESISANDGENETICDISEASE
By
JillWatkinsMiller
December,1998
Chairperson:Dr.MauriceS.Swanson » '
MajorDepartment:MolecularGeneticsandMicrobiology
Instabilityoftripletrepeatsequenceshasrecentlybeenrecognizedasthecauseof
anumberofneurological/neuromusculardiseases. Mostofthetripletrepeatexpansion
disorders(TREDs)aredominantlyinheritedandcanbeexplainedasa"gain-of-function"
mutationduetoanaberrantproteinproduct. Myotonicdystrophy(DM),whichalso
showsautosomaldominantinheritance,resultsfroma(CTG)repeatexpansioninthe3'
untranslatedregion(UTR)ofaproteinkinasegene(DMPK). Severalmodelshavebeen
proposedtoexplainhowa(CTG)nexpansioninthe3'-UTRresultsinanautosomal
dominantandvariablephenotype. WehavefocusedonthedominantRNAmutation
modelwhichproposesthatthe(CTG)nexpansionactsinadominantmanneratthelevel
oftheRNAtranscript. Theexpansioncoulddisruptthebindingofanessentialtrans-
actingfactorpreventingnormalprocessingoftheDMPKtranscript. Alternatively,the
expansioncouldactasanabnormalbindingsiteforanRNA-bindingprotein,thus
vii
sequesteringthisproteinawayfromitsnormalfunction. Thisprojectdescribesthe
isolationandcharacterizationoftwodifferent(CUG)nRNA-bindingactivities. Thefirst,
hNabSO,isasingle-strandedRNA-bindingproteinthatmaybeinvolvedinpoly(A)tail
lengthregulationandsplicesiteselection. Thesecondactivity,the(CUG)expansion
binding(EXP)proteins,areputativedouble-strandedRNA-bindingproteinswhose
functionsareunknown.
ThehNabSOproteinwasoriginallyisolatedinacross-speciestwo-hybridscreen
duringastudyoftheyeasthnRNP,Nab2p. Nab2pisimportantformRNA3'end
formationandnucleocytoplasmicexportofmRNAinyeast. Remarkably,thehNabSO
proteinisa(CUG)8-bindingfactorinvitro,andisthefirsteukaryotictripletrepeatRNA-
bindingproteintobecharacterized. ThehNab50/CUG-BPproteinisahighlyconserved
heterogeneousnuclearribonucleoprotein,whichislocalizedpredominantlytothe
nucleus. Itbindspoly(A)^RNAinvivobutdoesnotco-purifywiththemajorhnRNP
complexsuggestingtranscript-specificbindingactivity. WhilehNabSOisatripletrepeat
RNA-bindingprotein,italsoshowstranscript-specificcrosslinkingtoDMPKmRNAsin
HeLanuclearextractsbutnotother(CUG)n-containingtranscripts. DMPKtranscripts
withincreasednumbersof(CUG)nrepeatsshowelevatedbindingofhNabSOinaUV
Hghtinducedphoto-crosslinkingassay,althoughtheincreaseisnotproportionaltothe
correspondingincreaseinrepeatlength. Onthebasisoftheseresults,Iproposethat
hNabSOisapolyadenylationfactorinvolvedintheregulationofDMPKmRNA3'-end
formation.
InadditiontohNabSO,another(CUG)n-bindingactivitywasdiscoveredthatonly
photocrosslinkstoexpanded(CUG)repeatsof>20. Theexpansionbindingproteins
vm
(EXP)arespecificfor(CUG)nrepeatsanddonotcrosslinkto(CAG)nrepeatsortothe
double-strandedtransactivationregion(TAR)RNAelementofHuman
ImmunodeficiencyVirus(HIV). CrosslinkingofEXPproteinsto(CUG)nrepeatsof
variablesizeindicatesthatbindingisproportionaltorepeatsize,suggestingthattheEXP
proteinsmaybindtoanabnormalstructurecreatedbytheexpanded(CUG)repeat. The
existenceofhNabSOandtheEXPproteinsas(CUG)nrepeatRNA-bindingproteins
providessubstantialevidenceforthedominantRNAmutationmodelforthepathogenesis
ofmyotonicdystrophy.
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INTRODUCTION
TripletRepeatExpansionDisorders
HumanDNAcontainsanabundanceofsmallrepetitivesequencesinterspersed
throughoutthegenome. Althoughthepolymorphicnatureofsimplesequencerepeatshas
beenknownforsometime,repetitivesequenceshaveonlyrecentlybeenrecognizedasthe
causeofaclassofhumangeneticdiseases. Tripletrepeatexpansiondisorders(TREDs)
resultfromexpansionoftrinucleotiderepeatsequencesinthecontextofanaffectedgene
(ReddyandHousman,1997;PaulsonandFischbeck,1996). Priortoidentificationofthe
moleculardefect,thesediseaseswerelinkedbythegeneticphenomenonofanticipation,
whichischaracteristicallyanincreaseindiseaseseverityandadecreaseintheageofonset
witheachsuccessivegeneration. Themolecularbasisforanticipationwasfinallyelucidated
whenitwasnotedthatrepeatsizeincreasedgenerationallyaswell. Todatethereare12
differentdisordersthatareknowntoresultfromtheexpansionoftrinucleotiderepeat
sequences(Table1).
Thesedisordersfallintotwodistinctgroupsdependingonthelocationofthe
expandedrepeatwithintheaffectedgene. ForthefirstgroupofTREDs(HD,SBMA,
DRPLA,MJD,SCA1,2,6,and7),themutationoccursinthecodingregionofthegene
resultinginanenlargedpolyglutaminestretchandadominantphenotype. Itisbelievedthat
theexpandedpolyglutaminesdirectlycausetheneuropathologicaldiseasephenotypedueto
aberrantproteinpropertiesthatdevelopatacertainrepeatlength. Forexample,aderivative
1