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ThePennsylvaniaStateUniversity

TheGraduateSchool

EberlyCollegeofScience

DepartmentofBiochemistryandMolecularBiology

ROLESOFCCR4-NOTINUBIQUITINATIONANDTHELASTRESORT

MECHANISMOFRNAPOLYMERASEIIDEGRADATION

AThesisin

Biochemistry,MicrobiologyandMolecularBiologyby

AbhinayaSrikanth

©2016AbhinayaSrikanth

SubmittedinPartialFulfillmentoftheRequirementsfortheDegreeof

MasterofScience

August2016

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The thesis of Abhinaya Srikanth was reviewed and approved* by thefollowing:

JosephC.ReeseProfessorofBiochemistryandMolecularBiologyThesisAdviser

DavidS.GilmourProfessorofBiochemistryandMolecularBiologyCo-ChairGraduateStudiesScottE.LindnerAssistantProfessorofBiochemistryandMolecularBiology

*SignaturesareonfileintheGraduateSchool

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AbstractSeveral studies have explored the relationship between the cellular transcription

machinery, the stress response mechanism and mRNA decay factors. Despite the

identification of multiple factors that function in linking the three responses together,

studies are constantly being conducted to explore the extent of cellular responses to

various stress conditions. Cells mitigate the effect of stress damage by balancing

transcriptionandmRNAdecayeffectively.

The understanding of the transcription machinery covers various aspects of activation,

regulation,andrepressionundernormalcellconditions,aswellasproofreadingandrepair

response during a stress or damage-induced arrest. However, studies are still revealing

new mechanisms for these responses and the proteins involved in performing these

functions.Onesuchresponseistheselectiveubiquitinationanddegradationofthelargest

subunit of RNA polymerase II (Rpb1) upon DNA damage. Previous published work by

Jesper Svejstrup’s laboratory showed the processing of Def-1 and the Def-1 dependent

ubiquitinationanddegradationofRpb1inresponsetoDNAdamageinducedarrestofPolII.

Asafollow-uptothiswork,itwasshownthatthisdegradationisalast-resortmechanism

andcanbereversedbydeubiquitinationfactors(DUBs)likeUbp3anditscofactorBre5.

Inthisthesis,Ihaveexploredtheroleofonesuchproteincomplex,theCcr4-Notcomplex,

in transcription, mRNA decay and DNA damage response. The Ccr4-Not complex has

previouslybeen implicatedasa transcriptionalelongation factorandCcr4 is theprimary

deadenylase in yeast. In my study, I explored a novel function for Not4, one of the

componentsofthecomplex,inthestress-inducedubiquitinationofRpb1.Not4isrequired

fortheturnoverofRpb1underUV-inducedstressandtheabsenceofNot4leadstoalossof

ubiquitinationofPolII under4-NQO stress. Furthermore, theCcr4-Not complexwas also

showntohaveaphysicalinteractionwiththeDUBsthatreversetheubiquitinationofRpb1.

These results indicate a previously unknown role for the Not4 module of the Ccr4-Not

complexthatisrequiredfortheubiquitinationofRpb1andapossibleinteractionwiththe

DUBsUbp3/Bre5 to set up a novel stress-responseprocess thatwill be discussed in the

followingchaptersofthisthesis.

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TableofContentsLISTOFFIGURES..................................................................................................................................VILISTOFTABLES...................................................................................................................................VIIACKNOWLEDGEMENTS...................................................................................................................VIIISCOPEANDSIGNIFICANCEOFTHISPROJECT...............................................................................1CHAPTER1:BACKGROUND................................................................................................................21.1YEASTASAMODELORGANISM........................................................................................................................31.2THECCR4-NOTCOMPLEX................................................................................................................................41.3RNAPOLYMERASEII,TRANSCRIPTIONANDDECAY...................................................................................81.4STRESSRESPONSEINTHECELL....................................................................................................................111.5THEPOLYUBIQUITINATIONCODEANDPROTEASOME-MEDIATEDDEGRADATION..............................131.6RESCUEOFPOLIIBYUBP3/BRE5DEUBIQUITINATINGENZYME..........................................................181.7TURNOVEROFRPB1UNDER4NQOSTRESS..............................................................................................21

CHAPTER2:INVESTIGATINGTHEASSOCIATIONOFTHECCR4-NOTCOMPLEXWITHDUBUBP3/BRE5............................................................................................................................................242.1INTRODUCTION................................................................................................................................................242.2MATERIALSANDMETHODS..........................................................................................................................262.2.1Straingeneration....................................................................................................................................262.2.2YeastTransformation...........................................................................................................................262.2.3Harvestingcellsandproteinisolation...........................................................................................262.2.4Bradfordassaytodetermineproteinconcentrations............................................................272.2.5ProteinImmunoprecipitation...........................................................................................................272.2.6SDS-PAGEandproteintransfer........................................................................................................282.2.7Westernblottingandproteindetectionbyfluorescence......................................................282.2.8CellextractpreparationforRIP.......................................................................................................302.2.9RNAImmunoprecipitation.................................................................................................................312.2.10RealTimeReverseTranscriptase(RT)PCR...............................................................................32

2.3RESULTS............................................................................................................................................................362.3.1AnalyzingUbp3’sassociationwithitscofactorBre5..............................................................362.3.2ProteinImmunoprecipitationwithRNAPolII...........................................................................372.3.3DetectproteinassociationswiththeCcr4-NotComplex.......................................................382.3.4RecruitmentofproteinstomRNA...................................................................................................39

2.4DISCUSSION......................................................................................................................................................48CHAPTER3:NOT4ROLEASANE3RINGUBIQUITINLIGASEINPOLIIDEGRADATION513.1INTRODUCTION................................................................................................................................................513.2MATERIALSANDMETHODS..........................................................................................................................533.2.1Strainsandconditions..........................................................................................................................533.2.2Stress-inducingchemicals...................................................................................................................533.2.3CellGrowthandMG132/4NQOTreatment.................................................................................533.2.46AUtreatment.........................................................................................................................................543.2.5TrichloroaceticAcid(TCA)extractprep......................................................................................543.2.6SDSGelrunningandsoaking............................................................................................................553.2.7ECLVisualization....................................................................................................................................55

3.3RESULTS............................................................................................................................................................563.3.1MG132treatmentinTCAextractedsamples..............................................................................563.3.2MG132and4NQOtreatmentforIPsamples..............................................................................58

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3.3.26AUtreatment.........................................................................................................................................703.4DISCUSSION......................................................................................................................................................72

CHAPTER4:DISCUSSIONOFRESULTS..........................................................................................744.1PROPOSEDMODEL..........................................................................................................................................784.2FUTURESTUDIES.............................................................................................................................................80

REFERENCES..........................................................................................................................................83

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ListofFiguresFIGURE1.1:SUBUNITSOFTHECCR4-NOTCOMPLEXCOLORCODEDACCORDINGTOTHEIRFUNCTIONS........................................................................................................................................................6FIGURE1.2:CRYSTALSTRUCTUREOFPOLII.........................................................................................................10FIGURE1.3:MOLECULARMECHANISMFORDEGRADATIONOFTRANSCRIPTIONALLYSTALLEDRNA........12FIGURE1.4:GRAPHICALREPRESENTATIONOFPROTEINUBIQUITINATION.....................................................15FIGURE1.5:STRUCTUREOFUBP3ANDBRE5SHOWINGFUNCTIONALDOMAINS...........................................20FIGURE1.6:TURNOVEROFRPB1UNDER4NQOSTRESS....................................................................................22FIGURE1.7:GRAPHICALREPRESENTATIONOFRPB1TURNOVERUNDER4NQOSTRESS.............................23FIGURE2.1:INTEGRATIVEPLASMIDCONTAININGKANMXMARKERAND3HAOR13MYCTAG.................29FIGURE2.2:OUTLINEOFTHERNAASSAY.............................................................................................................34FIGURE2.3:PROTEINIMMUNOPRECIPITATIONUSINGTAGGEDSTRAINSFORUBP3ANDBRE5..................39FIGURE2.4:DIAGRAMSHOWINGTHEPROPOSEDMODELFORINTERACTIONOFUBP3/BRE5WITHPOLYMERASEANDTHECCR4-NOTCOMPLEX.........................................................................................................41FIGURE2.5:RESULTSSHOWINGTHERECRUITMENTOFUBP3ANDBRE5TOPYK1MRNA........................46FIGURE2.6:RESULTSSHOWINGTHERECRUITMENTOFUBP3ANDBRE5TOSKN7MRNA.......................47

FIGURE3.1:TCAPROCEDUREONSAMPLESGROWNWITHOUTMG132,ANDTREATEDWITHMG132...57FIGURE3.2:4H8IMMUOPRECIPITATIONSHOWINGTHELOADINGCONTROLLEVELSOFRPB1USING8WG16ANTIBODY......................................................................................................................................................60FIGURE3.3:IMMUNOPRECIPITATIONOFSTRAINSWITH4H8ANTIBODY,ANDPROBEDWITH4H8ANTIBODY......................................................................................................................................................................62FIGURE3.4:SAMPLEINPUTSPROBEDWITH4H8ANTIBODY,TREATEDWITHMG132,4NQOANDBOTHCHEMICALS.................................................................................................................................................64FIGURE3.5:IMMUNOPRECIPITATIONOFSTRAINSWITH4H8ANTIBODYANDPROBEDWITHANTI-HAANTIBODY.....................................................................................................................................................67FIGURE3.6:IMMUNOPRECIPITATIONOFSTRAINSWITHHAANTIBODYANDPROBEDWITH4H8ANTIBODY......................................................................................................................................................................68FIGURE3.7TCAPRECIPITATIONOF6AUTREATEDCELLSTOSTUDYUBIQUITINATIONOFRPB1INMUTANTSINTHECCR4-NOTCOMPLEX...................................................................................................................71

FIGURE4.1:DIAGRAMSHOWINGMODELOFASSOCIATIONOFUBP3/BRE5TORNAANDTHECCR4-NOTCOMPLEX...................................................................................................................................................79

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ListofTablesTABLE1:COMPONENTSOFCCR4–NOTINYEASTANDHUMANCELLS........................................................7

TABLE2:STATISTICALANALYSISFORRIPWITHPYK1ANDSKN7.............................................................45

TABLE3:PRIMERSEQUENCESUSEDFORTAGGINGANDDELETINGUBP3ANDBRE5..............................81TABLE4:LISTOFSTRAINSGENERATEDORUSEDINTHESTUDY...................................................................82

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Acknowledgements This thesis would not have been possible without the constant support and

encouragementthatIhavereceivedfrommyfamily.ToDr.GuruswamySrikanth,myfather

who was a constant force of inspiration and my biggest role model. To my mother,

KanakadharaSrikanth,theonepersonwhoalwaystookthetimetounderstandeverything

that I had to share andprovideher valuable insight. Tomy sister, Ahalya Srikanth,who

sharedalltheupsanddownsoflifelikeonlyabelovedsiblingcanandmybrother-in-law,

Ram Jalasutram,whose constant support andpositive attitudehelpedme through tough

times. To my husband, Kiran Sridhara, the reason I have been happy and successful is

becauseofyourunrelentingloveandcompleteconfidenceinme.Withoutallmypillarsof

support, I would never have endeavored to do any of the things I have, and I owe my

accomplishments to eachof you,who inyourownwayshavemademewho I am today.

Thisthesisandprojectwouldalsonothavebeenpossiblewithouttheexpertguidanceof

my mentor and advisor Dr. Joseph Reese, who taught me above all, how to be a good

scientist. Iwouldalso like to thank themembersofmy lab,DeborahReese, JasonMiller,

BrooksCrickard,VinodBabbarwalandDianeLibertforwelcomingmeintothelabfamily

andguidingmethroughGraduateSchool.

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Scopeandsignificanceofthisproject The purpose of this project is to understand the specific function of Not4 in the

regulation of RNA PolII under stress-induced polyubiquitination and degradation. The

Ccr4-Not complex is a myriad of functionally related proteins that link transcriptional

functionswithmRNAdegradation.Asdiscussedinlatersections,thiscomplexopensdoors

to inter-connectivityofstress-relatedNot4-dependentubiquitinationof theRpb1subunit

ofRNAPolymeraseII.ThispreviouslyunknownroleofNot4intheubiquitinationofRpb1

will add to the collection of functions of the complex as well as solve a piece of the

connectivity that links the transcriptionofgenes to thedecayofmRNAand to thestress

responseofthecell.

Manystudieshavebeendoneinallofthesefields–transcriptionregulation,mRNA

decay factors, stress response and overlapping factors.Work in our lab has shown that

when pausing or other events impede transcription, the Ccr4-Not complex promotes

elongation.Iftheblockisnotovercome,PolIIeitherassistsintranscription-coupledrepair

ordisengagesfromtheelongationsite.ThedetachmentofPolIIrequirestheremovalofthe

largestsubunitofPolII–Rpb1–byubiquitinationanddegradationbytheproteasome.

Initialstudieswereconductedtounderstandtheeffectof4-Nitroquinoline(4NQO)

stressontheturnoverofRpb1.Inwild-typecellstreatedwith4NQO,Rpb1wasdegraded

over thecourseof theexperiment.Thesameexperimentwasconductedwithmutantsof

theCcr4-Notcomplexanditwasobservedthatintheabsenceofnot4(inaΔnot4strain),the turnover of Rpb1was not observed. Not onlywasΔnot4 impairing the turnover ofRpb1,theeffectwasfoundtobecontributedbytheRINGdomainofNot4,whichpointstoa

functional role of Not4 in the turnover of Rpb1. This initial observation led to the

experimentsconductedinthisthesisthataimtobridgeagapbetweenthestressresponse

andtheRpb1degradationfactorsofthecell.

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Chapter1:Background The central dogma of Molecular biology describes the progression of genetic

informationfromtheDNAtothemessengerRNAandultimatelyresultsintheproduction

of theprotein that carries out the required function for the cell. In order to successfully

completethisdatatransferfromDNAtoprotein,severalkeyproteinsmustworktogether

efficiently.However,itisalsoessentialforthecelltoregulatetheprocessateverylevelto

ensure the balance of proteins, and for appropriate cellular response to stimuli.

Additionally,ifanerrorinthismechanismoccurs,theresultcouldprovedisastrousforthe

cell.Therefore,equally importantaretheproofreadingandrepair factorsthatensurethe

correctmessageisbeingrelayedandthecellcaneffectivelyrespondtodamageorstress.

Cell response to DNA damage and stress has been actively studied due to the

significanceoftheresponseinmaintaininggenomeintegrityandcellsurvival. Cellshave

evolved multiple mechanisms to ensure continued transcription following a stress or

damage. Gene transcription by the RNA Polymerase II (PolII) is affected by numerous

events such as stalling, pausing, arrest or evenbacktracking of PolII. To overcome these

hurdles, cells have specialized proteins and factors that assist Polymerase in completing

transcription successfully. One such multi-functional protein complex, the Ccr4-Not

complex,isconservedamongeukaryotesandiscomposedofninecoresubunits.Between

these subunits, the complex covers several functions such as transcription initiation,

deadenylation,mRNAturnover,transcriptioncouplednucleotideexcisionrepair(TC-NER),

deubiquitinationandothers(Reese2013).

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1.1 Yeastasamodelorganism

The experimental methods conducted in this thesis benefit from using a model

organism that is efficient and easy to manipulate. The model organism selected is

Saccharomyces cerevisiae, or Baker’s yeast, a single-celled eukaryote with most cellular

processesfunctionallyconservedamonghighereukaryotes.Theyeastgenomeconsistsof

5770genesthatareorganizedon16chromosomes.Theentiregenomeissequencedanda

completedatabaseisavailableonlineatwww.yeastgenome.org.

Apartfromthereadyavailabilityofgeneinformation,mutants,andinferredprotein

sequences,yeastareeasyandinexpensivetogrowwithadoublingtimeof90minutesfor

wildtype cells. They can also grow in a haploid condition, which makes them ideal for

knockoutgeneticmanipulationsthatwouldotherwisebedifficultinadiploidstate.There

are numerous mutants and tagged strains commercially available and others are easily

madeinthelaboratory.Yeastcanbemadewithseveralidentifyingmarkersandcanhence

have multiple gene deletions and tags within the same strain. These methods will be

employed for conducting the experiments described in this thesis and will be further

discussedineachchapter.

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1.2 TheCcr4-NotComplex

Theprimary complex in this study is theCcr4-Not complex. It is a highly conserved,

multi-functional ~1MDa protein complex that is composed of core protein components

with9subunitsandalarger~1.5MDacomplexwithadditionalproteinsubunits.The9core

subunits are Ccr4, Caf1, Caf40, Caf130, and Not1-5 as shown in figure 1.1 (Collart &

Panasenko2012),whileadditionalproteinslikeDhh1,Dbf2,Caf4,Caf16,andBtt1formthe

larger1.5MDacomplex(Reese2013).Thecomplexfunctionstoprovidemultiplelevelsof

gene regulation, in several crucial cellular processes like transcriptional initiation and

elongation,deadenylation,mRNAdecayandubiquitination.

In addition to co-purifyingwith RNAPolII, the Ccr4-Not complex also interactswith

other elongation factors, and mutations in the Ccr4-Not subunits were sensitive to

elongation inhibitors 6-Azauracil (6-AU) andmycophenolic acid (Reese 2013). Ccr4 and

Pop2/Caf1 are theprimarydeadenylases in yeast andact as a functionally separateunit

withCaf1actingasthelinkbetweenCcr4andtherestofthecomplex(Baietal.1999).Ccr4

is amember of the exonuclease III family of proteins, and as a deadenylase, has a 3’-5’

exonuclease activity on the Poly-Adenylation (Poly A) tail found on mRNAs, while Caf1

contains an RNaseD/DEDD domain and also has a 3’-5’ exonuclease activity (Chapat &

Corbo 2014). Although initially discovered for its role in activating ADH2 (Alcohol

Dehydrogenase2) inmediacontaininganon-fermentativecarbonsource,Ccr4wassoon

showntobearegulatoroftranscriptionaswellasmRNAdecay(Kruketal.2011).

Another significant function of the complex is ubiquitination by theNot4 protein,

discussed in further detail later. This subunit contains aRINGdomainwithE3ubiquitin

ligase activity, which is known to have several targets such as the histone demethylase

Jhd2, and leads to its degradation by the proteasome (Mersman, Du, et al. 2009).

Furthermore, the first fourNOTgenes,NOT1-4,were identifiedasnegative regulatorsof

TATA-lesspromoters(Collart&Struhl1994).

The complex has also been linked to the cellular stress response in a regulatory

manner.InastudyconductedbyTravenetal.(Travenetal.2010)itwasobservedthatin

cells lacking thePop2/Caf1 subunitof theCcr4-Not complex, adeletionofPuf5 (anRNA

binding protein that promotes mRNA deadenylation and associates with the Ccr4-Pop2

5

subunits)resultedintheincreasedsensitivityofthecelltoDNAreplicationstress.Puf5and

Pop2 were shown to act in the same genetic pathway and the repression of HO

endonuclease mRNA by Puf5 was eliminated in the absence of Pop2 (Goldstrohm et al.

2006). Another subunit, Dhh1, a DEAD-box RNA helicase, co-purifies with Pat1 (a

componentofp-bodies)andresults in thenuclear localizationofPat1uponUVstress. In

the absence of dhh1 this nuclear localization was abolished (Bahassou-Benamri et al.

2013).Dhh1wasalsoimplicatedincell-cycleDNA-damagecheckpointrecoveryattheG1/S

phase by doing a quality control check on mRNA (Bergkessel & Reese 2004) and cells

lacking Dhh1 were defective in release from arrest at the checkpoint.

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Figure 1.1: Subunits of the Ccr4-Not complex color coded according to their

functions.Thedeadenylases,Ccr4andCaf1indicatedinbluewithCaf1actingasthelink

betweenCcr4andtherestofthecomplex.Not4,theRINGE3ubiquitinligaseisindicatedin

yellowandDhh1 theRNAhelicase inpurple.With theexceptionofDhh1, the restof the

proteinsshownarecoresubunits.

Adaptedfrom:(Collart&Panasenko2012)

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Table1.ComponentsofCCR4–NOTinYeastandHumanCells.Tabletakenfrom:(Chapat&

Corbo2014)

Saccharomycescerevisiae

Homosapiens Domain PositionInsidetheComplex

Not1 CNOT1 HEATrepeatsMIF4G Scaffold

Not2 CNOT2 NOT-box ‘NOT’moduleC-terminalpartofNot/CNOT1

Not3 CNOT3 NOT-box Not4 CNOT4 RINGE3Ligase Ubiquitination

ModuleNot5 NOT-box Ccr4 CNOT6/CCR4a EEP,LRR ‘Deadenylase’module

centralpartofNot/CNOT1

CNOT6L/CCR4b EEP,LRR —Pop2/Caf1 CNOT7/CAF1a DEDD Deadenylase CNOT8/POP2/CAF1b DEDD —Caf40 CNOT9/hRcd1 Armadillorepeats Centralpartof

Not/CNOT1Caf130 — — C-andN-terminalpart

ofNot1— CNOT10 — N-terminalpartof

CNOT1(distinctmodule)

— C2ORF29/CNOT11 — —Dhh1 Rck/p54/DDX6* DEAD-box Centralpartof

Not1/CNOT1

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1.3RNAPolymeraseII,transcriptionanddecay

TheCcr4-Notcomplexwas initially identifiedassociatingwith theRNAPolymerase II

complexincellsandwashencedescribedasanelongationfactor.However,theassociation

of the Ccr4-Not complexwith PolII offers an ideal location for the protein to access the

DNA, RNA, and all the associated proteins that facilitate the multitude of functions

conductedbytheCcr4-Notcomplex.

RNA Polymerase II (PolII) is a very large protein complex – 0.5 MDa in yeast –

composedof12subunitsandessentialforcellularfunctionasindicatedinfigure1.2.Itis

anenzyme that transcribesDNAprotein-coding templates tomakemessengerRNAs that

are translated intoproteins. Italso transcribesseveralregulatoryelementssuchassmall

snoRNA,miRNAs, snRNAs, and siRNAs that are not translated into proteins (Woychik&

Hampsey2002). Its functiondictatescellulardifferentiation,adaptationtoenvironmental

changes, response to stress and generalmaintenance of the cell. Therefore regulation of

PolIIoccursatmultiplelevels.ThefirststepisduringinitiationwhenPolIIassembleswith

multiplegeneraltranscriptionfactorssuchasTFIIB,TFIID,TFIIE,TFIIFandTFIIH,toform

thepre-initiationcomplex.ThisallowsPolIItoaccesstheopenpromoterDNAandinitiate

RNAtranscription(Sainsburyetal.2015).

With the exception of Rpb4 and Rpb9, all other PolII subunits are necessary for the

survival of the cell. The largest subunits,Rpb1 and2 arehighly conserved and form the

centralclamparoundDNAfortranscription.Rpb1alsohasahighlyconservedC-terminal

tail (CTD), which is composed of the repeating sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser

(YSPTSPS)(Arndt&Kane2003).Thenumberofrepeatsvariesfromorganismtoorganism.

In yeast, there are26-27 repeatswhile inhumans there are52 repeats in theCTD.This

sequence is very important as it undergoes post-translationalmodifications during gene

transcriptionandimpactsthefunctionofotherfactorsandmodifiersthatarerecruitedto

its docking sites (Feaver et al. 1991). Two of themost significantmodifications are the

phosphorylation of Ser2 and Ser5. PolII enters the preinitiation complex in an

unphosphorylatedstate.Duringtranscriptioninitiation,theCTDhasapredominantlySer5

phosphorylation signature. This changes to a Ser2 phosphorylation during elongation

(Cramer2004).

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Of the12subunitsofPolII,10 formthecoreofpolymerase,while two–Rpb4and7-

form a dissociable heterodimer that has been linked to multiple post-transcriptional

processessuchasmRNAexport, translationanddecay(Harel-Sharvitetal.2010;Choder

2004).TheRpb4/7sub-complexhasalsobeen implicated inpromotingdeadenylationof

certain mRNA transcripts in the cytoplasm (Lotan et al. 2005). Additionally, it was

discovered that in order for the Ccr4-Not complex to bind to RNA PolII Elongation

Complexes(EC)invitro,theinteractionsneedstobestabilizedbythesub-complexRpb4/7

(Babbarwaletal.2014).

InordertounderstandtherolethatvariousfactorsplayinassociationwithPolII, it is

necessarytoreviewsomeofthesignificantstepsinthethreephases:Initiation,Elongation

and Termination. Initiation is carried out by a host of general transcription factors and

beginswithaTATA-bindingprotein(TBP)interactingwiththeTATAboxinthepromoter

region of a gene. A general transcription factor, TFIID alters DNA to prepare for

transcription and assemble the other factors. Another factor TFIIH unwinds DNA and

phosphorylates Ser5 of the CTD promoting the elongation phase (Woychik & Hampsey

2002). In the elongation phase, RNA PolII encounters frequent pauses, backtracks and

arrests due to a variety of reasons. Elongation factors assist in the continuation of

transcription,suchasTFIIS,anelongationfactorthatcausesPolIItocleavesthetranscript

in the3’ end to rescuePolII frombacktracking in theeventofanerrorormisalignment.

ThisallowsPolII to threadbackand forthtoreadjustandrecover fromthearrest. In the

eventofDNAdamageorotherbarrierslikeanucleosome,anotherimportantfactor,Spt4/5

helps inmaintaining theelongationbubbleaswell as stabilizing theemerging transcript

(Martinez-Rucoboetal.2011).

Cellularresponseandfunctionisacomplexcoordinationofproteinsthatinteractwith

each other. The synchronization of RNA PolII with the Ccr4-Not complex to facilitate

transcription elongation is a means by which the cell could further regulate the

transcription of a gene under precise conditions. In this thesis I explore themechanism

behindoneparticularcellularresponseinvolvingtheCcr4-NotcomplexandRNAPolII,and

DNA damage induced arrest of PolII. The requirement of the Ccr4-Not complex is

undeniable,andthemechanismandpurposeof theresponse isexplored in the following

chapters.

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Figure1.2:CrystalstructureofPolII.(a)Thepolymerasecorehasbeenshowndivided

into threesubassemblies, theRpb1subassemblycontainingRpb1,Rpb5,Rpb6andRpb8,

theRpb2subassemblycontainingRpb2andRpb9,and theRpb3subassemblycontaining

Rpb3, Rpb10, Rpb11 and Rpb12. The fourth subassembly consists of Rpb4 and Rpb7

protrudingfromthecore(PBDID1I6H).(b)Cartoonofthe12subunitsofeukaryoticRNA

PolymeraseII.

Figuretakendirectlyfrom:(Wild&Cramer2012).

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1.4Stressresponseinthecell

When the cell encounters a stress or damage to the DNA, the cell utilizes twomain

mechanisms of response. The first is a transcription-coupled repair mechanism –

Transcription Coupled Nucleotide Excision Repair (TC-NER) – that is initiated by the

recruitment of several factors like Rad4/Rad23 to recognize the lesion (Schärer 2007),

repairfactorslikeRad1/Rad10,andRad26torepairtheUVdamage.Thisusuallyoccursin

actively transcribed genes and is triggered by the stalled PolII (Svejstrup 2002) (Figure

1.3).Alternatively,thecellhasaGeneralGenomeRepair(GGR)mechanismthataddresses

damageinlocationsnotactivelytranscribed(Nouspikel2009).

In the event that the TC-NERmechanism is insufficient to resolve the DNA damage,

cellspossessaback-upmechanismtoovercometheproblem.Inyeast,uponDNAdamage,a

small protein –Def1 – that isnormally found in the cytoplasmundergoesubiquitination

and is partially processed in a proteasome-dependentmanner, to produce a functionally

activeprotein.ItistheninternalizedintothenucleuswhereitformsacomplexwithRad26

onthedamagedsiteandrecruitsfactorsthatubiquitinateanddegradethelargestsubunit

ofPolII–Rpb1.ThisresultsinthedisintegrationofthePolymerasecomplexandfreesup

theDNAforGGR(Wilsonetal.2013;Woudstraetal.2002).Thisprocesssalvagestheother

subunitsofPolII,whicharerecycledforfurtheruse.

In the Def-1 initiated last resort response, Rpb1 is monoubiquitinated by Rsp1 (as

shown in figure 1.3) and subsequently polyubiquitinated by the Elongin-Cullen complex.

TheElongin-CullenE3 ligase complex (Ela1-Elc1) interactsdirectlywithDef-1 via aCUE

(named after domain homology with Cue1 – coupling of ubiquitin to ER degradation)

domain-dependent manner and is recruited to PolII along with Ubc4/5 (Ubiquitin

conjugatingenzymes)(Wilsonetal.2013).Rpb1isthendegradedbytheproteasomeand

thedamagecausedtotheDNAisrepairedbyGGR.

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Figure1.3:Molecularmechanism fordegradationof transcriptionally stalledPolII.

Upon DNA damage the transcribing complex becomes irreversibly stalled. Blocking of

transcription might lead to cell death (C). To ensure continuation of transcription, the

damage is either repaired by TCR (A) or as a “last resort” mechanism RNAPII is

polyubiquitylated and degraded by the proteasome, an action which allows the GGR

pathwaytorepairthedamage(B).Imageunalteredfrom:(Karakasili2010).

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1.5ThePolyubiquitinationcodeandproteasome-mediateddegradation

To understand the mechanism of the stress response and ubiquitin-mediated

degradationofRpb1, it isnecessarytoappreciate the functionthatubiquitinplays in the

cell. A newly translated protein often undergoes several post-translationalmodifications

that allow the cells to control the protein’s function. Ubiquitin is one suchmodifier that

variesthefateandfunctionofthetargetproteintowhichitisappended,dependingonthe

siteofubiquitinationaswellasthenumberofubiquitinmoietiesappended.Thisubiquitin

code is read byUbiquitin reading proteins,which carry out the function dictated by the

code. Ubiquitination of a protein can result in multiple outcomes such as cell cycle

regulation, DNA repair, membrane protein trafficking, and proteasomal degradation of

proteins(Finleyetal.2012).

Ubiquitin is a 76 amino acid polypeptide that forms a chain through one of its 7

internal lysineresidues. Itcanalsomarkatargetproteinwhenit isappendedtoa lysine

residueonthetargetprotein.Mostoftheubiquitinationmarksresultinthedegradationof

the target protein but the primary mark that degrades proteins is through the K48

mediated poly-ubiquitination. The process of ubiquitination occurs as a cascading event

when an E1 (Ubiquitin activating enzyme) is chargedwith carboxylatemoiety at the C-

terminus of ubiquitin (Ub) linked to the catalytic cysteine of E1 by a thioester bond.

Following this, anE2protein (Ubiquitin conjugatingenzyme)associateswith theE1and

ubiquitin is transferred to the E2. This charged E2 then interactswith an E3 (Ubiquitin

ligase)andtheubiquitinmoiety iseitherdirectly transferredtothetargetprotein,orvia

theE3asanintermediate(Komander&Rape2012;Herideetal.2014).Figure1.4shows

thetransferofaubiquitinmoietyfromanE1toanE2andthentothesubstrateviatheE3.

In yeast, themachineryhas ahierarchical organizationwithonlyoneE1 (Uba1), 11E2s

(Ubc1-8,10,11,13)and60-100E3s.ThisisbecausetheE3proteinsrecognizethetarget

proteinsandarehencespecifictothetargets(Finleyetal.2012).

Ubiquitination can result in multiple outcomes depending on the number of

ubiquitinmoietiesattached–monoubiquitinationorpolyubiquitination–aswellasthesite

ofubiquitination.Polyubiquitinationoccurswhenubiquitin istransferredfromacharged

E2 to a lysine on the target protein and subsequent ubiquitinmolecules are attached to

14

lysine48on theprecedingubiquitin to formamulti-ubiquitinchain.Thischainactsasa

messagefortheproteasomemachinerytodegradethetargetedprotein(Finleyetal.2012;

Herideetal.2014).

The proteasome is a 2.5 MDa protease with 33 distinct subunits and is highly

conservedamongalleukaryotes.Itsprimaryfunctionistodegradeproteinsmarkedwith

thelysine48chainubiquitination.It isorganizedintotwosubunits, the26Scoreparticle

thatcontainstheproteolyticactivesites,andthe19Sregulatoryparticlethatrecognizesthe

substrate.The core is composedof twoαand twoβ ringsof seven subunits each.Theα

ringsflanktheβringsinaα-β-β–αstructure,withtheproteaseactivesitespresentinthe

β rings. The Regulatory particle is comprised of a lid structure containing the ATPase

subunitsRpn3,-5-9,and-11,-12,aswellasabasethatcontainstheATPasesRpt1-6and

non-ATPasesRpn1,-2,-10(Glickmanetal.1998).Rpn10containsahydrophobicpatchthat

binds to Ubiquitin chains (Fu et al. 1998). Proteins entering the proteasome core are

unfoldedandaDUB(DeubiquitinatingEnzyme)removestheubiquitinmarksforrecycling.

Thisunderstandingoftheubiquitinpathwaycanbeappliedtoexploringtheroleof

theCcr4-Notcomplexintheubiquitinationandproteasome-mediateddegradationofRpb1.

As discussed in the following section, Not4 is a RING E3 ubiquitin ligase and it is

functionallyrelevantinthestress-responsedegradationofRpb1.

15

Figure1.4:Graphicalrepresentationofproteinubiquitination.Ubiquitinactivationby

E1 in an ATP-dependentmanner followed by transfer of the ubiquitin to the ubiquitin-

conjugating enzyme (E2), and finally covalent attachment to substrate proteins via

ubiquitin ligases (E3).A singleubiquitinmolecule generatesmonoubiquitinatedproteins

andrepeatedroundsofubiquitinationresultsinmulti-orpolyubiquitinatedproteins

Unalteredimagefrom:(Finleyetal.2012).

16

1.6Not4asaRINGE3ubiquitinligase

Yeast cells have 44 different E3s that contain a RING (Really Interesting New Gene)

domain. As previously mentioned, the RING domain binds to the E2 and transfers the

ubiquitintothetargetbyinducingsubtlechangestotheE2.

TheubiquitinationmoduleoftheCcr4-NotcomplexistheNot4protein.Not4isanE3

ubiquitin ligasewithaRINGdomainontheN-terminus,acentralRRM(RNARecognition

Motif)andanunstructuredC-terminus(Bhaskaretal.2015).Not4functionswithubiquitin

conjugatingenzymesUbc4/5,andhasbeenreported toubiquitinatemultiple targets like

Jhd2, the H3K4 histone demethylase in yeast (Douglas PMersman, Du, et al. 2009) and

Cdc17,thecatalyticsubunitofpol-αinyeast(Haworthetal.2010)Not4isalsorequiredfor

thestress-inducedtranscriptionofRNR(RibonucleotideReductase)genes–RNR2,3and4.

ItwasobservedthattheTATABindingProtein(TBP)andPolIIarenotrecruitedtotheRNR

genesintheabsenceofNot4(Mulder,Inagaki,etal.2007;Mulderetal.2005).

The function of Not4 as an E3 in the process of ubiquitination of Rpb1 is unclear.

However, it is known that Rpb1 is degraded through a proteasome-dependent manner.

Not4 is also known to associate with the proteasome to regulate levels of H3K4

trimethylation(Laribeeetal.2007).Lossofnot4wasshowntodecreaseglobalandgene-

specificH3K4trimethylation.ThiswasdemonstratedbyNot4’sabilitytopolyubiquitinate

Jhd2, and target Jhd2 for proteasome-mediated degradation (Douglas P Mersman,

Harmeyer,etal.2009).AnothersubstratefortheubiquitinationactivityofNot4isEgd2,a

complexthat isassociatedwiththeribosomalexit tunnel(Panasenkoetal.2006)and its

ubiquitinationisrequiredforregulationofnascentproteintargeting.

The presence of multiple substrates for the ubiquitination activity of Not4 and its

associationwiththeproteasomecouldindicatethatRpb1isalsoasubstrateforNot4under

DNAdamageinducedarrest.However,Not4mayalsofunctionindirectlyintheturnoverof

Rpb1byubiquitinatinganaccessoryproteinlikeDef1.Def1isknowntobeproteolytically

processed in the cytoplasm, in response to Rpb1 arrest and is required for the

ubiquitinationofRpb1.Not4alsotargetsothersubstratesinthecytoplasmsuchasRsp7A,

asmallribosomalprotein.Anothernotablesubstrate isaribosome-associatedchaperone

protein NAC (Nascent polypeptide-Associated Complex) that plays a role in removing

17

translationalarrestproductsbyNot4-mediateddegradationbytheproteasome(Dimitrova

etal.2009).

Multiple studies have also shown the possible role of Not4 in cotranslational quality

control (QC). AlthoughNot4 does not ubiquitinate the arrest products, it accumulates in

response to factors that induce translational QC (Halter et al. 2014), and Not4 deletion

leadstoanaccumulationofpolyubiquitinatedandaggregatedproteindeposits(Panasenko

2014).

18

1.7RescueofPolIIbyUbp3/Bre5Deubiquitinatingenzyme

Although the process of RNA Polymerase II arrest and ubiquitination of Rpb1 is a

process to recover theremainingproteins inPolIIandrepairing thedamage to theDNA,

the cell possesses a method to rescue polymerase as a last resort mechanism.

Deubiquitinases(DUBs)arealargegroupofproteasesthatplayanantagonisticroletothe

ubiquitin machinery and reverse the fate of proteins designated for destruction by the

proteasome. DUBs also functionwith the proteasome to release ubiquitinmoieties from

proteins ready for degradation. As previously discussed, RNA PolII undergoes

ubiquitination and degradation as a last-resortmechanism in response to DNA damage.

YeastUbp3targetsPolIIasitssubstratetorescueitfrompossibledegradation(Kvintetal.

2008).However,Ubp3alsoplaysarole inNucleotideExcisionRepair(NER)bytargeting

Rad4 for degradation. Rad4 and Rad23 function to detect and respond to DNA damage

Ubp3 was shown to be a negative regulator of a cellular response to UV irradiation by

interacting with Rad4, however the actual degradation of Rad4 may require additional

factors(PengMao&Smerdon2010).ItispossiblethatUbp3/Bre5mayalsoplayarolein

rescuingPolIIfromdegradationbytargetingfactorsthatubiquitinateanddegradePolII.

Ubp3 plays a crucial role in removing ubiquitin chains from PolII. This function

rescues Rpb1 from proteasome-mediated proteolysis in the event of DNA damage that

cannotberepairedeffectivelythroughTC-NER.Deletionofubp3resultsinhigherlevelsof

ubiquitinatedPolIIthatarealsodegradedfasteruponUVtreatment(Kvintetal.2008).An

x-ray crystal structure of Ubp3, as shown in figure 1.5, reveals that the protein forms a

hetero-tetrameric complexbetweenBre5andUbp3.Bre5 consistsof anuclear transport

factor2-likedomain(NTF2)attheN-terminusandaRNARecognitionMotif(RRM)atthe

C-terminus. Ubp3 consists of a UCH (ubiquitin carboxyl-terminal hydrolase) catalytic

domainontheC-terminusandaBre5bindingdomainclosetotheN-terminus.Bre5forms

ahomodimerthroughtheNTF2domainandassociateswithUbp3viatheBre5bindingsite

(Lietal.2007).

Other functions of Ubp3 include transcription initiation, silencing, protein

degradationandretrogradetransport(Moazed&Johnson1996;Soléetal.2011;McCullock

et al. 2006). Ubp3 interacts with transcription factor TFIID, as well as ubiquitin ligase

19

Bul1/Rsp5.Ubp3was found topromote cellular resistance toDNAdamagingagents ina

way that isgeneticallyantagonistic toBul1gene(Bilslandetal.2007).Similarly, it isalso

involvedinthedegradationofRad4,aproteinthatrespondstoDNAdamagebyUVdirectly

andfacilitatesNucleotideExcisionRepair(NER)(PengMao&Smerdon2010).Itisnotyet

clearifthisfunctionisagenome-wideeffectorifUbp3targetsparticulargenesinastress-

specific way. However, the role of Ubp3 and Bre5 is not restricted to just DNA damage

repair, Ubp3 also acts as a transcriptional activator of Gal4 and Gcn4 by preventing the

degradation of TATA-binding protein Tbp1/Spt15. The authors showed that upon

inductionoftheGal1andHis3genes,Ubp3isrecruitedtothepromoterofbothgenesand

isrequiredtoactivatethegenes(Chewetal.2010a).Inadditiontothis,Ubp3isrecruited

toosmoresponsivegenestomodulatetranscriptionalinitiationaswellaselongation(Solé

etal.2011).

20

Figure 1.5: Structure of Ubp3 and Bre5 showing functional domains. (a) Domain

structureofUbp3andBre5showingimportantsites–Bre5bindingdomain,catalyticsite,

NTF2domainandRNARecognitionMotif.(b)Ribbonstructureoftheinteractionbetween

two units of Bre5 (shown in two shades of blue)with the two units of Ubp3 (shown in

shadesofred).(c)Aspace-fillingmodelshowingtheBre5homodimerandtheN-terminus

ofUbp3.

Imagesunalteredfrom:(Lietal.2007)

21

1.8TurnoverofRpb1under4NQOstress

Inpreviousexperimentsconductedinthelab(Libert&Reese2014),theturnoverof

Rpb1uponUVstresswasobservedandtheresultsofthisexperimentweredocumentedin

yeast strains expressing either wild-type or deletion mutants in the Ccr4-Not complex

members. The hypothesiswas that Rpb1 turnoverwould be diminished as compared to

wild-type strains, in the absence of not4, under 4NQO stress. The experimental results

supported the hypothesis, suggesting a previously undiscovered role for Not4. Other

subunitsoftheCcr4-Notcomplexwerealsotestedanddidnotshowthiseffect.

Moresignificantly,theexperimentwasrepeatedwithadditionalmutantsinthenot4

RINGdomainbyintroducingplasmidscontainingvariantsofnot4intoaΔnot4background;

acompletedeletionoftheRINGdomaindeactivatesthefunctionofnot4completely,anda

point mutant in the RING domain (I64A) which was identified as a key residue for

functionality of the RING domain. It was observed that the RING domain deleted Not4

proteinimpairedtheturnoverofRpb1nearlyasmuchasthedeletionofnot4itself,while

the RING domain point mutant (I64A) showed an intermediate phenotype. The gel

experiment is shown in figure 1.6, where the cell extracts from each strain (wild-type,

Δnot4,Δccr4,Δdhh1)weretreatedwith100ug/uLcycloheximidetoinhibitfurtherproteinbiosynthesis.Eachlanerepresentsatime-pointinthecourseofthe4NQOtreatmentfrom0

min or untreated sample, to 30, 60, 90 and 120 min of treatment with 6ug/uL 4NQO,

alongsideanuntreatedsampleatthefinaltimepoint.ThesampleswereprobedforRpb1

levelsusinganantibody toRpb1.Theresults fromthisexperimentaregraphed in figure

1.7.TofurtherconfirmtheroleofNot4intheturnoverofRpb1,aplasmidcontainingNot4

wassubstitutedintheΔnot4strain.TheturnoverofRpb1wasrecoverednearlyaswellas

wild-typeimplyingthatNot4hasafunctionalroleinthestress-responseturnoverofRpb1

(figure1.7).Errorbarswereunavailableforthisdata.

ThisnovelroleofNot4intheturnoverofRpb1under4NQOstresswasakeyfactor

thatledtoquestionsthatareaddressedinthisthesis.Thisexperimentlaunchedanumber

of hypotheses regarding the role of the Ccr4-Not4 complex in a complicated stress-

responsemechanism.

22

Figure1.6:TurnoverofRpb1under4NQOstress.CellsweregrowninYPDandtreated

in log phase growth. At time 0 min., all samples were treated with 100 ug/mL

cycloheximide(CHX)andindicatedcellswith6ug/mL4NQO.Rpb1levelsdroppedinwt,

Δccr4,andΔdhh1strainsoverthecourseoftheexperiment.Δnot4strainsweredefectivein

thisturnoverofRpb1.

Figureunalteredfrom:(Libert&Reese2014).

23

Figure1.7:Graphical representationofRpb1 turnoverunder4NQO stress: The top

graph shows the turnover of Rpb1. Δnot4 strains have a nearly consistent level of Rpb1

comparedtowildtypestrainandothers.ThesuddenincreaseinRpb1levelsintheΔdhh1

strainmaybeduetoexperimentalinconsistencies.Thesecondgraphshowstherescueof

theΔnot4phenotypebyincorporatingaplasmidcontainingwtNot4,Not4withadeletion

intheRINGdomain,andapointmutationintheRINGdomain(I64A).Resultsshowthatthe

RINGdomainisrequiredfortheturnoverofRpb1.Errorbarsareunavailableforthisdata.

Extractedunalteredfrom:(Libert&Reese2014)

24

Chapter2:InvestigatingtheassociationoftheCcr4-NotcomplexwithDUBUbp3/Bre5

2.1Introduction

Protein-proteininteractionsfacilitateaquickandeffectiveresponseforthecell.The

interactioncanchangedependingonexternalstimuliandinternalresponses.Thepurpose

of this study is to understand howUbp3/Bre5 interactswith the Ccr4-Not complex and

PolII,andtheimplicationofthisassociation.InthecaseofPolII, thereareseveral factors

that are connected to the complex at each stage in transcriptional initiation, elongation,

arrest and transcription termination. As previously described, the Ccr4-Not complex

interacts with the transcription machinery for various functions, as does the DUB

Ubp3/Bre5.Inordertounderstandhowthesetwoproteincomplexesfunctiontogether,it

is necessary to know how they associate with each other and the polymerase. This

association could shed some light on themethod of recruitment of these factors during

stressresponse.

The association of the Ccr4-Not complex to Ubp3 was first visualized in a

preliminary experiment (unpublished work Babbarwal, V) where the deletion of Not4

resultedinthepulldownofseveralproteinssuchasRad26,Ubp3,Def1andCdc48withthe

Ccr4-Notcomplex.Rad26asdescribed inearliersections isa factor that functions inTC-

NERandalsoassociateswithDef1intheeventofalast-resortdegradationofRpb1inDNA

damage response.Def1 isubiquitinatedandprocessed in the cytoplasmand internalized

intothenucleuswhereitisrequiredfortheubiquitinationofRpb1(MarcusDWilsonetal.

2013). Cdc48 is a cell cycle regulator that is thought to play a significant role as a

proteasomeadapterintheubiquitin-proteasomesystem(UPS).ItsfunctionsvaryfromER-

associatedproteindegradation(Stolzetal.2011),controlofE3ligaseactivityforenzyme

regulation (Yen et al. 2012) and even during mitochondrial stress response (Heo et al.

2010). Interestingly, Cdc48 also interacts with Ubp3 to function in starvation-induced

ribophagy (Ossareh-Nazari et al. 2010). The DUB Ubp3 has been previously shown to

interact with the transcription machinery where ubp3 mutant cells are defective in

expressionofosmoresponsivegenes(Soléetal.2011),howeveritsassociationtotheCcr4-

Not complex is novel. The information provided in this thesis does not confirm a direct

25

associationbetweenUbp3/Bre5andtheCcr4-Notcomplex,andit is likelytheinteraction

couldbemediatedbyotherproteins(likeCdc48).Cdc48maynotbedirectlylinkedtothe

ubiquitination of Rpb1, but instead may associate with factors that lead to Rpb1

destruction.

Additionally, according to the domain structure of Bre5, there is a defined RNA

recognitionmotif (RRM),which does not have any knownRNA targets. Ubp3 itselfmay

have anunidentifiedRNAbindingdomain andwas identified in a study looking atRNA-

bindingproteins(Tsvetanovaetal.2010).AspreviousexperimentshaveshownthatUbp3

and Bre5 form a complex together and associate with RNA PolII, it is reasonable to

speculate that the interaction may be through the emerging RNA transcript, or may be

stabilizedby theRNAorperhaps thedeubiquitination function requires interactionwith

mRNA.

ToassesstheinteractionofUbp3anditscofactorBre5withtheCcr4-Notcomplex,

protein immunoprecipitationswereperformedwithstrainscontainingtaggedversionsof

Ubp3andBre5,aswellasdeletionsofeitherofthetwogenesinthecomplex.Furthermore,

to investigate recruitment of Bre5 tomRNA,RNA immunoprecipitations (RIP)were also

performed with the tagged strains to investigate the recruitment of Ubp3 and Bre5 to

specific mRNA. The purpose of these experiments was to determine whether the

interaction with the Ccr4-Not complex might regulate the recruitment of Ubp3/Bre5 to

mRNA. Specific mRNA candidates were chosen based on previous work that identified

RNAsthatassociatewiththeCcr4-Notcomplex(J.E.Miller,J.C.Reese,unpublished)under

stressedandunstressedconditions.

ThedetailsoftheinteractionoftheseproteinswitheachotherandtomRNAcould

answeranumberofquestionsaboutthemodeoffunctionofUbp3/Bre5andtheCcr4-Not

complex,andleadustoasknewquestionsaboutthepurposeoftheinteraction.Onemodel

isthatUbp3/Bre5couldusetheemergingtranscriptasascaffoldtoberecruitedtoPolII.

TherecruitmentcouldalsobeamethodofselectivityfordeterminingwhichmRNAsareto

be degraded or maintained during a stress response. These experiments and the

observationsarepresentedintheupcomingsectionsofthischapter.

26

2.2MaterialsandMethods

2.2.1Straingeneration

Ubp3 and Bre5 were tagged using common yeast tagging methods via PCR

amplificationofa taggingcassette.UsingapFA6aplasmidwithdifferentmarkers (figure

2.1) (Bähler et al. 1998),Ubp3was taggedwith a13-Myc tag andBre5with a3-HA tag.

Further, strainswere createdwithdeletions inBre5andUbp3 to formcombinations–a

Δbre5Ubp3-MycstrainandaΔubp3Bre5-HAstrain.Alongwiththese,Ialsomadestrains

withdeletionsofproteinsintheCcr4-Notcomplextoexploretherelationshipbetweenthe

complex and the DUBs. For this experiment, I made Δccr4 and Δnot4 mutants with the

taggedUbp3-Mycstrain.

2.2.2YeastTransformation

Yeast cell transformation of the tagging cassetteswas performed to generate the

desiredmutantstrains.AnovernightcultureofBY4741strainofSaccharomycescerevisiae

wasseededandthengrowntoanOD600of1.0.Thecellswereharvestedandwashedin

ddH2O, then resuspended in 1X lithium acetate (0.1 M Lithium Acetate). For the

transformation step, 1-3ug of cassette DNA, 50ug salmon sperm carrier DNA, and 50ul

yeastcellsuspensionweremixedina1.5mlsterilemicrofugetube.300ulPLATE(1mlv/v

10Xlithiumacetate,1mlv/v10XTris-EDTA(TE)pH8.0at21°C,8ml50%v/vPEG3350)

wasaddedandthetubewasvortexedfor10sec.Afterincubationat30°Cfor30min.,the

tubeswere placed at 42°C for 15min. The cellswere then spun at 4300 X g for 2min,

resuspendedinYPD,andincubatedinasterileglasstubeonarollerfor4-5hours.Around

200uLofthecellswasplatedoneachoffourselectiveplatesandincubatedat28°Cfor2-4

days. Colonies were re-streaked onto selective plates and PCR tested using two sets of

primers.Onesetwasdesigned toanneal to the insert toverify thestrains,and theother

was designed to anneal to the ORF to confirm that the strains contain the desired gene

deletions.

2.2.3Harvestingcellsandproteinisolation.

Cellswereinoculatedinto200mlrichmediaYPDandgrownat30˚CuntilanOD600

of1.0-2.0wasreached.Cellswerethencentrifugedat4000Xg,5mininaSLC6000rotorat

27

4°C. Cells from each bottle were resuspended in 3mls ice-cold dH2O containing 1 mM

Benzamidine-HCland0.25mMPMSFandcombinedintoasingle10mlscrew-toptubeand

werefurthercentrifugedat1500Xgor10minutesat4°C.Thepelletwasresuspendedin2

volumesof0.2MNaClWholeCellExtractbuffer(WCE)(25mMTris-HClpH7.5at21°C,0.2

M NaCl, 5 mMMgCl2, 2 mM EDTA, 10% v/v glycerol, 0.1% v/v triton-X100 – protease

inhibitors were added fresh 3µg/mL of leupeptin and aprotinin, 2ug/mL pepstatin A,

1ug/mLchymostatin,1mMbenzamidine-HCl,1mMPMSF).Acid-washedglassbeadswere

addedinequalvolumeasWCEandtubeswerevortexedrapidlyusingVortexGenie-IIfor

30secswith2minrestoniceforatotalof8times.Cellsuspensionwastransferredtoa

freshtubeusingaPasteurpipetandavoidingthebeads.Additional2mLofWCEwasadded

tothebeadstowashthemandthiswascombinedwiththeextractandthiswascentrifuged

in the J6 centrifuge for15minutes at 1300X g SS-34 rotor at 4°C.The supernatantwas

transferredintoafresh10mlscrewtoptubeandcentrifugedinaSS34rotorat38000Xg

for 30 min at 4°C. The final supernatant was carefully removed and used for protein

analysisandimmunoprecipitation.

2.2.4Bradfordassaytodetermineproteinconcentrations

Astandardcurvewaspreparedusing0,2,4,8,15,and20mg/mLBSAmixedwith

1mL 1X Bio-Rad Protein Assay Dye Reagent (dye, phosphoric acid, methanol) (Bio-Rad

#5000006). OD595 readings were plotted into a graph using Microsoft Excel. OD 595

readings from1uL samplemixedwith1mL1XBio-RadProteinAssayDyeReagentwere

made.Usingthestandardcurvegraphtogenerateanequationforthecalculationofprotein

concentrationfromODmeasurements,concentrationofproteinsintheunknownsamples

wasdeterminedbyinterpolationfromthegraph.

2.2.5ProteinImmunoprecipitation

1.0-2.0mgofproteinofeachextractwasbroughttoafinalvolumeof1mLin0.15M

NaClIPbuffer(25mMTris-HClpH7.5at21°C,0.15MNaCl,5mMMgCl2,2mMEDTA,10%

glycerol,0.1%NP40–proteaseinhibitorsaddedasbefore).Extractswereincubatedonice

for10minandwere thencentrifugedat13000Xg,4°C for10min.Thesupernatantwas

transferredtofreshtubesand2μLofpolyclonalantibody(1.0mg/mLconcentrationraised

28

inrabbit)wasaddedtoeachtube(antibodydependsontheassay–detailedinformationis

provided in the results section). The mixture was incubated at 4°C with end-over-end

mixing (Labquake) for about 1-3 hrs. The tubes were centrifuged again for 10 min at

13000Xg,4°Candthesupernatantwastransferredtoafreshtube.30μLofa50%slurryof

protein A sepharose beads (washed thoroughly in 0.3MNaCl IP buffer)was added and

againthetubeswereincubatedat4°Covernightwithend-over-endmixing.Thefollowing

morning, the tubes were centrifuged at 60 X g (lowest speed), 4°C for 2 min, and the

supernatantwas carefully removed. The beadswerewashed three timeswith 1.0mL IP

buffer0.15MNaClbymixingfor5minat4°Cend-over-end(labquake)andcentrifugingat

lowspeed(60Xg2min)andremovingthewashsolutioneachtime.Afterthefinalwash,

40µLof1.5XSDSloadingbufferwasaddedandthesamplewasheatedat90-100°Cfor4°C

min.Supernatantwasseparatedfromthebeadsbyspinningat600Xgandtransferredtoa

freshtube.

2.2.6SDS-PAGEandproteintransfer

Standard 6% SDS-PAGE gels were run and transferred according to the protocol

describedinpublishedliterature(Mahmood&Yang2012).

2.2.7Westernblottingandproteindetectionbyfluorescence

Thede-stainedmembranewasblockedfor1hrin5%milksolution(5gpowdered

milk,100mLTBST)thenincubatedwithaprimaryantibodyata1:1000dilutionin2%milk

solution overnight at 4oC. The primary antibody used was mouse monoclonal 8WG16

(manufactured by Covance) for detecting Rpb1. Afterwashing twice for 5minwith 2%

milk solution, themembranewas incubated at room temperature in darknesswith Cy5-

labeled anti-mouse (8WG16) secondary antibodies at a 1:3000 dilution for 1-2 hrs. The

membranewaswashedoncefor5minandtwicefor10minin2%w/vmilksolution,then

twicefor5mininTBSTbeforebeingscannedonaTyphoon8600ataPMTvoltageof500V

todetectfluorescence.

29

Figure 2.1: Integrative plasmid containing kanMXmarker and 3HA or 13Myc tag.

TheseplasmidswereusedtogeneratetaggedBre5andUbp3respectively.Imageextracted

from:(Bähleretal.1998)

30

2.2.8CellextractpreparationforRIP

1Lyeastculturesofeachstrainweregrownat30°CovernightinYPD(2%peptone,

1%yeastextractand2%dextrose)supplementedwith0.02mg/mladeninesulfatetoan

OD600ofapproximately0.9.Formaldehydewasaddedto~1%w/vfinalconcentrationand

incubated for 15 minutes, and the cultures were quenched with glycine ([final

concentration]=136mM)for5minutes.Cellswereharvestedbycentrifugation(4000Xg

for5minsat4˚C) inSLC6000rotorandthepelletwasresuspendedin20mlice-coldSTE

(TE plus 50mM NaCl) containing protease inhibitors (0.5mM phenylmethylsulfonyl

fluoride(PMSF);1mMbenzamidine-HCl)andtransferredtoasterile50mlcentrifugetube.

SampleswerespuninaJ6rotorat2000Xgfor15minutes,thesupernatantwasremoved

onceagainandthepelletwasresuspendedin18mlcoldSTE.Cellswerealiquotedinto24

microfugetubes(0.9mleach)andspunat10Kfor2minutesat4oC.

Eachcellpelletwasresuspendedincold250μlFA-lysisbuffer(50mMHEPES/KOH

pH7.5,150mMNaCl,1%TritonX-100,0.1%sodiumdeoxycholate)andcompleteprotease

inhibitors were added just prior to use (2ug/ml leupeptin; 3ug/ml aprotinin; 2ug/ml

pepstatin A; 1ug/ml chymostatin; 1mM benzamidine-HCl, 0.5mM PMSF). To the

suspension,300ulofglassbeadswereaddedandvigorouslyvortexed4timesbyhandfor

40seceachsample,andplacedonicebetweenbursts.Tubeswereplacedontheshakehead

vortexer and continuously shaken on high speed at 4°C for an additional 45mins. Two

hundredmicrolitersofFALysisbufferwasaddedtoeachtubeandtheextractseparated

from thebeadsby centrifuging.Total cell extract volumewas combined for each sample

andbroughttoabout1.8mlsbyaddinglysisbufferifneeded.

The sample was sonicated to liberate the RNA from the crosslinked material.

Sonication also breaks up RNA into smaller fragments. Samples were placed in a 15ml

polystyrenetubeandcoveredwithmetalcaps.Tubeswereplacedinice-coldwaterinthe

Bioruptor and set to 2 pulses (30 sec on and 30 sec off). Cell suspension is clarified by

centrifuging at 13000 X g for 15 mins at 4oC. Supernatant was transferred to a fresh

eppendorf tubeandcentrifugedagain for30minsat13000Xgand4oC.Clarifiedextract

wasanalyzedforproteinconcentrationusingBradford’sassayasdescribedearlier.

31

2.2.9RNAImmunoprecipitation

2.5mlsofWCEwasdilutedwithequalvolumeofFALysisbufferandplacedonice

for 5mins. 500ul of 10XMgCl2/CaCl2 (25mM/5mM) solutionwas added andmixed. To

each tube, 250ul RNAse-freeDNase I (concentration 4U/ul)was added and the solution

wasmixedgentlybyinversion.Sampleswereincubatedfor90minutesat30˚C(waterbath)

and mixed gently by inversion a few times. To stop the DNase treatment after the

incubation, 250ul of RNAse free 0.5 M EDTA was added. Sample was aliquoted into 4

microfugetubesandspunat13000Xgfor10minat4oC.Supernatantswereremovedand

dividedintotwoparts.Two100ulaliquotsweresetasideasinputsamples(each1/60of

the total) and the restwas incubated overnightwith 30ul of 50% protein A sepharose-

antibodycomplex,prepared independentlyby incubatingwith lysisbuffer-washedbeads

with20ulofantibodyper200ulofbeadsfor2-3hrsonend-over-endrotation.Beadswere

pelletedbycentrifugation(268Xg2min4oC),aspiratedandwashedasfollows.

Sampleswerewashedwith1mlofFA-lysisbufferoncewith5minincubation,and

twicemorewith10minincubations.Thiswasfollowedbytwo5minwashesin1mlofFA-

wash2(0.5MNaCl,50mMHEPES/KOHpH7.5at21°C,1mMEDTA,1%v/vTritonX-100,

0.1%w/vsodiumdeoxycholate;0.5MNaCl,0.5MPMSF,1mMbenzamidine-HCl).Finally,

beads are washed in 1mlWash buffer 3 (0.25 M LiCl; 1% v/v NP-40; 1% w/v sodium

deoxycholate; 1 mM EDTA; 10 mM Tris-HCl, pH 8.0 at 21°C, 0.5 M PMSF, 1mM

benzamidine-HCl)for5minandthen2minincubation.Ice-cold1XTEpH8.0wasaddedto

the beads andwashed for 5mins. An additional 0.5ml TEwas added, the tubesmixed

gently, and spun to remove the liquid immediately. Immune complexes were eluted by

adding450ulofElutionBuffer(25mMTris,pH7.5at21°C,1mMEDTA,0.2MNaCl,0.5%

w/vSDS) tobeads, vortexedbriefly tomix and incubatedat65°C for20minutes.Tubes

were vortexed gently and spun at 600 X g for 3 min. Input samples were processed

simultaneouslywiththeIPandwashedasabove.

Three microliters of 10 mg/ml Protease K was added to the IP samples and

incubated at 65 °C for 4-5 hours to digest protein and to reverse the protein-nucleotide

cross-linking.Tothe100ulof inputsamples350ulofelutionbufferand10ulof10%SDS

wasadded,followedwith3μlof10mg/mlProteaseKandincubatedat65oCfor4-5hours.

32

Tothis,400ulAcid-phenol(pH4.8)/chloroform(1:1)wasadded,vortexedfor15sec,spun

for4minutesat13K,and380ulofaqueousphasewasrecovered.RNAwasprecipitatedby

adding10ulof2mg/mlglycogen,20ulof3MNaOAc(pH5.0)andthen1ml100%ethanol.

Tubeswereinvertedmultipletimestomixwellandplacedat–80˚Cfor1-2hours.Sample

waspelletedbycentrifugingathighestspeedfor8-10minsandthenwashedoncewithice-

cold 75% v/v ethanol. Pellets were resuspended in DEPC treated water and the

concentrationwascalculatedusingaNanodropspectrophotometer.

ToremoveanyadditionalDNA,asecondDNasetreatmentwasdonebyadding8ul

DEPCwater,10ul10X(MgCl2-CaCl2buffer)DNase Ibuffer,1ulRNasin (RNase Inhibitor),

2.5ul10URNAse-freeDNaseIandincubatedat37˚Cfor20mins.Samplewasacidphenol

chloroform-isoamylalcohol(PCIAA)treatedandRNAwasprecipitated inthepresenceof

20ul of 3 M sodium acetate and 10ug glycogen with 550ul 100% ethanol. Tubes were

incubatedat -20˚Covernight.Thetubeswerespunatmaximumspeedfor8-10minsand

thepelletswerewashedoncewith80%v/vethanolandair-dried.ThefinalRNApelletwas

resuspended in DEPC-treated water and once again the concentration was measured

spectrophotometrically.Theprocedureisoutlinedinfigure2.2.

2.2.10Real-TimeReverseTranscriptase(RT)PCR

For thequantitativemeasurementofRNA immunoprecipitatingwith theproteins,

4ul of each RNA sample was incubated with random primers (0.01ug/ul, (Promgea

#C1181)) in a 30ul total volume for 10minutes at 70°C. The sampleswere then briefly

transferredtoroomtempandthenincubatedonicefor5minutes.Amastermixcontaining

ddH2O, AMV (Avian Myeloblastosis Virus) buffer, 1mM dNTPs, and 1 unit/ul AMV RT

(Promega#M510F)wasaddedto thereactioncontainingRNAandprimers.Thesamples

werethenincubatedat42°Cfor1hourtosynthesizecDNAfromtheRNAtemplate.cDNA

was then serial diluted so that therewere twodifferent concentrations in order to later

measure amplification efficiency of the real time reaction. Amastermix of forward and

reverseprimers,andQuantaPerfeCTaSYBRGreenSuperMixwasaddedto4ulofdiluted

cDNA (in duplicate) in a 96-well plate. The Applied Biosystems StepOnePlus instrument

wasusedtocarryoutReal-TimePCR.Tomeasuretheenrichment,%IPwasmeasuredas

theaveragevalue2^(CtMeanInput-CtMeanIP)betweenbothdilutions.

33

IPwithMycandHAwasdoneintheabovelistedstrainsandtheRNAwasisolated

afterreversingthecross-linkingovernightasdescribedinthemethods.Astandardcontrol,

no-RT(ReverseTranscriptase)PCRwasdonetodeterminethattheRNAsampleswerenot

contaminatedwithDNA.ThiswasusedasatemplateforPCRtoamplifyarandomhouse-

keepinggene(PDA1)withhighcopynumberandrunoutonastandardagarosegel(1.2%).

Once the samplewas determined to be free of DNA, 4ul of the RNAwas loaded in two

different concentrations 1:1 and 1:2 in DEPCwater and loaded in technical triplicate to

increaseaccuracyofPCR.The inputsampleswerealsodiluted1:5and1:10and4ulwas

loadedintriplicatealongsidetheIPsamplesonthesame96wellPCRplate.Amastermix

as described in the earlier sectionwas preparedwith primers toSKN7 orPYK1 and 8ul

addedtoeachwell.Threewellswereloadedwith4ulDPECwaterasnegativecontroland

combinedwiththemastermix.

FromthePCRresults,CT(CycleThreshold)wasrecordedandthedatawasanalyzed

inMicrosoftExcel.ThemeanCTof triplicates is takenandefficiencyofPCR is calculated

betweenthetwoconcentrationsofeachsample.FromthemeanCTthe%IPisestimatedby

firstmeasuring:

C=2^(INCT–IPCT)

Usingthisvaluewiththeappropriatedilutionfactor(DINorDIP)tocalculate%IP:

%IP=C*(DIP/DIN)*100

The%IP foreachsample iscalculated fromtheCTvaluesof the IP for thestrainand the

InputCTvaluesandadjustedforthedilutionfactorofthetwosets.The%IPforeachsample

isplottedonagraph,intheorder:Wild-typeornegativecontrol,Dhh1orpositivecontrol,

dualtaggedUbp3-13MycBre5-3HA,Δbre5Ubp3-13Myc,Δubp3Bre5-3HA.The%IPofeach

strain allows us to compare the difference between immunoprecipitation of a particular

sampleorproteinversustheInputorbackgroundsample.Theantibodyuseddetermines

which protein is studied and the %IP shows the percent over background

immunoprecipitatedwiththemRNA.

34

FormaldehydeCrosslinkingproteintoDNA&RNA

Celllysiswithbeadbeatingandsonication

DnasetreatmentI

ReversecrosslinkingproteinaseKandDnasetreatmentII

ProteinAconjugatedprimaryantibody

Beadwashingandprotein-RNAelution

RNAanalysisbyRealtimeRTPCR

cDNA

35

Figure 2.2: Outline of the RNA Assay: Cells are cross-linked using formaldehyde and

lysed. Nucleotide-protein conjugates are released by sonication and DNA is removed by

DNase treatment. Protein-A beads conjugated to primary antibody is used to target the

protein-of-interest and pull down the protein crosslinked to RNA. By reversing the

crosslinking, and an additional DNase treatment, RNA is released from the protein of

interest. RT and Realtime PCR is used to generate cDNA and determine quantitative

analysisoftheRNA-proteininteractions.

36

2.3Results

Thepurposeof this studywas toexplore theextentof interactionbetweenUbp3,

Bre5,theCcr4-NotcomplexandRNAPolII.Understandingtheprotein-proteinassociation

is key to constructing a model for how Not4 functions in the ubiquitination and

degradationofRpb1under4NQOstress.TheassayforproteinIPisqualitativeandserves

to show the physical interaction of the proteins immunoprecipitated and the proteins

probed for.Theassaydoesnot account fordirector indirect associationof the proteins;

hencetheresultisincomplete.ThehypothesisisthattheCcr4-Notcomplexmayalsoplaya

roleinthelast-resortrescueofRpb1byassociationwithUbp3andBre5.Thisimpliesthat

theroleofNot4 isnot justasaRINGE3ubiquitin ligase,butalsoasaregulatorofstress

response to DNA damage by controlling whether PolII will resume transcription, or be

dismantledbyGGRspecificfactors.

To explore the interaction between the Ccr4-Not complex and the Ubp3/Bre5

complex, the sampleswere RNA Immunoprecipitated (RIP). Thismethod is used for the

identificationofmRNAsthatassociatewithUbp3andBre5.IfUbp3andBre5arerecruited

tothesamemRNAasCcr4,itcouldshedsomelightonthemethodofinteractionofthetwo

protein complexes. Ubp3/Bre5 could associate with the Ccr4-Not complex or to PolII

throughthemRNA.

2.3.1AnalyzingUbp3’sassociationwithitscofactorBre5

The first step is to determine if the tagged proteins, Ubp3 andBre5, still interact

witheachotherinanimmunoprecipitation.Usingthedualtaggedstrain,proteinimmuno-

precipitation(IP)wasperformedononeofthetwoproteins–Ubp3orBre5–followedbya

westernblotfortheother.ImmunoprecipitationwassetuptoeitherMyc(forUbp3)orHA

(for Bre5) and the blot was incubated with antibody to Bre5 (HA) or Ubp3 (Myc)

respectively,toobservetheinteractionofBre5toUbp3andviceversa.Itisexpectedtosee

a band for Bre5 in the assay with Ubp3 and a band for Ubp3 in the assay with Bre5,

confirmingthatthetwoproteinsdoassociatewitheachother.

Figure2.3(a)showstheblotprobedforBre5-3HAwithananti-HAantibodyinthe

toprowandUbp3-13Mycprobedwithananti-Mycantibodyinthebottomrow.TheInputs

fromeachsamplearetotheleftandtheIPsaretotheright. Infigure2.3(a), it isevident

37

thatbothUbp3andBre5immunoprecipitatedwitheachotherinthedualtaggedstrainin

lane8.Asexpected,noneoftheotherIPlanes(5-7)showanyproteinbandsbecausethe

strainslackBre5orUbp3.Thecorrespondinginputlanesshowtheproteinconcentration

ineachstrainloadeddirectlyfromthecellextractwithoutperforminganIP.Theinputsin

thelowerpanelappeartoshowadecreasedabundanceofBre5intheubp3mutantdespite

adjustingtheconcentrationsloadedusingthevaluesfromtheBradford’sassay.Thiseffect

was observed on multiple replicates and may indicate a possible decrease in the

expression,orstabilityofBre5intheabsenceofUbp3.

TheinputsamplesshowanumberofsmallerbandsbelowtheprimaryUbp3protein

bandwhichmaybepartiallydegradedspeciesofUbp3.Thebandingpatternseeninthetop

panelofinputsamples(2.3alanes3,4)butnotintheIPwithBre5(lane8)suggeststhat

the degradation of Ubp3 could be in the C-terminus region (opposite to the tag), and

appearstoaffectthebindingofBre5interactionwithUbp3(referfigure1.5aforUbp3and

Bre5interaction).

2.3.2ProteinImmunoprecipitationwithRNAPolII

OnceUbp3andBre5areshowntobeassociatingwitheachother,thenextstepof

verification is to confirm that they associatewith RNA PolII as previously observed and

whattheeffectofadeletionofoneofthetwoproteins(Ubp3andBre5)wouldbeonthis

interaction.Followingsimilarmethodsasforthepreviousimmunoprecipitations,WCEwas

incubatedwithPolIIantibody(8WG16)andwesternblottedforUbp3orBre5.Additionally,

theassaywasdoneinmutantsstrainsΔccr4andΔnot4todetermineiftheseproteinsplay

anyroleintheinteraction.

From theblots in figure2.3b,bothUbp3andBre5 immunoprecipitatedwithPolII

(figure2.3blane12),andintheabsenceofBre5,Ubp3isstillrecruitedtoPolII(lane11).

Onthecontrary,intheabsenceofUbp3,noBre5wasdetectedintheIPwithPolII(lane10).

ThiscouldsuggestthatUbp3mightbeessentialforthestabilizationoftherecruitmentof

Ubp3-Bre5complextoRNAPolII.ItappearsthatneitherCcr4norNot4arerequiredforthe

recruitmentofUbp3-Bre5complextoRNAPolII(lanes8and9).OnlyUbp3wastaggedin

thesestrainsanditisassumedthatBre5isalsorecruitedtoPolIIwithUbp3.Thisdoesnot

ruleoutotherinteractionsviatheCcr4-Notsubunit.Theanalysisrequiresmoresignificant

38

experimentation with other members of the Ccr4-Not complex to determine the role it

playsintheassociation,aswellastodetermineiftheassociationisdirectorindirect.

2.3.3DetectproteinassociationswiththeCcr4-NotComplex

Tocompletetheassociationanalysis, thefinalpieceofthepuzzle istounderstand

how Ubp3-Bre5 interacts with the Ccr4-Not complex and what the impact of deleting

proteincomponentswillbeon this interaction.Because theΔccr4andΔnot4 strainsonly

haveataggedUbp3protein,thisexperimentonlylookedattheinteractionofthecomplex

(Ubp3-Bre5) as a whole assuming the two proteins stayed associated with each other.

PolyclonalantibodiestoNot4oftheCcr4-Notcomplexwereusedtoimmunoprecipitatethe

Ccr4-Not complexandeitherMycorHAantibodieswereused tovisualizeUbp3orBre5

respectivelybywesternblot.

In the assay with Not4, (figure 2.3c) interestingly, both Ubp3 and Bre5 were

detected in thedual taggedstrain,but in theabsenceofBre5onlya faintband forUbp3

wasdetectedintheblot(lane11figure2.3c).Bre5immunoprecipitateswithNot4evenin

the absence ofUbp3 (lane10 figure2.3c). Additionally, in theUbp3-13MycΔccr4 strain,

Ubp3wasstilldetectableindicatingthatCcr4isnotnecessaryfortheinteractionofUbp3-

Bre5with the Ccr4-Not complex. This result shows that Bre5may be necessary for the

associationofUbp3totheCcr4-Notcomplex,andBre5eitherrecruitsUbp3totheCcr4-Not

complexorstabilizestheinteraction.

The immunoprecipitation assay only accounts for association of the proteins, but

doesnotdetermineiftheassociationisdirectorindirectthroughanotherproteinorother

factor.Usingjusttheinformationfromthegels,iftheassociationisdirect,amodelcanbe

postulated from the interactionpattern.Theoverallproposedassociation,basedononly

theresultsfromtheassayisdescribedinfigure2.4,whereUbp3isrequiredfortheDUBto

be recruited topolymerase, andBre5 is necessary tobridge the gapwithCcr4-Not. This

figureisincompletesinceBre5maystillassociatewithPolII,andtheinteractionscouldbe

indirectlymediatedbyotherproteinsornucleicacids.

39

Input IP

2.3a

12345678

Ubp3Bre5

Input IP

123456789101112

2.3b

IP:8WG16(Rpb1)Western:Ubp3-MycWestern:Bre5-HA

Ubp3Bre5

40

Figure 2.3: Protein immunoprecipitation using tagged strains for Ubp3 and Bre5.

Input samples to the left indicate the concentration of each protein in the strains. (a)

InteractionofUbp3withBre5.Ubp3wastaggedwitha13-MyctagandBre5witha3-HA

tag. Strains lacking Ubp3 and Bre5 do not show the association. (b) IP with antibodies

against polymerase shows Ubp3 and Bre5 associating with polymerase. (c) IP with

antibodiestoNot4oftheCcr4-NotcomplexalsoshowsassociationofUbp3andBre5.

2.3c

Input IP

123456789101112

Ubp3Bre5

IP:Not4Western: Ubp3-13Myc

Bre5-3HA

41

Figure2.4:DiagramshowingtheproposedmodelforinteractionofUbp3/Bre5with

PolIIandtheCcr4-Notcomplex.Fromthegelsintheproteinimmunoprecipitationassay,

Ubp3isseenassociatingwithPolII, irrespectiveofdeletionofnot4orccr4.Althoughit is

notknownwhetherBre5 independently interactswithPolII, in thismodel, it is assumed

thatitsinteractionisthroughUbp3.PreviousstudieshavealreadyshownthattheCcr4-Not

complexassociateswithPolII,andtheIPassaysinthisthesisindicatethatBre5associates

withtheCcr4-Notcomplexevenintheabsenceofubp3.

42

2.3.4RecruitmentofproteinstomRNA

Sincetheproteinimmunoprecipitationdoesnotaccountforindirectassociation, it

is possible that Ubp3-Bre5may interactwith PolII or the Ccr4-Not complex via another

protein, or even by recruitment to mRNA. To test the theory that Ubp3 and Bre5 are

recruitedtoRNA,analteredmethod forprotein immunoprecipitationwasusedtodetect

relativeRNArecruitmentusingRNAImmunoprecipitation(RIP).Inthismethod,mRNAis

crosslinked to proteins and nucleic acids (both DNA and RNA) using formaldehyde and

isolatedfromcellculturesandpurified.TheRNAisbrokenintofragmentsassociatedwith

RNAboundproteinsandtheseproteinsareimmunoprecipitatedusingantibodiesspecific

to their tag.Onceprecipitated, the crosslinking is reversedand theRNA is isolated.This

RNA is reverse transcribed to cDNA using reverse transcriptase. Using Real-Time

quantitative PCR, the RNA copies for a selected gene are quantified andmeasured. The

moreanmRNAisenrichedforinaparticularimmunoprecipitate,themoresignificantthe

recruitmentof theprotein is to themRNA.By immunoprecipitatingtaggedproteins from

thedifferentstrains:Ubp3-13Myc,Bre5-3HA,Δubp3Bre5-3HA,Δbre5Ubp3-13Myc,itcan

be determined if Ubp3 or Bre5 or both associate with mRNA, and if either protein is

necessaryfortheinteraction.BecauseitisnotknownwhichmRNAinparticulartheymay

target(ifany), thesampleswereexperimentallyassayedformRNAsthatareenrichedby

RIPexperimentsinvolvingcomponentsoftheCcr4-Notcomplex.

Inpreviousexperimentsperformedinthelaboratory,RIPwasdoneforcomponents

of the Ccr4-Not complex – like Ccr4 and Dhh1 – and followed up with genome-wide

sequencingofthemRNAtodeterminethemRNAtargetsforCcr4andDhh1(J.E.Miller,J.C.

Reese,paperinreview).TheexperimentidentifiedseveralprominenttargetsforCcr4and

Dhh1amongthemRNAtranscripts,amongwhichtwowerealsotestedinthisstudy–SKN7

andPYK1.ThesemRNAswerechosenbecausetheywerehighlyenrichedtargetsforCcr4.

Because it is possible that Ubp3/Bre5 may interact with the Ccr4-Not complex, these

targetsweregoodinitialcandidatestoestablishwhetherUbp3and/orBre5arerecruited

to mRNA. To estimate the recruitment of Ubp3 and Bre5, Dhh1 was used as a positive

controlbecauseit ishighlyrecruitedtobothmRNAstudiedinthisthesis.Untaggedwild-

typesampleswereusedasabackgroundmeasure.

43

In three independent experiments, the results showed a few consistent results

regardingtherecruitmentofUbp3toSKN7andPYK1.However, theexperimentdoesnot

have a positive control for anHA-taggedRIP, hence the results forRIPwith anti-HA for

Bre5 recruitment is inconclusive. Because the tags used for detecting both proteins are

different,13MyconUbp3and3HAonBre5, theoutputbetween the twoantibodiesvary

considerably.Additionally,theefficiencyoftheantibodies,MycorHA,coulddeterminethe

efficiency of the RIP, however, the amount of antibody used was determined to be

saturated.ThestructureofUbp3-Bre5dimercouldalsoalterthepositioningofthetagand

the accessibility of the antibody. The experiment is not designed to compare the exact

recruitmentofUbp3andBre5,butinsteadtosimplydetermineiftheproteinsarerecruited

tomRNA.Theerrorbarson thegrapharecalculated fromthestandarddeviationacross

threereplicatesperformedforeachofthestrains.

Fromfigure2.5aand2.6a,thebargraphsshowtheresultsoftheRNAIPwithanti-

MyctoPYK1andSKN7respectively.Dhh1asthepositivecontrol(indicatedinred)forthe

RIP showed statistically significant recruitment (p-values 0.034 and 0.017) toPYK1 and

SKN7respectively(t-testp-valuesarelistedintable2).TherecruitmentofUbp3toPYK1in

the dual-tagged strain (grey bar figure 2.5a) is also significant (p-value 0.017), implying

that protein complex as a whole may be recruited to the PYK1 mRNA. Ubp3 does not

appeartobesignificantlyrecruitedtoPYK1 in theΔbre5strain(2.5a,yellowbar,p-value

0.148).

In figure 2.6a, recruitment of Ubp3 to SKN7 is not statistically very significant in

eitherthedualtaggedstrain(greybar)ortheΔbre5mutantstrain(yellowbar)(p-values

0.064and0.088).Thisresultmaybeabiologically importantphenomenonregardingthe

recruitmentofUbp3tospecificmRNA(suchasPYK1)andnottoothers(likeSKN7).

TherecruitmentofBre5-HAtoPYK1andSKN7isshowninfigures2.5band2.6bwith

thep-valuesandstandarderrorslistedintable2.Inthedualtaggedstrain,Bre5doesnot

show a statistically significant recruitment to PYK1 (2.5b grey bar, p-value 0.060). The

recruitmentisalsonotsignificantintheΔubp3strain(2.5bdarkbluebar,p-value0.155).

Duetothelackofapositivecontrol, therecruitmentofBre5cannotbeinferredfromthe

RIP experiments. Similarly, in figure 2.6b, Bre5 is not significantly recruited to SKN7 in

44

eitherthedualtagged(greybar,p-value0.059)ortheΔubp3mutantstrains(darkbluebar,

p-value0.440).

Thefullscopeofthisprojectwasnotattained,buttheexperimentshaverevealeda

possiblespecificrecruitmentofUbp3/Bre5tocertainmRNAinaBre5-dependentmanner

thatmayhelpexplaintheprotein’sfunctionbetter.Thedifferenceinrecruitmenttothetwo

mRNA,aswellastheroleofBre5intherecruitmentneedtobefurtherexplored.Additional

experimentationwithexpansiontoothertargetmRNAandstressinducedRIPprocedures

mayexpanduponthegenome-widerecruitmentofUbp3andBre5tomRNA,aswellasthe

roleofBre5andUbp3inidentifyingmRNA.

45

Table2.StatisticalanalysisforRIPwithPYK1andSKN7.mRNAIP-antibody

PYK1-Myc %IP Standarderror T-testpvaluesWildtype 1.663483608 4.05013132 0Dhh1:Myc 26.60451337 3.816128681 0.034919562 Ubp3:Myc Bre5:HA 24.74532598 5.921914282 0.017189921 Δbre5 Ubp3:Myc 42.26902168 11.42898272 0.148348464 Δubp3 Bre5:HA 6.390076158 3.261459592 0.12492601

PYK1-HA %IP Standarderror Wildtype 4.478132969 0.306823078 0Dhh1:Myc 4.96299142 0.0 0.332640948 Ubp3:Myc Bre5:HA 9.654530083 3.724662054 0.06016574 Δbre5 Ubp3:Myc 2.760567699 0.638473238 0.00975957 Δubp3 Bre5:HA 9.062466494 3.263107344 0.155699447

SKN7-Myc %IP Standarderror Wildtype 1.878999801 1.625451355 0Dhh1:Myc 29.88442351 7.895574033 0.017459373 Ubp3:Myc Bre5:HA 81.87877472 18.95356103 0.064954902 Δbre5 Ubp3:Myc 18.35589584 10.13602043 0.088364468 Δubp3 Bre5:HA 4.859551045 2.038253023 0.424224192

SKN7-HA %IP Standarderror Wildtype 3.973459462 0.528847677 0Dhh1:Myc 4.312019802 0.0 0Ubp3:MycBre5:HA 1.414849118 0.065473554 0.059304907 Δbre5Ubp3:Myc 1.271837056 0.636505881 0.013203349 Δubp3Bre5:HA 5.973404012 1.940091062 0.440246479

The table shows the RIP samples immunoprecipitated with the tag Myc or HA and

quantified for the mRNA PYK1 or SKN7. The %IP values show the relative percent of

recruitment of the sample when compared to input samples. The standard error was

calculated from three independent experiments and the p-values were calculated

statisticallyusingstudentst-test.

46

Figure2.5:ResultsshowingtherecruitmentofUbp3andBre5toPYK1mRNA.

TheY-axis shows thepercent IP.TheWildtype strainactsas thebackgroundsignal

andtheDhh1-Mycstrainwasusedasapositivecontrolbasedonpreviousworkthat

identifiedthe interactionofDhh1withPYK1mRNA.(a)InanIPwithanti-Myc,dual

taggedUbp3andBre5showhighenrichmentwithPYK1mRNA(thirdbar).Even in

theabsenceofBre5,therecruitmentisnotdiminished(fourthbar).(b)Inthereverse,

with anti-HAantibodies, the recruitmentofBre5 tomRNA is significant in thedual

taggedstrain(thirdbar)andsomewhatsignificantintheΔubp3mutant(fifthbar).

A

B

0

2

4

6

8

10

12

14

16

% IP

PYK1 anti-HA

Wt

Dhh1:Myc

Ubp3:Myc Bre5:HA

Δbre5 Ubp3:Myc

Δubp3 Bre5:HA

-5

0

5

10

15

20

25

30

35

% IP

PYK1 anti-Myc

Wt

Dhh1:Myc

Ubp3:Myc Bre5:HA

Δbre5 Ubp3:Myc

Δubp3 Bre5:HA

47

Figure2.6:ResultsshowingtherecruitmentofUbp3andBre5toSKN7mRNA.TheY-

axis shows the percent IP using the untaggedwildtype strain as background. Dhh1-Myc

strainwasusedasapositivecontrolbasedonpreviousworkthatidentifiedtheinteraction

of Dhh1 and SKN7 mRNA. (a) In the IP using anti-Myc antibody, Ubp3 shows very high

enrichmenttoSKN7mRNAinthedualtaggedstrain(thirdbar).IntheabsenceofBre5,the

recruitment ofUbp3 is slightly diminished but still significant (fourth bar). (b) In the IP

withanti-HA,thereisnosignificantenrichmentinanyofthesamples.

A

B

0

1

2

3

4

5

6

7

8

9

% IP

SKN7 anti-HA

Wt

Dhh1:Myc

Ubp3:Myc Bre5:HA

Δbre5 Ubp3:Myc

Δubp3 Bre5:HA

0

10

20

30

40

50

60

70

80

90

% IP

SKN7 anti-Myc

Wt

Dhh1:Myc

Ubp3:Myc Bre5:HA

Δbre5 Ubp3:Myc

Δubp3 Bre5:HA

48

2.4Discussion

Protein-proteinassociationsandstudyoftheirrecruitmenttootherfactorshelpto

understandhowthesefactorsinteractwitheachotherandcarryoutfunctionswithinthe

cell.ThepurposeofthissectionwastodeterminehowtheCcr4-Notcomplexinteractswith

the Ubp3/Bre5 complex, and to begin to understand how this interaction regulates the

functionofbothproteincomplexes.

Theresults shown in figure2.3outline thebasicphysicalassociationofUbp3and

Bre5toeachother,theCcr4-Notcomplex,andtoRNAPolII.Becausetaggingproteinscan

alter their structure, interfere with function and affect interaction with other proteins,

protein immunoprecipitation was performed with known functional associations. Ubp3

andBre5areknowntoformacomplextogetherandthiscomplexisconsideredessential

for their functions. Theprotein immunoprecipitations indicate that the taggedUbp3 and

Bre5associatewitheachotherasexpected.Theonlyoddphenomenonobservedinseveral

blotswasthedecreaseintheintensityofthebandforBre5-3HAwhenUbp3wasdeletedin

the input samples (figure 2.3a input lane 3). This may indicate a decrease in relative

abundanceofBre5intheΔubp3mutantbuttheinferenceis inconclusivesinceitwasnot

consistentinfigure2.3b.

ThepresenceofpartiallydegradedspeciesofUbp3 (seen inall the input samples

butnotimmunoprecipitated)couldbebiologicallyrelevanttounderstandtherecruitment

oftheUbp3/Bre5complextoPolIIaswellasthefunctionofUbp3.TheC-terminusofUbp3

containsthecatalyticdomain(figure1.5a)andtherecouldbearoleforBre5inpreventing

thecleavageof theC-terminus.Bre5maydissociate fromUbp3 toallow its cleavageand

inhibititsfunctionasaDUB.

The self-association experiment was followed up with an inquiry into the

interaction of Ubp3/Bre5with theRNAPolII. In addition to the strains used previously,

twomorestrainswithmutantsintheCcr4-Notcomplexwereincluded.Thedualpurposeof

thisexperimentwastodeterminewhatroleUbp3andBre5playedintheinteractionwith

PolII, as well as the possibility of Ccr4 and Not4 affecting this interaction. Despite the

possibility of a decreased abundance of Bre5 in the Δubp3 Bre5-3HA strain, it was

determinedthatBre5isnotessentialfortheinteractionofUbp3withPolII,andneitherare

49

Not4 and Ccr4. Although it cannot be concludedwhether Bre5 interactswith PolII, it is

likely thatUbp3 can interactwithPolII independent of Bre5.As discussed in the results

section, the assay does not concludewhether the association of Ubp3-Bre5with PolII is

directorindirectthroughanotherproteinormRNA.

Onehypothesiswas thatUbp3/Bre5couldberecruited toRNAPolIIvia theCcr4-

Notcomplex.Althoughitisnotpossibletoentirelydeletethecomplex(asitisessentialfor

cell survival), Not4 and Ccr4 were selectively deleted to observe the outcome on the

recruitmentofUbp3/Bre5toPolII.WhileneitherproteinaffectedtherecruitmentofUbp3,

thesestrainsdonotcontainataggedBre5forobservation.Tofollowupontheinteraction

withtheCcr4-Notcomplex, theassaywasperformedwithNot4andprobedforBre5and

Ubp3. Itappears thatUbp3maynotassociatewithNot4robustly in theabsenceofBre5,

butBre5doesassociatewithNot4evenintheabsenceofUbp3.Thisresultcouldindicatea

role for Bre5 in the recruitment or stabilization of Ubp3/Bre5 to the Ccr4-Not complex.

Again,theassociationisnotconfirmedtobeadirectonesincetheIPisdonewiththeentire

cellextractandcouldbemediatedbyotherfactors.

BecauseBre5 andUbp3 associatewith both PolII and the Ccr4-Not complex, it is

interestingtoexplorethepossibilitythattheUbp3/Bre5complexisalsobeingrecruitedto

mRNA.TheCcr4-NotandPolIIassociatewithmRNAanditislikelythatmRNAmaybridge

some of the interactions between these various proteins. Additionally, theRRMonBre5

andpreviousstudiesonUbp3suggesta linkwithmRNA.ByselectingmRNAthatCcr4 is

already known to bind, it is possible to see if Ubp3 and Bre5 also followed similar

recruitment patterns. Although the assay does not determine if the recruitment of

Ubp3/Bre5tomRNAisdirect,itprovidesinsightintothepossiblefunctionsofthecomplex.

Both PYK1 and SKN7 are prominent targets for Ccr4 recruitment under stressed and

unstressed conditions. The idea is to follow this experimentwith a genome-wide study,

similar to theoneconductedwithCcr4,and lookat similarities in therecruitmentof the

twocomplexes(discussedinchapter4).

TherecruitmentofUbp3toPYK1issignificantasindicatedbythep-valuesintable2

but less so for SKN7. The Δbre5 mutant does not appear to significantly decrease the

recruitmentofUbp3toPYK1.TheseobservationsmayindicatethatUbp3’sassociationwith

PYK1isindependentofBre5yetitsassociationwithSKN7mayrequireBre5.Itsuggestsa

50

dynamic relationship between Ubp3 and Bre5 which may assist in the identification of

specificmRNAandrecruitmenttomRNAinastress-specificmanner.

On the other hand, Bre5 does not appear to be recruited to SKN7andPYK1 in a

similar manner. Bre5 is not significantly recruited in any of the strains, indicating that

theremaybeadifferentmechanismfor therecruitmentofUbp3andBre5tomRNAIt is

possiblethatUbp3mayberecruitedtoPolIIortotheCcr4-NotcomplexthroughmRNAor

maybeselectivelyrecruitedtoparticularmRNAtoidentifythetranscriptthatneedstobe

upregulated.Ubp3couldfunctioninasimilar,antagonisticwaytotheCcr4-Notcomplexby

targetingmRNAthatareessentialforstressresponseandcounteringtheubiquitinationof

Rpb1 on those transcripts. This is an efficient method for the cell to regulate the

transcriptionofgenesnecessaryforsurvival,whileterminatingthetranscriptionofother

genesthatarenotessential.ItisstillunclearwhattheroleofBre5isintherecruitmentof

Ubp3orintheidentificationofmRNAforstress-regulation.

51

Chapter3:Not4’sfunctionasanE3RINGubiquitinligaseinPolIIdegradation

3.1Introduction

TheresponsetoDNAdamagedependsonseveralfactorssuchastheseverityofthe

damage, the type of damage, the location and the effect of the response. As mentioned

earlier, in extreme cases when the damage is not repaired by transcription-coupled

machinery,cellsresorttoamechanismwhereinthelargestsubunitofRNAPolymeraseIIis

ubiquitinated and degraded in a proteasome-dependent manner. This disengages the

elongationcomplex(EC)fromtheDNAwithminimaldegradationofotherproteins,giving

waytothegeneralgenomicrepairmachinerytofixthedamage.

Several factors have been implicated in the process of ubiquitination of Rpb1. In

yeast,mostnotableareRad26andDef1(Woudstraetal.2002).Def1isitselfubiquitinated

and processed by the proteasome upon DNA damage and transported into the nucleus

wherein it interactswith Rad26 and assists in the ubiquitination of Rpb1 (Wilson et al.

2013).However,Def1isnotapartoftheubiquitinationmachinery,henceitmustinteract

withanE1,E2and/orE3tofacilitateubiquitinationofitselfandRpb1.AccordingtoWilson

et al (Wilson et al. 2013), Def1 recruits the Elongin-Cullen complex, which is an E3

ubiquitin ligase shown tomediate the ubiquitination and degradation of PolII under UV

stress (Woudstra et al. 2002; Yasukawa et al. 2008). However it is likely that the

mechanismofresponsemayvarydependingonthedamageandotherfactors.Henceit is

likelythattheproteinsinvolvedintheresponsemayalsodiffer.Oneproteinthatmaybe

involvedwith theprocessofubiquitinationofRNAPolII isNot4.AsaRINGE3Ubiquitin

ligase that is already associated with the Elongation Complex and part of the Ccr4-Not

complex,itispossiblethatNot4mayplayaroleinubiquitination.

BecauseNot4hasaroleintheturnoverofRpb1asdescribedinprevioussectionsof

thisthesis,itisimportanttoknowifthisisduetoitsroleinubiquitinationofRpb1,ordue

toanalternatefunctionintheturnoverofRpb1.ToexploretheroleofNot4asaubiquitin

ligase in conjugatingubiquitinmoieties toRpb1, immunoprecipitation experimentswere

done todetermine if thepolymerasewasundergoingubiquitination in thepresence and

52

absenceofNot4aswellasothersubunitsoftheCcr4-Notcomplex.Theseexperimentsare

discussedindetailinthischapter.

53

3.2MaterialsandMethods

3.2.1Strainsandconditions

In order to effectively visualize ubiquitin substrates, all strains previously made

weretransformedwithaplasmidcontainingaHAtaggedubiquitinspecies–pRG426(2u

URA3). Strainswere already lacking pdr5, a pleiotropic drug resistance transporter that

confersmulti-drugresistanceinyeast(Balzietal.1994).Theabsenceofpdr5ensuresthat

anydrug-inducedstresswillbemoreefficient,withoutthedrugbeingtransportedoutof

thecell.AdditionofMG132,whichisaproteasomeinhibitor,preventsthedegradationof

proteins upon stress-induced ubiquitination. Strains were created with the Δpdr5

background and the following gene deletionsweremade by standard approaches in the

Ccr4-Notcomplex–Δnot4,Δnot5,Δdhh1andΔccr4.

3.2.2Stress-inducingchemicals

Apeptidealdehyde,MG132or carbobenzyl-Leu-Leu-Leu-aldehyde,wasoneof the

first classesofproteasome inhibitorsdevelopedand is themostwidelyused (Kisselev&

Goldberg 2001). These aldehyde inhibitors are slow binding but reversible. MG132

functions by inhibiting the chymotrypsin-like site; the pharmacophore region interacts

with a catalytic residue to form a covalent adductwhile the peptides associatewith the

activesite in thebindingpocketof theenzyme.MG132actsasaproteasome inhibitor to

ensurethattheproteinisnotdegradedbeforeitcanbeobserved.

4-Nitroquinoline (4NQO), one of the chemicals that is used for stress induction, is a

mimic of UV damage on cells. It is a quinolone derivative that is a known tumorigenic

compound and induces DNA lesions. These lesions are normally repaired by the cell’s

NucleotideExcisionRepair(NER)machinery(LaVoieetal.1983).

6-Azauracil(6AU)isatranscriptionelongationshut-offdrugthatworksbydecreasing

GTP and UTP pools (Exinger & Lacroute 1992). This method is a very effective way of

haltingtheElongationmachineryandmimickingastress-inducedarrest.

3.2.3CellGrowthandMG132/4NQOTreatment.

Yeastcultureswereinoculatedinto50mLminimalnitrogenbasemediawithamino

acidmix lackingUracil andwere grown at 30˚C until anOD600of 1.0. Sampleswere as

54

follows:WildtypeΔpdr5,Δnot4Δpdr5,Δccr4Δpdr5,Δnot5Δpdr5andapositivecontrolofa

proteasomemutantpre1,2.Theproteasomemutantinhibitstheproteasomeandwasused

as a positive control for the visualization of ubiquitinated proteins and to check the

efficiencyofMG132.Foreachofthesesamples,fourseparate50mLculturesweregrown,

except for the proteasome mutant where only two cultures were grown because these

strainsdonotrequiretheproteasomeinhibitorMG132.Twosetsofsamplesweretreated

with50uMMG132permLofsamplefor1hourtoinhibitthefunctionoftheproteasome.

OneoftheseMG132treatedsamplesandathirdsamplewasfurthertreatedwith6ug/mL

4-Nitroquinoline1-oxide(4NQO)for1houraftertheMG132treatmenttocausetheDNA

damage-inducedarrestofRNAPolII.Thefourthsamplewasleftuntreatedascontrol.For

the proteasomemutant, one flaskwas treatedwith 4NQO and the otherwas untreated.

Cultureswereharvested as described earlier. Assayswere set up as described in earlier

sections.

3.2.46AUtreatment

Cellsweregrownina50mLculturetoanOD600of1.0and100ug/mlof6-Azauracil

(6-AU)wasaddedfor2hourstoonesetofculturestoinhibittranscriptionelongationby

decreasingtheGTP(GuanosineTriphosphate)andUTP(UridineTriphosphate)production.

Theother culturewas leftuntreatedas control. Cellswereharvestedby theTCAextract

preparation.

3.2.5TrichloroaceticAcid(TCA)extractpreparation

ThisprocedurewasusedinsteadoftheIPextractpreparationtopreparewildtype,

Δnot4,Δccr4,andΔnot5cellextractsfollowing6-AUtreatment.Allstepswerecarriedoutat

roomtemperatureunlessotherwiseindicated.Cellsweregrownandtreatedwith6-AUto

inhibittranscriptionelongation,10mLofculturewasremovedandspun10minat600Xg.

Thepelletwasresuspendedin1mL20%v/vTCA,spunfor2min600Xg,aspiratedand

resuspendedin200ul20%v/vTCA.Twohundredandfiftymicrolitersofglassbeadswere

addedandthe tubesvortexedtwice foroneminutebeforebeingplacedonashakerhead

vortexer for 10min. An additional 400ul 5% v/v TCAwas added before the lysatewas

separatedfromthebeadsbycentrifugation.Thetubewasspun2400Xgfor10min,then

thepelletwaswashedin1mL0.25MTrispH7.4at21°C.Thetubeswerespunat2400Xg

55

for5min,thesupernatantaspirated,thenthepelletwasresuspendedin100ul0.25MTris

pH7.4at21°Cand150uL3XSDS-PAGEloadingbuffer.Finally,thesampleswereboiled4-5

min,spunat11000Xgfor10min,and200-300ulofsupernatantwastransferredtoanew

tube.

3.2.6SDSGelrunningandsoaking

Sixpercentacrylamidegelswerepouredaspreviouslydescribedandrunin1XTris-

glycine SDS at 30mA per gel for 45min. Because heavily ubiquitinated proteins tend to

moveslowly throughgels, theyaredifficult to transferontomembranes.To increase the

efficiencyofthetransfer,gelsweresoakedfor20minsinasoakingbufferwith1%SDS,5%

2-mercaptoethanol, 63 mM Tris-HCl, pH 6.8 prior to setting up transfer (Mimnaugh &

Neckers 2005). After soaking, the gel was transferred onto a 0.2um reinforced

nitrocellulosemembraneusingthetanktransfermethod(1XTrisGlycinetransferbuffer-

25mMTris,190mMglycine20%methanol).

3.2.7ECLVisualization

Thesenitrocellulosemembraneswereblockedin5%w/vmilk inTBSTfor1hour

and set in primary antibody overnight at a 1:1000 dilution in 2%w/vmilk in TBST as

previouslydescribed.Theblotwaswashedtwicefor5minin2%w/vmilkandTBSTand

incubatedin1:1000dilutionofHRP-(horseradishperoxidase)conjugatedsecondaryanti-

mouseantibodyfor1hour.Theblotwaswashedagaintwicein2%w/vmilksolutionand

twiceinTBST.

FortheECL(EnhancedChemiluminescence),akitfromThermoScientificwasused.

Togeneratethesubstrate,5mlofreagentAand5mlofreagentBweremixedandtheblot

wasincubatedinthissolutionfor1minwithgentletiltingandshaking.Theblotwasthen

removedanddrainedofexcessliquidandplacedonaglassplatewiththeproteinsideup

andtheblotwascoveredwithathinplasticfilmwrap.Theplatewasthenexposedtox-ray

filminadarkroomformultipleexposures(30sec,1,3,5and10mins)dependingonthe

intensityofthebands.

56

3.3Results

3.3.1MG132treatmentinTCAextractedsamples

A preliminary set of samples including awildtype,Δccr4,Δdhh1,Δnot4,Δnot5and

pre1,2 (proteasomemutant)was grown under the conditions described in the previous

section (3.2.3) and a second set of these samples (except for pre1,2) was treated with

proteasomeinhibitorMG132topreventthedegradationofRpb1andallowtheobservation

of the ubiquitinated proteins. These cell cultures were not immunoprecipitated with

antibodies to either ubiquitinated proteins or PolII, butwere extracted by TCA protocol

anddirectlyrunonageltodetectthequantityofPolIIproteinineachsample,theeffectof

addition of MG132, and extent of ubiquitination and degradation of PolII. Ubiquitinated

proteinshavehighermolecularweightsandrunslowerthanthenativeproteinonthegel.

Polyubiquitinated species like Rpb1 are expected to show a streak or ladder above the

native protein. It is predicted that the assay for the detection of ubiquitinated proteins

wouldshowasmearingofhighermolecularweightproteinsindicatingubiquitinatedRpb1

under 4NQO stress induced conditions. From the experiment shown in the introductory

figure1.7theΔnot4strainshouldbedeficientintheubiquitinationofRpb1underthesame

4NQOstresswhencomparedtotheotherstrains.Asapositivecontrolfortheadditionof

MG132todisabletheproteasomefunction,theblotincludesaproteasomemutantpre1,2

topreventthedegradationofpoly-ubiquitinatedproteins.

Thepanelsinfigure3.1showthesameblotundertwodifferentexposures.Theblot

inthetoppanelwasexposedtothefilmfor30secondsandthebottomfor1minute.Wild-

type BY4741 sample was run without any proteasome inhibitor or 4NQO added. The

remainingsamples:wildtype,Δccr4,Δdhh1,Δnot4andΔnot5 eachcontainapdr5deletion

thatpreventsdrugexportandincreasestheeffectivenessofMG132asreportedinvarious

studies(Balzietal.1994;Yenetal.2012;Haworthetal.2010).Onesetofthesesamplesis

treated with MG132 to observe the effect of the proteasome inhibitor on the mutants.

Finally,thelastsampleisthepre1,2usedasapositivecontrolforproteasomeinhibition.

57

Figure3.1:TCAprocedureonsamplesgrownwithoutMG132,and treated

with MG132. Wild-type BY4741 sample was run without any proteasome

inhibitoradded.Bothpanelsarethesamesetofsampleswithdifferentexposures

(30secand1min).GelsshowthelevelofPolIIinthedifferentsamplesusingthe

antibody8WG16.

MG132 --+-+-+-+-+ -

123456789101112

123456789101112

Exposure:30s

1min

MW(kbp)200150200150120

Rpb1

Rpb1

58

Thebands seenbelow theprimaryRpb1bandare likely tobepartiallydegraded

species ofRpb1and the additionofMG132 to the sampleswasnot sufficient toprevent

this. However, it appears that the addition of MG132 may increase the visibility of the

higher species that are modified species of Rpb1. Since this assay looks at all Rpb1

modifications, it is not conclusive because Rpb1 undergoes modifications other than

ubiquitination(Chenetal.2009).

TheamountofRpb1ineachofthesamplesvariesduetotheapproximationofthe

volumeofTCAsamplesadded to thegel.The secondpanel (higherexposure) shows the

amountofsmearingoftheprotein,predictedtobemodifiedspeciesofRpb1.Becausethe

effect of MG132 and the pdr5 deletion appears to assist in the visualization of the

polyubiquitinated species, this drug was used in subsequent experiments to visualize

polyubiquitinatedRpb1.TheblotshowsthatalthoughthelevelsofRpb1areinconsistent,

thereisnosignificantlossofRpb1inthemutantstrains.

3.3.2MG132and4NQOtreatmentforImmunoprecipitatedsamples

To observe the effectiveness of the antibody to detect Rpb1, an

immunoprecipitationassaywasperformedwith the4H8antibody(Mousemonoclonal to

RNA polymerase II CTD repeat YSPTSPS phospho S5) and probed for Rpb1 using the

8WG16 antibody. This antibody 4H8 was selected because it recognizes both the

phosphorylatedandunphosphorylated formsof theRNApolymerase II (BiolegendCat.#

904001Applicationnotes). In figure3.2, the twopanelsshowthedifferentstrainsunder

MG132 treatment, 4NQO treatment, and both Mg132 and 4NQO added. As described

earlier,additionof4NQOmimicsUVdamageontheDNA,causingPolIItobearrestedand

Rpb1 to be ubiquitinated. The wildtype and pre1,2 samples were loaded in duplicates

because the sampleswere divided into two gels and these two sets of samples act as a

transfercontrol.Allthesampleswereloadedinequalconcentrations,determinedfromthe

Bradford’sassay.ThepanelsshowthequantityofRpb1appearstobeslightlydecreasedon

addition of 4NQO, possibly because of the effect of 4NQO leading to the degradation of

Rpb1.However,theprimarypurposeofthisblotistoverifythattheΔnot4isnotdeficient

inRpb1concentrations.Fromthelowerpaneloffigure3.2,lanes7-10,itappearsthatthe

concentrationofRpb1isrelativelyconsistentintheΔnot4strain.

59

ThepurposeofaddingMG132wastoinhibittheproteasomeandallowforabetter

visualization of ubiquitinated species. To fully solubilize MG132 in the culture, it was

dissolved in 1 mL cell culture medium and placed in a hot water bath at 40°C for 15

minutesandaddedtotheflasksontheshaker.UltimatelyMG132wasnotessentialforthe

detectionofpolyubiquitinatedspecies,perhapsduetotheefficiencyofthe4H8antibodyin

detectingtheubiquitinatedspeciesandtheΔpdr5mutationinthestrains.

60

Figure3.2:4H8immunoprecipitationprobedwith8WG16antibodyshowing

the levels of Rpb1. Each strain was treated with MG132 or 4NQO or both

chemicals and immunoprecipitated with 4H8 antibody. The blot was then

incubated with 8WG16 antibody. The blots show the amount of Rpb1 antibody

being pulled down by the 4H8 antibody and levels of the protein under each

circumstance.

4NQOMG132

-+-+-+-+-+-+-+----++--++--++

-+-+-+ -+-+----++ --++

4NQOMG132

1234567891011121314

12345678910

Rpb1

Rpb1

61

Infigure3.3,immunoprecipitationwithanti-Rpb1antibody4H8wasperformedand

the blots were probed with (4H8).The major band that is visible in all the lanes was

identified as Rpb1,while the bands below it are possibly partially degraded products of

Rpb1.ItislikelythattheantibodyrecognizesthepolyubiquitinatedRpb1speciesthatare

visible as a smearing and banding pattern above the primary Rpb1 band. The smearing

pattern shows some interesting information about the different strains as well as the

chemicals added. As expected, the presence of 4NQO increases the appearance of these

polyubiquitinatedspecies.Unfortunately,additionoftheproteasomeinhibitorMG132does

not improve the detection of polyubiquitinated species, but the detection of thesei

ubiquitinatedspecieswasnothinderedintheabsenceofMG132.

The wildtype and Δnot5 strains show similar patterns, with an increase in

polyubiquitinated species upon addition of 4NQO. The Δccr4 strain, on the other hand,

shows an overall decrease in the Rpb1 levels (lanes 7-10), but in higher exposures (not

shown here), this strain also shows the presence of polyubiquitinated species upon

addition of 4NQO. Interestingly, the Δnot4 strain does not have any visible smearing of

proteinsabovetheprimaryband(lanes21–24).Thiscouldbetheexpectedresultofloss

of polyubiquitination of Rpb1 in thenot4mutant. The 8WG16 antibody blot (figure 3.2)

doesnotdetect thesehigherbandspossiblybecause8WG16maybeweakeratdetecting

Rpb1thanthe4H8antibody.

62

Figure3.3: Immunoprecipitation of sampleswith 4H8 antibody, andprobedwith 4H8

antibody. Samples treated with MG132, 4NQO or both, were immunoprecipitated with the

4H8 antibody andprobedwith the same antibody. Loading concentrationswere adjusted by

valuescalculatedfromtheBradford’sassay.

123456789101112131415161718192021222324

4NQOMG132

-+-+-+-+-+-+-+----++--++--++

-+-+-+-+-+----++--++

Rpb1

63

To follow up on the verification of overall Rpb1 protein concentrations in the

strains,Figure3.4showstheinputsamplestreatedwithMG132,4NQOorbothchemicals

and probed with the anti-Rpb1 antibody (4H8). The input samples are not

immunoprecipitatedwithanyantibodyandarejustloadedwithequalconcentrationofcell

extractforeachstrain.Thereisarecurrenceofwildtypeandpre1,2samplesinthetwogel

columnsbecauseallthesamplesdidnotfitintoonegelandtheserepeatedsamplesactasa

controlfortransferofsamplesfromthegeltothemembrane.Inbothexposures,thehigher

order modified Rpb1 species is visualized in each sample set, there appears to be an

increase in themodifiedRpb1species(highermolecularweightandsmearing in thegel)

withthetreatmentofthecellswith4NQOinthepresenceofMG132.However,intheΔnot4

strains, there is amarked absence of the highermolecularweight species in any of the

lanes.Additionally,theΔccr4strainalsoappearstoshowmodificationofRpb1toalesser

degreethanwild-typeorΔnot5strains.

64

Figure 3.4: Sample inputs probed with 4H8 antibody, treated withMG132, 4NQO and

both chemicals. Inputswere loadedwith equal concentrations determined from Bradford’s

assay.Theblotswereprobedwith4H8antibody.Thetoppanelshowstheblotsexposedfor30

secsandthebottomfor1min.

-+-+-+-+-+-+-+----++--++--++

-+-+-+-+-+----++--++

4NQOMG132

123456789101112131415161718192021222324

123456789101112131415161718192021222324

MW(kbp)

200150

120100

200150

12010085

Exposure:30sec1minRpb1

Rpb1

65

To confirm that the smearing observed in the inputs is ubiquitinated Rpb1,

immunoprecipitationswereperformedusing the4H8antibody forRpb1 (figure3.5)and

anti-HA antibody for ubiquitinated proteins (figure 3.6), and probed with the anti-HA

antibody and anti-Rpb1 (4H8) antibody respectively. All the strains contained the

ubiquitin-HA plasmid that over-expressed Ub-HA. Under normal growth conditions,

proteinsareregularlyubiquitinatedanddegradedaspartofhouse-keepingwithinthecell.

Consequently,allstrainsexcept for theΔnot4 strainshowedubiquitinationofproteins in

strainsthatwereleftuntreated,asseeninfigure3.5,lanes1,3,11,15and17.

In figure 3.5, the immunoprecipitation with 4H8 antibody pulls down Rpb1 and

other proteins associated with it. Probing with anti-HA visualizes all the ubiquitinated

proteinsthathaveimmunoprecipitated,inadditiontoRpb1.Therefore,itisnotconclusive

from this IP alone that the protein modifications seen in the blot are indeed

polyubiquitinatedRpb1.Infigure3.6,allubiquitinatedproteinswereimmunoprecipitated

withtheanti-HAantibodyandtheblotwasprobedforRpb1usingthe4H8antibody.The

smearing in this blot also shows the ubiquitinated species associatedwith Rpb1 that is

detectedbytheantibodyand it iscomparable to theblot in figure3.5 indicatingthat the

smearingseeninfigure3.5couldalsobeRpb1poly-ubiquitination.

Inbothblots,thereappearstobeanincreaseinthesmearingofubiquitinatedRpb1

inthewildtypeandnot5mutantstrainsupon4NQOaddition(lane4,12and18fromboth

figures 3.5 and 3.6). The ccr4mutant shows intermediate effect with lesser smearing

overall (lanes 7 – 10) and the not4 mutant does not appear to show any higher order

ubiquitinatedspeciesofRpb1(lanes21–24).MG132actsasaproteasomeinhibitorand

shouldallowforbetterdetectionofubiquitinatedproteinsbeforetheyaredegradedupon

treatmentwith4NQO,buttheeffectwasnotvisibleinthesamples(figure3.5and3.6lanes

6,10,14,20).

Both the IP and Input samples (figures 3.4, 3.5 and 3.6) show higher molecular

weightproteins(presumablypolyubiquitinatedspeciesofRpb1)upon4NQOadditioninat

least three replicates (only one replicate of each strain is shown in each figure).

Interestingly, Δccr4 strains showed an intermediate phenotype for the ubiquitination

judging by the amount of smearing detected in lanes 7-10 figures 3.4, 3.5 and 3.6. The

amountofsmearingwasdecreasedinallreplicatesofΔccr4whenvisiblycomparedtothe

66

wildtypeornot5mutantsamples,butwasnotabsentasinthecaseoftheΔnot4strain.The

results implythatNot4isrequiredforthepoly-ubiquitinationofRpb1upon4NQOstress

andtheabsenceofCcr4mayaffecttheroleofNot4insomeway.Alternatively,Ccr4may

also play a supporting role in this function. The absence of Not5 does not have any

significant effect on the ubiquitination of Rpb1. These results are further discussed in

Chapter4.

67

Figure3.5: Immunoprecipitationofstrainswith4H8antibodyandprobedwithanti-

HAantibody.Sampleswere loaded on the gel in equal concentrations deduced from the

Bradford’sAssay.Blotswereprobedwithanti-HAantibodytodetectubiquitinatedspecies.

Each strain contains aplasmid-expressedubiquitin taggedwithHA. Sampleswere treated

withMG132,4NQOorboth.

4NQOMG132

-+-+-+-+-+-+-+----++--++--++

-+-+-+-+-+----++--++

123456789101112131415161718192021222324

Rpb1

68

Figure 3.6: Immunoprecipitation of strains with HA antibody and probed with 4H8

antibody.SampleswererunonagelwithequalconcentrationsdeducedfromtheBradford’s

Assay.Blotswereprobedwith4H8antibodytodetectubiquitinatedRpb1species.Eachstrain

contains a plasmid-expressed ubiquitin taggedwith HA and the strainswere treatedwith

MG132,4NQOorbothdrugs.

123456789101112131415161718192021222324

Rpb1

4NQOMG132

-+-+-+-+-+-+-+----++--++--++

-+-+-+-+-+----++--++

69

Theresultsoftheimmunoprecipitationindicateavery interestingphenomenonin

theubiquitinationofRpb1.TheassayidentifiesanyUbiquitin-HAboundproteinsandthe

presenceofabandineachoftheΔnot4strains(lanes21-24figure3.5)couldindicatethat

theRpb1detectedispossiblymono-ubiquitinatedPolIIandisidentifiedviatheHAtagged

ubiquitinboundtoit.Ithasalreadybeenshownbeforethattheprocessofubiquitinationof

Rpb1occursinatwo-stepprocess;firstasingleubiquitinmarkisaddedontoRpb1which

servesmultiple purposes as described in the introduction section 1.5, and then Rpb1 is

polyubiquitinatedeitheratthesamesite,oradifferentone.

Alternatively,thepresenceofabandintheΔnot4correspondingtoRpb1couldbe

becausetheIPwasdoneundernativeconditionsandRpb1couldbepulleddownindirectly

through a different subunit of PolII that may be ubiquitinated. Studies have shown the

ubiquitinationofRpb2,Rpb7,Rpb6andRpb7detected ina large-scalepost-translational

modification(PTM)screeninyeast(Beltraoetal.2012).

70

3.3.26AUtreatment

Inorder tounderstandhowNot4 functions, thestrainswere testedusinganother

chemical 6-Azauracil (6-AU). As described in section 3.2.4, 6-AU leads to a reduction of

intracellularGTP levelswhichblocks transcriptional elongation.Adding6-AUserves two

purposes:Ontheonehand,itpreventsnewproductionofRpb1thatcouldaltertheresult

showing the degradation of Rpb1, and on the other hand it acts as a stress itself by

arrestingPolIIontheDNA,causingasituationsimilartoDNAdamageinducedstress.The

purposeoftheexperimentwastoobserveiftheubiquitinationofRpb1couldbedetected

upon6-AUtreatment,andifNot4wasrequiredforthisubiquitinationaswell.

The experiments for 6AU treatment were done using TCA precipitation and only

observed the amount of ubiquitinated smearing in each strain. As shown in figure 3.7,

strainsshowanincreasedsmearinguponadditionof6AUinlanes4and10.Thebandseen

inbetweenlanes2and3isanoverflowofsamplefromlane2.Theproteasomemutantin

lanes 1 and 2 do not showmuch increase in ubiquitination upon addition of 6-AU. The

smearingbelowtheRpb1bandmaybepartiallydegradedproductsduetotheadditionof

6-AU.However, both theΔnot4 strain and theΔccr4 strain showed little or no smearing

(lanes 6 and 8); indicating that once again Not4 is required for the ubiquitination and

subsequentdegradationofRpb1.Additionally,itappearsthattheΔccr4alsoisdeficientin

theubiquitinationanddegradationofRpb1,implyingthatCcr4mayalsoplayaroleinthis

function, as observed in the experiments conductedwith4NQO.Not5 on the other hand

doesnotaffectthefunctioningofNot4sincethesmearingofubiquitinatedproteinsinthe

Δnot5strainissimilartothatofthewild-typestrain.

71

Figure3.7TCAprecipitationof6AUtreatedcellstostudyubiquitinationofRpb1in

mutants intheCcr4-Notcomplex.Theorderon theblot from left to right:Proteasome

mutant,wildtypeΔpdr5,Δpdr5Δnot4,Δpdr5Δccr4andΔpdr5Δnot5withandwithout6AU.

Smearing in the lanes may be an indication of ubiquitinated species as the smearing is

increasedinthesampleswith6AUtreatment.

-+-+-+-+-+6AU

12345678910

Rpb1

72

3.4Discussion

ThemainhypothesisofthisthesiswasthatNot4isrequiredforthestress-induced

poly-ubiquitination of Rpb1. The results from this chapter indicate a lack of poly-

ubiquitinatedspeciesintheabsenceofNot4,signifyingthatthehypothesisisvalid.While

Not4may not be the only protein involved in this process, it is likely that Not4 plays a

functionalroleeitherbydirectlyubiquitinatingRpb1or indirectlyubiquitinatinganother

factorthatisresponsibleforthisprocess.Onealternativehypothesismentionedpreviously

wasthatNot4couldleadtotheproteasome-mediatedprocessingofDef1,whichcausesit

tobeinternalizedintothenucleuswhereitaidsintheubiquitinationofRpb1.

It is also suggested from the results that Ccr4 may also play an accessory or

supporting role in theubiquitinationanddegradationofRpb1, resulting inadecreaseof

poly-ubiquitinated species in the Δccr4 strains. In addition to its primary role as a

deadenylaseinthecell,deletionofCcr4alsoleadtoanincreaseddensityofRNAPolIIalong

thegene.ThiswasobservedtoalargerextentintheΔnot4strainimplyingthatbothCcr4

and Not4 assist PolII in traversing the gene during transcription (Kruk et al. 2011).

Surprisingly, deletionofNot5didnothaveany significant effect on theubiquitinationof

Rpb1, although deletion of Not5 has been shown in other studies to be lethal when

combined with the deletion of Not4 (Villanyi et al. 2014). Not5 also plays a role in

transcription regulation aswell as in the tri-methylation of histoneH3K4 (Collart et al.

2013)indicatingthatitsrolemaybeconnectedwithNot4(Mulder,Brenkman,etal.2007).

The mechanism of the stress-mediated ubiquitination and degradation is in

responsetoDNAdamageinducedarrestofthetranscriptionmachinery.Toreplicatethose

conditionsexperimentally,thestrainsweretreatedwith4NQO.Thisprovedtobeeffective

in preliminary experiments (shown in the introduction figure 1.7)whereRpb1 turnover

was decreased in Not4 mutants when compared to wild-type strains. This line of

investigation was pursued by tagging Ubiquitin with a HA tag to visualize it better.

Immunoprecipitating with anti-HA antibody resulted in an overabundance of

ubiqutitylatedspeciesbeingpulleddownandaninefficiencytodetectRpb1.Hencetheonly

reasonablewaytoidentifyubiquitinatedRpb1istoimmunoprecipitateRpb1usingthe4H8

antibodyandthenprobeforubiquitinatedspecies.

73

This techniqueproved tobe effective and showeda lackofpoly-ubiquitinationof

Rpb1notonlyintheIPbutalsointheinputsthemselves, indicatingthatthelossinpoly-

ubiquitinationwasnotanartifactof the IPprocess.Additionof theproteasome inhibitor

MG132alsoincreasedthedetectionofpoly-ubiquitinatedRpb1beforeitwasdegradedby

theproteasome.ThistoodidnothaveanychangeintheNot4mutant.Therequirementof

Not4isundeniable,howeverthefunctionisstillunclear.

Furthermore,theΔnot4mutantrevealedaninterestingdetailregardingtheprocess

ofubiquitinationofRpb1.OnesuggestionfromtheresultisthatRpb1isubiquitinatedeven

intheΔnot4strain,butthelowermolecularweightofthebandimpliesitmaybeamono-

ubiquitinated protein. Therefore, while it can be deduced that Not4 is required for the

polyubiquitinationofRpb1,Not4isnottheonlyE3ubiquitinligasethatplacesitsubiquitin

markonthepolymerase.Alternatively,Rpb1couldbepulleddownindirectlywithanother

subunitofPolIIthatisubiquitinated.Post-translationalmodificationsofRpb2,Rpb7,Rpb6

andRpb7weredetectedusingmass-spectrometryanditwasidentifiedthattheseproteins

arealsoubiquitinated(Beltraoetal.2012).

74

Chapter4:Discussionofresults The results shown in the experiments described in this thesis suggest a new

dynamicinthecoordinationofcellularresponsetoDNAdamage.Alltheproteinsdescribed

havemultiplefunctionsthatoverlap.Thisthesisdevelopsanewroleforthiscomplexinthe

stress-responsivedegradationof the largest subunitofRNAPolymerase II.TheCcr4-Not

complex’s previously identified functions in transcriptional elongation andmRNA decay

suggestthatitsnewrolemaybeonepartofanintricateresponsethatiswell-regulatedby

thecell.

The Ccr4-Not complex has been identified as a key player in multiple cellular

functions ranging from transcriptional elongation to ribophagy and mRNA decay. The

differentsubunitsofthecomplexcoordinatetheirfunctionswitheachother.Thedeletion

ofonesubunitcanleadtothealterationofthefunctionofanothersubunit.Ccr4isnotonly

theprimarydeadenylase,butalsohas secondaryphenotypes in transcriptionelongation.

Onesuchphenotypewasobserved(Kruketal.2011)inboththeΔccr4andΔnot4strains,

and resulted in thebuild-upofRNAPolII along thegene.Thisbuild-up could trigger the

recruitmentofothercellularresponsefactorssuchastheGGRorTCNERfactors.Theresult

ofPolIIcollectionalongthetranscriptmaybebecauseofthelossofpolyubiquitinationof

Rpb1duetothedeletionofNot4asseenintheresultsinthisthesis.Thepartialdecreasein

polyubiquitination of Rpb1 in the Δccr4 strain that was also seen in the western blot

experimentscouldbeasecondaryeffectwhereCcr4assists inpromotingelongation,and

lossofCcr4resultsinthestallingorarrestofPolIIalongthetranscript.TheCcr4-Notalso

functions in the proteasome-mediated degradation of Yap1 (Gulshan et al. 2012),

ubiquitination of Jhd2 (Douglas P. Mersman et al. 2009) and modulates Ubc4p/Ubc5p-

mediated stress responses in vivo (Mulder, Inagaki, et al. 2007). These functions of the

Ccr4-Notcomplexnotonlypositionitinastrategicwaytoassistinstressresponseatthe

transcriptional level,butalso indicate that the complexhasbeenpreviously identified in

stressresponse functions.Thissuggests that thehypothesisandresultspresented in this

thesiscouldaddtothepreviouslyknownfunctionsoftheCcr4-Notcomplexandbroaden

the understanding of both the complex and other regulatory factors involved in cellular

stressresponse.

75

MultiplestudieshaveshowntheroleofUbp3intheremovalofubiquitinationmarks

fromseveralproteins.InadditiontoRpb1(Kvintetal.2008),Ubp3-Bre5alsofunctionsin

transcriptional activationby reversing theubiquitinationofTbp1 (TATA-bindingprotein

1)(Chewetal.2010b).Itplaysaroleinheatresistancebydestabilizingmisfoldedproteins

(Ölingetal.2014).However,Ubp3alsoplaysanantagonisticroleinthenegativeregulation

ofseveralfactors.ItplaysaroleinUVresistancebytargetingRad4fordegradation.Ubp3

interactswiththe26SproteasomeandlossofUbp3resultsintheUVresistanceincells(P.

Mao & Smerdon 2010). Ubp3 is also involved in ribophagy and is required for specific

pathways thatactivateundernitrogenstarvation inyeast(Ossareh-Nazarietal.2014). It

wasshownthatLtn1,anE3ligase,actsantagonisticallytoUbp3.Ubp3isitselfatargetfor

phosphorylationunderosmostress,andisrecruitedtoosmoresponsivegenestomodulate

transcription(Soléetal.2011).

Althoughtheproteinsstudiedinthisthesisallfunctionindependentlyofeachother,

they are interconnectedmore than just by physical interaction. RNAPolII interactswith

Ubp3/Bre5andtheCcr4-Notcomplexphysicallyandfunctionally.TheCcr4-Notcomplexas

describedearlier,assistsintranscriptionandmRNAdecay,andfromtheresultsshownin

thisthesis,Not4isalsoessentialforRpb1poly-ubiquitination.PolIIalsointeractswiththe

Ubp3/Bre5 complex which functions to reverse the poly-ubiquitination of Rpb1. This

suggests that theremaybea coordinateddegrade/rescuemechanismbetween theCcr4-

Not complex and Ubp3/Bre5. Both Ubp3/Bre5 and Not4 play opposing roles in the

degradation of Rpb1, and both proteins are recruited to PolII either independently or

dependentonsometertiaryfactorsuchasmRNA,oranasyetundiscoveredprotein.It is

alsopossiblethatthestress-inducedarrestofPolIIresultsinamassrecruitmentofmany

proteinsincludingUbp3/Bre5.

Several studies have looked into the coordination of stress-response and gene

transcription(deNadaletal.2011)andspecificgeneexpressionresponsestostresswere

seen in culturedhumancells (Murrayetal.2004).Evenwith the studiesdone in the lab

with the Ccr4-Not complex (Miller, Reese, unpublished) it was observed that the

deadenylase Ccr4 is recruited to a group of mRNA under unstressed conditions, and a

specific subset of mRNA under stressed conditions. It is predicted that a similar

recruitmentofUbp3-Bre5tospecificmRNAmayexist,leadingtoacoordinatedpromotion

76

of gene-transcription by deubiquitinating Rpb1. This specific stress response may be

initiated by targeting mRNA required for the specific response and the recruitment of

Ubp3-Bre5toPolIImaybethroughtheidentificationofspecificmRNA.

To followupon the idea that the recruitmentof theseproteins toPolIImayhave

mRNA as the common denominator, the recruitment of these protein complexes to a

commonmRNAwasexplored.Interesting,Ubp3isrecruitedtooneofthemRNAsstudied

that the Ccr4-Not complex is also highly recruited to (as measured by the associated

protein–Dhh1).WiththepreviousdeductionofthecoordinatedrolesofUbp3/Bre5and

the Ccr4-Not complex, it is likely that the coordination could be intermediated through

mRNA. In our other studies on the Ccr4-Not complex, it was discovered that Ccr4 is

selectively recruited to a certain subset ofmRNA under unstressed conditions, and this

subsetisdiminisheduponhydrogen-peroxideinducedstress.Thehypothesisisthatsince

Ccr4istheprimaryyeastdeadenylase, it targetsmRNAthatarenon-essential forthecell

uponstress.Inorderforthecellstorecoverfromthestress,Ccr4isnolongerrecruitedto

mRNA that are necessary for cell survival. This led to a potential similar hypothesis for

Ubp3/Bre5, except in theoppositedirection.Ubp3/Bre5 rescuesRpb1 fromdegradation

uponstress-inducedarrest.Whenthecellencountersstress,itrequiresanup-regulationof

certainmRNAandadown-regulationofothers(Gasch2002).Ubp3/Bre5couldidentifythe

essentialmRNAandbeselectivelyrecruitedtorescuePolIIandresumetranscription,while

theCcr4-Not4complexcouldassistinthisregulationbytargetingthecorrespondingdown-

regulatedmRNAfordegradation.Simultaneously,Not4could facilitate the terminationof

transcriptiononthesemRNAbytargetingRpb1fordegradation.

It is also likely that the interactionwith RNA could be independent of its role in

polymeraserescuefromdegradation.BothUbp3andBre5havebeenknowntobeinvolved

in ribophagy as discussed earlier (Kraft et al. 2008). This function may require an

association with RNA to regulate the degradation of mature ribosomes. Alternatively,

because the immunoprecipitation assays indicated that Ubp3/Bre5 may be associating

with theCcr4-Not complex (figure2.3), their interactionmayalsobe independentof the

polymerase. Ccr4-Not plays various roles in the cytoplasm and Ubp3/Bre5 could have

alternative functions in mRNA decay. Figure 4.1 shows some possible interactions of

Ubp3/Bre5withRNAandthepredictedfunctionoftheseinteractions.

77

WhileNot4was shown tobenecessary for thepolyubiquitinationofRpb1, itwas

also observed that Rpb1 appears to bemonoubiquitinated even in the absence of Not4

suggestingthatthemechanismofubiquitinationanddegradationofRpb1maybeamulti-

step process involving other ubiquitin ligases. Proteins are ubiquitinated for several

reasons and functions as discussed earlier, andRpb1 showedmonoubiquitination under

unstressedconditions, implyingthat thisubiquitinationmaybe independentofNot4,but

maybe required for the subsequentpolyubiquitinationbyNot4.Oneproposed theory is

that the Elongin-Cullen complex, which was shown to also be required for the Def-1

mediated degradation of Rpb1 (Marcus D. Wilson et al. 2013), may place the initial

ubiquitinmark onRpb1, andNot4maybe responsible for the poly-ubiquitinationmark.

Bothproteinsfunctionwiththesamesubstratebutunderdifferentconditions.Themono-

ubiquitinmarkbytheElongin-CullincomplexmayserveasatargetforNot4,whichwould

thentargettheRpb1fordegradation.

78

4.1ProposedModel

In the model described below, two possible interactions of Ubp3/Bre5 with the

Ccr4-Notcomplexareillustrated.Inthefirstimage,theinteractionofUbp3/Bre5withthe

Ccr4-Not complex and mRNA is described by the bridging of the two with Ubp3/Bre5.

Additionally,Ubp3/Bre5recruitment toRNAPolymerase II isdescribedasa recruitment

viaeithertheCcr4-Notcomplex,orthemRNAtranscript.

It is possible that the interactionofUbp3/Bre5 to theCcr4-Not complex couldbe

throughtherecruitmenttotheemergingmRNAtranscript.ThepriorstudieswiththeCcr4-

Notcomplexshowa largegroupofmRNAthatCcr4isrecruitedto,andthissetofmRNA

changesduringthecellularstressresponse.Onepossibility isthatunderstress-response,

theCcr4-NotcomplexisrecruitedtomRNAeithertocontrolthedegradationofthemRNA

or toregulate the transcriptionbyRpb1degradation.Subsequently,Ubp3/Bre5mayalso

berecruitedtospecificmRNAalongwiththeCcr4-Notcomplextopreventthedegradation

ofRpb1.It isalsopossiblethattherecruitmentofUbp3andBre5maybeindependentof

mRNA,orcouldvarydependingonotherfactors.ThenecessityofBre5fortherecruitment

ofUbp3tomRNAalsoneedstobefurtherexplored.TherecruitmentofUbp3toSKN7and

PYK1 from figure 2.5 and2.6 showed thatUbp3maybe recruited to certainmRNA in a

Bre5dependentmanner,andnottoothers.

AnotheralternativeshowninthesecondfigureisthattheassociationofUbp3/Bre5

to the Ccr4-Not complex may be independent of its role with RNA PolII. The Ccr4-Not

complex plays multiple roles, such as deadenylation, which requires association with

mRNA.ItispossiblethatUbp3/Bre5mayberecruitedtomRNAatthisstagetocounterthe

deadenylation. This too may be in a mRNA specific manner, and could act as a stress

responseinthecell,preventingcertainmRNAfrombeingdegraded.

Afinalalternative isthattherecruitmentofUbp3/Bre5andtheCcr4-Notcomplex

maybeentirely independentof eachother.Ubp3/Bre5maybe recruited toPolII via the

mRNAduringstressresponse,or in itsrole inribophagy(Kraftetal.2008).Additionally,

Ubp3/Bre5couldalsoplayanasyetunstudiedrolebyitsassociationwithmRNA.

79

Figure4.1:DiagramshowingapossiblemodelofassociationofUbp3/Bre5toRNA.

(a)Fromtheimmunoprecipitationstudiesshowninfigure2.3, it is likelythatUbp3-Bre5

associateswithbothPolIIand theCcr4-Notcomplex.Ubp3 isalsorecruited tomRNA,so

this figure incorporates both interactions to produce a hypothetical interaction ofUbp3-

Bre5withpolymerasethroughmRNAtranscript.(b)Bre5wasalsoshowntoberequired

fortheassociationofUbp3-Bre5complexwiththeCcr4-Notcomplex.BothSKN7andPYK1

are targets for the Ccr4-Not complex, and Ubp3 is also recruited to these mRNAs. This

recruitmentishypothesizedinthesecondfigure.

Ccr4-Not

RNAPolII

Ubp3

Bre5

mRNA

Ccr4-NotmRNA

A

B

80

4.2FutureStudies

The association of Ubp3/Bre5 with PolII and the Ccr4-Not complex could be

indirectlymediatedbyotherproteinsornucleic acids.Treating the samples toRNaseor

DNase and repeating the protein immunoprecipitation assays would determine if the

associationisindirectlythroughRNAorDNA.Touncoverthefullpotentialoftheproposed

hypothesis regarding theselectiverecruitmentofUbp3/Bre5 tomRNA, it isnecessary to

conductagenome-wideanalysisoftherecruitmentofUbp3tomRNAandcomparethese

results with the set of results from Ccr4 recruitment. Following this up with a stress-

induced assay would shed some light on the cell’s stress response. Additionally, if the

degradationor rescueofRpb1 isdependenton themRNAtranscript, futureexperiments

could be conducted using purified proteins – RNA PolII, Ubp3/Bre5 and the Ccr4-Not

complex–inanin-vitrostudytounderstandtheeffectofeachproteinontheubiquitination

and deubiquitination of Rpb1. This assay would also confirm if Not4 can directly

ubiquitinateRpb1.

Furthermore, since it has been shown in previous studies that the Elongin-Cullin

complex (Ela1-Elc1) can also ubiquitinate Rpb1 under stress-induced arrest (Marcus D

Wilsonetal.2013),itisnecessarytopursuethefunctionaldifferencebetweentheElongin-

CullincomplexandNot4.Itislikelythatthemethodorsiteofubiquitinationisdifferentin

either case; or the type of stress-induced arrest could determinewhich protein complex

induces the ubiquitination and degradation of Rpb1. It is also likely that theUbp3/Bre5

complexmayinteractwiththeElongin-Cullincomplex.Aubiquitinmarkononeresidueof

Rpb1 could recruit the Ccr4-Not complex to PolII, while a ubiquitin mark on another

residuecouldrecruittheUbp3/Bre5complexinstead.Moreresearchneedstobedonein

ordertounderstandthefunctionsoftheseproteincomplexes.

81

Tables

Table3:PrimersequencesusedfortagginganddeletingUbp3andBre5

Protein

tagged/deleted

Primerlocation Primersequence

TaggingBre5 5’endprimerplus

strand

5’-AATGGAACACGTTCTCATAGAAAGCAA

CCCCTAAAAAGAAAGGACCGGATCCCCGG

GTTAATTAA-3’

3’endprimerminus

strand

5’-ATACAGTTTCTTGTTTCAATTTTTTAG

ATTTTAATTAGCGGTTTTGAATTCGAGCT

CGTTTAAAC-3'

DeletingBre5 5’endprimer 5’-GCATTTGAAGTCATACCCTCGAATAGA

AGTATCAAATAAAAGAAACGGATCCCCGG

GTTAATTAA–3’

3’endprimer 5’-TTTTATTATTTTTTCAATTTTTCTTTT

TAAAAGGCTTGTGGTTGAGAATTCGAGCT

CGTTTAAAC-3'

DeletingUbp3 5’endprimer 5’-ACCATCATCCAGGTACCGCTTTCCTTT

CCATCATCATTAAAAAAACGGATCCCCGG

GTTAATTAA–3’

3’endprimer 5’-CTATATTATTTTTTATGTATTTTGTCT

ATAATACCACCCCCCGTCGAATTCGAGCT

CGTTTAAAC-3'

TaggingUbp3 Upstream 5’-TCTGATTCGAGGACTGCCTATATTTTA

ATGTATCAAAAGAGAAATCGGATCCCCGG

GTTAATTAA

Downstream 5’-CCAAGCATAAAGTGTAACTCTGTTTCT

CTGTCTGTCTCTATTTCTGAATTCGAGCT

CGTTTAAAC

82

Table4:ListofstrainsgeneratedorusedinthestudyStrainName DetailsJR1711 MATaUBP3-13MYC::kanMX∆bre5::URA3

his3∆1leu2∆0met15∆0JR1710 MATalpha∆ubp3::kanMXBRE5-3HA::HIS3

leu2∆0met15∆0ura3∆0JR1712 MATaUBP3-13MYC::kanMXBRE5-3HA::HIS3

leu2∆0met15∆0ura3∆0BY4741 MATa;his3Δ1;leu2Δ0;met15Δ0;ura3Δ0BY4742 MATα;his3Δ1;leu2Δ0;lys2Δ0;ura3Δ0JR1688 MATaUBP3-13MYC::kanMX

his3∆1leu2∆0met15∆0ura3∆0JR1700 MATalpha∆ubp3::kanMX

his3∆1;leu2∆0;lys2∆0;ura3∆0;JR1612 MATahis3∆1;leu2∆0;lys2∆0;ura3∆0;ccr4∆::NATmX;JR1588 MATahis3∆1;leu2∆0MET15?;lys2∆;ura3∆0

not4∆::NATMxJR1552 MATahis3∆1;leu2∆0;MET15?;ura3∆0

not5∆::HIS3;

83

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