The Biology of RNA-Protein Complexes
PROGRAM AND ABSTRACTS
October11thͲ14th2017,ThonͲDittmerPalais,Regensburg,Germany
GENERAL INFORMATION
Throughout this program, the numbers next to the titles refer to corresponding oral or poster abstract numbers in the abstract section of this book. Please refrain from citing the abstracts in bibliographies. The information contained in this abstract book should be treated as personal communicationandshouldbecitedonlywiththeexpressedconsentoftheauthor.
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CoverPicture:TobiasBeck Ͳ Layout:JanMedenbach Ͳ Print: Offset Haas
2 MEETING SPONSORS
3 SCIENTIFIC ORGANIZERS
Prof. Dr. Thomas Dresselhaus Cell Biology and Plant Biochemistry University of Regensburg [email protected]
Prof. Dr. Dina Grohmann Microbiology & Single Molecule Biochemistry University of Regensburg [email protected]
Dr. Jan Medenbach Biochemistry I University of Regensburg [email protected]
Prof. Dr. Gunter Meister Biochemistry I University of Regensburg [email protected]
Prof. Dr. Herbert Tschochner Biochemistry III University of Regensburg [email protected]
4 ABOUT THE SFB960
Ribonucleoproteins (RNPs) are cellular multicomponent assemblies whose biological functions dependontheinteractionbetweenRNAandproteins.ThedynamicprocessofRNPassemblyand regulationofRNPfunctionrepresentsthemaininterestoftheCollaborativeResearchCentreSFB960. TheSFB960issupportedbytheDeutscheForschungsgemeinschaft(DFG)sinceJuly2011establishing astronglocalscientificnetworkattheUniversityofRegensburg.Itcurrentlyunites13researchand two core/service projects focusing on biogenesis and regulation of RNPs which are all directly or indirectlyinvolvedinthesynthesisorregulationofribosomes.Multisubunitcomplexesinteracting with the rRNA synthesizing machineries, with argonauteͲcontaining complexes or with nascent or mature ribosomes are especially interesting. This enables comparative analyses, which will reveal commonprinciplesandspecificaspectsofimportantRNPͲrelatedprocesses.
To further the knowledge of how ribosomes and other RNPs are synthesized and regulated, the SFB960 organizes international scientific meetings on a regular basis. The symposium 2017 "The biology of RNAͲprotein complexes" is already the third international conference organized by members of the SFB960, in which leading scientists of the RNP field exchange their thoughts and presenttheirongoingworkinahopefullystimulatingenvironment.
5
6 INDEX
Schedule...... 8 OralAbstracts...... 15 PosterAbstracts(AͲZ)...... 63 ListofParticipants...... 134 VenueInformation&Maps...... 154
7 SCHEDULE
Wednesday,October11th
13:00 Registrationstarts:ThonͲDittmerͲPalais,1stfloor KeynoteLectures
15:30 WelcomeAddress 15:45 RoyParker:RNPGranulesinHealthandDisease 16:15 Reinhard Lührmann: CryoͲEM snapshots of the highly dynamic spliceosome 17:00 AlanHinnebusch:Anetworkofmolecularinteractionsrestrictsstable preinitiationcomplexassemblytooptimaltranslationstartcodonsin vivo 17:45 DinneratThonͲDittmerͲPalais 18:30 PostersandDrinks–atthe‘großerDollingersaal’ Evenposternumbers
8 Thursday,October12th
Session:Transcription Sessionchair:DinaGrohmann 09:00 WelcomeAddress:Prof.Dr.UdoHebel,PresidentoftheUniversityof Regensburg 09:10 Evgeny Nudler: Structural Principles of TranscriptionͲcoupled DNA Repair 09:35 Stefan Knauer: Exploring RNA Polymerase Regulation by NMR spectroscopy 09:50 AlbertWeixlbaumer:RegulationofTranscriptionElongationbyNusA –biochemicalandstructuralstudies 10:05 DavidDulin:AnetworkofpausestrapsthebacterialRNApolymerase anditsnascenttranscriptonthepromoter 10:20 FinnWerner:MolecularMechanismsofTranscriptioninArchaea 10:45 coffeebreak 11:15 DylanTaatjes:NonͲcodingRNAs,Mediator,andMediatorkinases 11:40 Steven Hahn: Fuzzy FreeͲforͲAll: Retention of Fuzzy Binding in the MultiͲDomain Interactions of Transcription Activator Gcn4 and MediatorSubunitMed15 12:05 Zoltan Villanyi: Ccr4ͲNot is at the core of the gene expression circuitry 12:20 Christoph Müller: Structural biology of RNA polymerase I and III transcription 12:45 Lunchbreak
9 Session:RNAprocessing Sessionchair:JanMedenbach 14:00 JulianKönig:iCLIPͲbasedmodelinguncovers3’splicesitedefinition: howU2AF65specificityreliesonregulationbycoͲfactors 14:15 ŠtĢpánka VaŸáēová: N6Ͳmethyladenosine demethylase FTO targets preͲmRNAsandregulatesalternativesplicingand3’endprocessing 14:40 Michaela MüllerͲMcNicoll: Cellular differentiation state modulates themRNAexportandretentionactivitiesofSRproteins 15:05 DanielaLazzaretti:InsightsintospecificRNArecognitionmediatedby human Staufen1 from the crystal structure of its minimal RNAͲ bindingregionincomplexwithaphysiologicaldsRNAtarget 15:20 coffeebreak 15:50 Oliver Mühlemann: Dissecting the functions of SMG5, SMG7 and PNRC2innonsenseͲmediatedmRNAdecayofhumancells 16:15 RemcoSprangers:TheRrp4Ͳexosomecomplexrecruitsandchannels substrateRNAbyauniquemechanism 16:30 Michael Sattler: Decoding regulatory proteinͲRNA interactions in generegulationusingintegratedstructuralbiology 17:00 SponsoredSeminar: NicolasPiganeau Whole Transcriptome Analysis: Choosetherighttoolforthejob 17:30 Guidedtouroftheoldtown startingrightoutsidetheThonͲDittmerͲPalaisonHaidtplatz 19:00 ConferenceDinnerat‘HausHeuport’
10 Friday,October13th
Session:Ribosomebiogenesisandfunction Sessionchair:SébastienFereirraͲCercaandJorgePerezͲFernandez I–FundamentalaspectsofribosomebiogenesisinEukaryotes 09:00 Ed Hurt: Mechanism of ribosome biogenesis and its link to nucleocytoplasmictransport 09:25 Brigitte Pertschy: Coordination of distant 40S ribosomal subunit maturationevents 09:40 Susan J. Baserga: High throughput discovery of novel regulators of humanribosomebiogenesis 10:05 CameronMackereth:AnoveldsRNAͲbindingmodulefromanuclear prolylisomerase II–Ribosomebiogenesisanddisease 10:20 Nicholas Watkins: The 5S RNP provides the link between ribosome biogenesisandthetumoursuppressorp53 10:35 Antje OstareckͲLederer: Interaction of hnRNP K and rpS19 and its functioninerythroidmaturation 10:50 coffeebreak III–Ribosomefunction 11:20 DanielNWilson:RibosomeͲtargetingantibioticsandmechanismsof bacterialresistance 11:45 Axel Innis: Discovery and characterization of ribosomeͲarresting peptidesbyHighͲthroughputInverseToeprinting 12:00 Nenad Ban: Extensions, extra factors and extreme complexity: Ribosomalstructuresprovideinsightsintoeukaryotictranslation 12:25 KristynaPoncova:ThesmallribosomalproteinRPS3anditsfunctions 12:45 Lunchbreak 11 IV–Translationcontrolandqualitycontrol 13:45 GeorgAuburger:EfficientPreventionofNeurodegenerativeDiseases byDepletionofStarvationResponseFactorAtaxinͲ2 14:00 Nagammal Neelagandan: Mutant TDPͲ43 selectively affects translationofspecificmRNAsinculturedcorticalneuronsandmotor neuronͲlikecells 14:15 Keynote LectureͲNahum Sonenberg: Ribosome quality control of premature polyadenylated mRNAs by a unique E3 ubiquitin ligase andRNAͲbindingprotein 15:00 coffeebreak Session:nonͲcodingRNAs Sessionchair:MarkusKretz 15:30 LeemorJoshuaͲTor:MadaboutU:regulatingthelet7preͲmiRNA 15:55 Peter Sarnow: From bench to clinical trial: microRNA 122 as an antiviraltargetforhepatitisCvirus 16:20 ClausͲD.Kuhn:TheroleofpiRNAsinplanarianregeneration 16:35 SaraK.Eisenbart:AnickelͲregulatedsmallRNArepressesexpression ofmultiplemajorvirulencefactorsinHelicobacterpylori 16:50 LisaͲKatharina Maier: SelfͲtargeting and gene repression with the CRISPRͲCassystemofHaloferaxvolcanii 17:05 Markus T. Bohnsack: METTL16 is a N6Ͳmethyladenosine (m6A) methyltransferase that targets preͲmRNAs, lncRNAs and the U6 snRNA 17:20 SvenDiederichs:lncRNA&RibonucleoproteinsinCancer 17:45 UtzFischer:MechanisticdissectionofUsnRNPbiogenesisanditsrole indisease 18:10 DinneratThonͲDittmerͲPalais 18:45 PostersandDrinks–atthe‘großerDollingersaal’ Oddposternumbers 12 Saturday,October14th
Session:RNAsystemsbiology Sessionchair:AndreaBleckmann 09:00 Vicent Pelechano: GenomeͲwide study of transcription complexity andribosomedynamics 09:25 Jörg Vogel: GradͲseq discovers the third domain of small RNAͲ mediatedgeneregulationinbacteria 09:50 Nicholas Jaé: Functional characterization of hypoxiaͲregulated lncRNAsinendothelialcells 10:15 Kathi Zarnack: HighͲthroughput screening for splicing regulatory elements 10:30 coffeebreak 11:00 AwardCeremony:PosterprizesGunterMeister Sponsored by the Federation of European Biochemical Societies (FEBS), the SFB960, and the Faculty of Biology and PreͲClinical MedicineoftheUniversityofRegensburg Session:RNAsystemsbiology(continued)
11:15 JernejUle:RegulationofgeneexpressionbyproteinsboundtoLINEͲ derivedRNAelements 11:40 DorotheeStaiger:iCLIPdeterminesthetargetrepertoireandbinding landscapeoftheclockͲregulatedRNAͲbindingproteinAtGRP7 11:55 Cynthia Sharma: Functional characterization of the CRISPRͲCas9 systemofthefoodborneͲpathogenCampylobacterjejuni 12:20 Closingremarks
13
14
ORAL ABSTRACTS
15 O11
RNPGranulesinHealthandDisease
AnthonyKhong1,BrianaVanTreeck1,TylerMatheny1,BhalchandraRao1,DavidS.W.Protter1,Yuan Lin2,SaumyaJain1,JoshuaR.Wheeler1,MichaelRosen2,andRoyParker1
1UniversityofColoradoandHHMI,Boulder,CO,USA,2DepartmentofBiophysics,HowardHughesMedicalInstitute, UniversityofTexasSouthwesternMedicalCenter,Dallas,Texas75390,USA
StressgranulesarenonͲmembranousmRNPassembliesthatformwhentranslationislimitingandare related to pathologicalaggregates observed in neurodegenerative diseases. Analysis of stress granules from yeast and mammalian cells identifies a diverse proteome within stress granules includingmanyATPͲdependentremodelingcomplexes.RNAͲSeqalsorevealsadiversetranscriptome within stress granules. Genetic experiments demonstrate that numerous remodeling complexes affectboththeassemblyanddisassemblyofeukaryoticstressgranules.Suchremodelingcomplexes mayalsoplayaroleintheassemblyanddisassemblyofRNAͲproteinaggregatesthatforminsome humandiseases.
UsingRNPgranulesasanexample,weprovideevidenceforanassemblymechanismoflargecellular structures wherein specific proteinͲprotein, RNAͲRNA, or proteinͲRNA interactions create an assembly,whichisstabilizedbypromiscuousinteractionsofintrinsicallydisorderedregions(IDRs)of assembledcomponents.SuchIDRsmayremaininadynamicweakinteractionand/ortransitionto more stable amyloidͲlike assemblies in the context of long lived assemblies. This synergistic assemblymechanismilluminatesRNPgranuleassembly,andexplainswhymanycomponentsofRNP granules, and other large dynamic assemblies, contain IDRs linked to specific proteinͲprotein or proteinͲRNA interaction modules. We suggest assemblies based on combinations of specific interactionsandpromiscuousIDRsarecommonfeaturesofeukaryoticcells.
16 O21
CryoͲEMsnapshotsofthehighlydynamicspliceosome
ReinhardLührmann
MaxͲPlanckͲInstituteforBiophysicalChemistry,DepartmentofCellularBiochemistry,AmFassberg11,37077Göttingen, Germany
ThespliceosomecatalysestheremovaloftheintronfromnuclearpreͲmRNAsandassemblesinitially into a preͲcatalytic ensemble, termed complex B, which contains the snRNPs U1, U2 and the U4/U6.U5 triͲsnRNP and numerous nonͲsnRNP proteins. For catalytic activation the spliceosome undergoes a major structural rearrangement, mediated by the Brr2 RNA helicase, yielding the activated spliceosome (Bact complex). The final catalytic activation of the spliceosome requires an additionalrestructuringstepbytheRNAhelicasePrp2,generatingtheB*complexwhichcatalyses thefirststepofthesplicingreaction,yieldingtheCcomplex.Subsequentlythecatalyticcenterofthe spliceosomehastoberemodeledbytheRNAhelicasePrp16togeneratetheC*complexasapreͲ requisiteforsecondstepcatalysis.
Usingcryoelectronmicroscopywehaveinvestigatedthe3DstructureofthehumanU4/U6.U5triͲ snRNP complex and several purified spliceosomal complexes (in collaboration with Holger Stark; MPIbpc,Göttingen).OurtriͲsnRNPmodelrevealshowthespatialorganizationofBrr2RNAhelicase preventsprematureU4/U6RNAunwindinginisolatedhumantriͲsnRNPsandhowtheSad1protein likelytethersBrr2toitspreͲactivationposition,farawayfromitsU4/U6RNAsubstrate.TheCryoͲEM structureofthehumanBcomplexrevealsthatmosttriͲsnRNPproteinsundergosignificantstructural rearrangementsduringintegrationofthetriͲsnRNPintotheBcomplex.Theseincludeformationofa partiallyͲclosedPrp8conformationthatcreates,togetherwithDim1,a5’splicesitebindingpocket, displacementofSad1,andrearrangementofBrr2suchthatitnowcontactsitsU4/U6substrateand ispoisedforthesubsequentspliceosomeactivationstep.ThemoleculararchitectureofseveralBͲ specificproteinsuggeststheyareinvolvedinnegativelyregulatingBrr2,positioningtheU6/5’sshelix and/orstabilizingtheBcomplexstructure.Ourdataalsoindicatesubstantialdifferencesintheearly activationpathwaysbetweenhumanandyeastspliceosomes.TheCryoͲEMstructureofthepurified S.cerevisiaeBactcomplexrevealshowthefirststepreactants(i.e.the5'splicesiteandthebranch siteadenosine)aresequesteredbyproteinpriortocatalysisandprovideinsightsintothemolecular remodeling events that must be facilitated by Prp2 in order to generate a catalytically active B* spliceosome.Finally,IwillalsopresenttheCryoͲEM3DstructureofahumanC*complex,whichhas undergonethePrp16Ͳmediatedremodelingstep.Ourcombinedstudiesshowthatthespliceosomeis anextremelydynamicmolecularmachinewhichundergoesdramaticlargeͲscalestructuralchanges duringoneroundofpreͲmRNAsplicing.
17 O31
Anetworkofmolecularinteractionsrestrictsstablepreinitiation complexassemblytooptimaltranslationstartcodonsinvivo
AnilThakur1,JinshengDong1,ColinAitken2,PilarMartinMarcos3,FujunZhou2,CharmKarunasiri2, JagpreetNanda2,LauraMarler1,JyothsnaVisweswaraiah1,TanweerHussain4,JoseL.Llácer4,Michael Zeschnigk5,MercedesTamame3,JonR.Lorsch2,V.Ramakrishnan4,andAlanG.Hinnebusch1
1LaboratoryofGeneRegulationandDevelopment,EuniceKennedyShriverNationalInstituteofChildHealthandHuman Development,NIH,Bethesda,MD20892;2LaboratoryontheMechanismandRegulationofProteinSynthesis,EuniceK. ShriverNationalInstituteofChildHealthandHumanDevelopment,NIH,Bethesda,MD20892,USA;3InstitutodeBiología FuncionalyGenómica(IBFG)CSIC/UniversidaddeSalamanca,Salamanca,Spain;4MRCLaboratoryofMolecularBiology, CambridgeCB20QH,UK;5InstituteofHumanGenetics&EyeCancerResearchGroup,FacultyofMedicine,University DuisburgͲEssen,Essen,Germany
Inthescanningmechanismoftranslationinitiation,apreinitiationcomplex(PIC)comprisedofthe 40S ribosomal subunit, initiation factors 1, 1Aand 3, and a ternary complex (TC) of eIF2ͲGTP and initiatortRNAiattachestomRNAandscanstheleaderforan AUGcodoninoptimalcontext.eIF1 stimulatesrecruitmentofTCwithtRNAiboundinametastablestate(POUT)suitableforscanning,and opposestransitiontoamorestablePINstateatthestartcodon,thusnecessitatingeIF1releaseon AUGrecognition.StructuresofreconstitutedPICsrevealedanopenconformationofthe40Ssubunit atanearͲcognateAUCcodon,andamoreclosedconfigurationatAUG.Intheclosedstate,eIF1shifts itspositiononthe40SanditsɴͲhairpinloopsaredistortedtoavoidaclashwithtRNAi.Substitutions ineIF1loopͲ2thatremoveionicrepulsionorcreateattractionwithtRNAiincreaseinitiationatboth UUGcodons(SuiͲphenotype)andAUGsinpoorcontextinvivo,andstabilizeTCbindingatUUGstart codons in reconstituted PICs in vitro. Thus, the loopͲ2/tRNAi clash destabilizes the PIN state to disfavorsuboptimalstartcodons.eIF2ɴcontactseIF1andtRNAiexclusivelyintheopencomplex,and substitutionsattheeIF2ɴ/eIF1interfacelikewiseconferSuiͲphenotypes,indicatingthateIF1:eIF2ɴ interactionsalsoimpederearrangementtoPINatsuboptimalstartsites.Incontrast,theunstructured
NͲterminaltail(NTT)ofeIF1Acontactsthecodon:anticodonhelixonlyintheclosed/PINstate,and substitutingitsconservedbasicresiduessuppressesinitiationatUUGcodons(SsuͲphenotype)and destabilizes TC binding at UUG codons in vitro. Ribosome profiling of the NTT substitution eIF1AͲ R13P reveals decreased initiation at AUGs in poor context genomeͲwide, implicating the NTT in selectingsuboptimalstartcodons.Similargenetic/biochemicalfindingsidentifyrolesforconserved residues in ribosomal proteins at the mRNA exit (uS7) and entry (uS3) channels in stabilizing the closed/PIN conformation via contacts with eIF2ɲ (uS7) or mRNA (uS3), to enable recognition of suboptimalstartsites.TheeIF3isacentralplayerinPICrecruitmenttomRNA,andweusedmodel mRNAslackingcontactswiththe40SentryorexitchannelsinreconstitutedPICstoidentifyacritical roleforeIF3ainstabilizingPIC:mRNAinteractionsattheexitchannel,andanancillaryroleatthe entrychannelthatisfunctionallyredundantwithuS3residuesthatcontactmRNA.Weproposethat eIF3and40SproteinscollaboratetofixthemRNAintheexitandentrychannelsoftheribosomeand therebystabilizetheclosed/PINconformationatthestartcodon.Thus,multipleinteractionswithin thePICservetostabilizetheopenorclosedstatesandsettheproperstringencylevelforinitiationat nonͲoptimumstartcodonsinvivo. 18 O41
StructuralPrinciplesofTranscriptionͲcoupledDNARepair
VenuKamarthapu1,FangfangZheng2,LiqiangShen2,BritneyMartinez1,VitalyEpshtein1,YuZhang2, andEvgenyNudler1
1DepartmentofBiochemistryandMolecularPharmacology,HowardHughesMedicalInstitute,NewYorkUniversitySchool ofMedicine,NewYork,NewYork10016,USA2KeyLaboratoryofSyntheticBiology,ChineseAcademyofSciencesCenterfor ExcellenceinMolecularPlantSciences,InstituteofPlantPhysiologyandEcology,ChineseAcademyofSciences,Shanghai, 200032,China
Nucleotideexcisionrepair(NER)isanevolutionarilyconserved,multistepprocessthatcandetecta wide variety of DNA lesions.RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitmentofNERenzymestothedamagesitetherebyexpeditingtherecognitionofDNAdamage byNERcomponents–theprocessknownastranscriptionͲcoupledDNArepair(TCR).Recently,we havedescribedanewpathwayforTCRinE.coli,whereUvrDplaysthecentralrolebypullingstalled RNAPbackwards,awayfromthesiteofDNAdamage,therebyallowingrepairenzymestogainaccess to DNA lesions.Transcription elongation factor NusA and small molecular alarmone ppGpp play importantroleinthisprocess.HerewepresentastructuralmodelfortherecruitmentofUvrDbyE. coliRNAPtoenableTCR.WemapandfunctionallyvalidateRNAPͲUvrDinteractionsbyacombination of XͲray crystallography, crosslinkingͲcoupled mass spectrometry (XLͲMS), fast photoͲoxidation of proteins (FPOPͲMS), and genetic analysis. We identify the extensive interacting surface between UvrD and RNAP and propose the model of UvrDͲmediated RNAP backtracking based on the positioningofUvrDwithrespecttotheDNAscaffoldintheelongationcomplex.Wefurtheridentify the“ɴpincer”moduleofEcRNAPandtheCͲterminaldomainofUvrD(EcUvrDͲCTD)asoneofthe main contacting points between the two enzymes. Disrupting those interactions by mutations in RNAPorUvrDseverelycompromisesTCRinvivo.
19 O51
ExploringRNAPolymeraseRegulationbyNMRspectroscopy
StefanH.Knauer,PhilippZuber,BenjaminDudenhöffer,BirgittaM.Wöhrl,KristianSchweimer,and PaulRösch
LehrstuhlBiopolymereundForschungszentrumfürBioͲMakromoleküle,UniversitätBayreuth,Bayreuth,Germany
MultisubunitRNApolymerases(RNAPs)transcribeallcellulargenomesandarehighlyregulatedby numerous transcription factors. Only little information about dynamics, conformational rearrangements, and transient interactions of RNAPs is available, which, however, is crucial for a comprehensiveunderstandingoftranscriptionregulationinatomicdetail.
Here we present approaches to study Escherichia coli RNAP (400 kDa) by NMR spectroscopy in solution to gain insights into its dynamics and interaction with transcription factors. First, we developed an efficient procedure to assemble active RNAP from separately produced subunits, allowingthespecificlabelingofindividualconstituents[1].Using[1H,13C]ͲlabeledIle,Leu,andVal methyl groups in deuterated proteins as probes (methyl group labeling) we recorded [C,H] correlationspectraofcompleteRNAPandofmethylgrouplabeledɴ’subunitwithinreconstituted, deuterated RNAP, setting the basis for the study of transcription factor/nucleic acid binding [1]. Further,wedevelopedapproachestodeterminetheRNAPbindingsurfacesoftranscriptionfactors and to follow conformational changes within those factors upon RNAP binding using [1H,13C]Ͳ labeled methyl groups of Val, Leu, and Ile in deuterated transcription factors as probes [2]. This allowedus,forexample,todeterminetheRNAPbindingsurfaceoftheNͲterminaldomain(NTD)of NͲutilizationsubstance(Nus)AandthustocreateamodeloftheNusAͲNTD:RNAPcomplex.Finally, weusedtheseapproachestoexploretherecruitmentmechanismofthetransformerproteinRfaHto thepausedtranscriptionelongationcomplex(TEC)bydirectlyfollowingRfaHbindingtotheTECand bydirectlyobservingthestructuraltransformationofitsCͲterminaldomain.
References [1]J.Drögemüller,M.Strauß,K.Schweimer,B.M.Wöhrl,S.H.Knauer,andP.Rösch,SciRep.5,10825(2015) [2]J.Drögemüller,M.Strauß,K.Schweimer,M.Jurk,P.Rösch,andS.H.Knauer,SciRep.5,16428(2015)
20 O61
RegulationofTranscriptionElongationbyNusA–biochemicaland structuralstudies
XieyangGuo,AlexanderMyasnikov,JamesChen,GaborPapai,MariaTakacs,AlbertWeixlbaumer
IGBMCͲInstituteofGeneticsandMolecularandCellularBiologyDepartmentofIntegratedStructuralBiologyIllkirch, France
During the first step of gene expression, a universally conserved enzyme called RNA Polymerase (RNAP)transcribesRNAfromaDNAtemplateineverylivingcell.Transcriptionisafundamental,yet incompletely understood process. Crystal structures of RNAP core as well as RNAP elongation complexes(ECs)haverevolutionizedourunderstandingoftranscription.Thisgroundworkpavedthe waytoaddressmorecomplexquestionsregardingtheregulationoftranscriptionandtheinfluence oftranscriptionfactors.
Transcriptionalregulationimpactseveryaspectofbiologyincludingagrowinglistofhumandiseases. Onewaytoregulatetranscriptionisthroughaprocesscalledtranscriptionalpausing,whichplaysa majorroleinproͲandeukaryotes.PausingisatemporaryinterruptionofRNAextensionduringthe elongationphase.Importantly,pausingcanbefurthermodulatedbytranscriptionfactors.
We will report on our progress using biochemical and structural approaches to gain mechanistic insightsontheregulationoftranscriptionbyaconservedbacterialtranscriptionfactorcalledNusA. NusAisinvolvedinstabilizingpausedstatesofRNAPandalsohasastimulatoryroleintranscription termination. We determined single particle cryoͲEM reconstructions of functional elongation complexesofE.coliRNAPandNusAandwilldescribetheimplicationsforpausingandtranscription termination.
21 O71
AnetworkofpausestrapsthebacterialRNApolymeraseandits nascenttranscriptonthepromoter
DavidDulin,AnssiM.Malinen,DavidL.V.Bauer,JacobJ.W.Bakermans,MartinKaller,Zakia Morichaud,IvanPetushkov,KonstantinBrodolin,AndreyKulbachinskiyandAchillefsN.Kapanidis
FAUErlangenͲNürnberg,OxfordUniversity
Transcription in bacteria is controlled by multiple molecular mechanisms that ensure precise regulation of gene expression. Initial RNA synthesis by promoterͲbound RNAP holoenzyme was recentlyidentifiedtobeinterruptedbypauses,whichdeterminantsandrelationshipwithproductive RNAsynthesisandabortiveinitiationremainpoorlyunderstood.Here,weemployedsingleͲmolecule FRETtodisentanglethepausingͲrelatedreactionpathwaysofinitialtranscription.Wepresentfurther evidencesthatregionʍ3.2,constitutesabarrierat6ntRNA(ITC6),haltingtheRNAP.TheITC6pause is a physiological sensor that directs the holoenzyme either towards productive transcription or towardsalongͲlivedunscrunching–scrunchingpathwaythatdoesnotnecessarilyleadtotranscript release(andinsteadisassociatedwithblockingoftranscriptioninitiation).Thisdemonstratesthat abortive RNA release and the loss of scrunched DNA conformation are not as tightly coupled as previouslythought.Theabovemolecularmechanismrenderstheproductivityofinitialtranscription sharplydependentandpotentiallyregulatedbyNTPconcentration.
22 O81
MolecularMechanismsofTranscriptioninArchaea
FinnWerner
UCLISMB
Wearefollowingamultidisciplinaryapproachtostudythemolecularmechanismsoftranscription usingbotheuryͲandcrenarchaealmodelsystems,rangingfromthemolecularͲtothesystemslevel.I willpresentrecentinsightsintothemechanismsunderlyingtranscriptionprocessivity,andtheway bywhichvirusinfectionͲinducedtranscriptionfactorsappropriatethehosttranscriptionapparatus.
23 O91
NonͲcodingRNAs,Mediator,andMediatorkinases
JonathanD.Rubin,CharliB.Fant,MeaganEsbin,ZacharyPoss,WilliamOld,RobinD.Dowell,DylanJ. Taatjes
UniversityofColorado,Boulder
The human Mediator complex regulates RNA polymerase II (pol II) activity genomeͲwide, and has beenimplicatedinbindingoffunctionallyrelevantnonͲcodingRNAs.Wehavebeguntoexplorehow nonͲcoding RNAs might interact with and/or regulate Mediator function. Our results suggest MediatorisapromiscuousbinderofRNAs,analogoustothePRC2complex.MediatorcontrolspolII functioninpartbyrelayingregulatorysignalsfromDNAͲbindingtranscriptionfactors(TFs)tothepol IIenzyme.Conversely,MediatorcanregulateTFfunctionthroughtheMediatorͲassociatedkinases CDK8 and CDK19, as DNAͲbinding TFs are common substrates for Mediator kinases. Using a combination of approaches, including SILACͲbased phosphoproteomics, GROͲSeq, and in vitro transcription assays, we have begun to uncover basic mechanisms by which the human CDK8 module,andCDK8kinaseactivityperse,regulatespolIItranscriptionatvariousstages.TheCDK8 moduleappearstofunctioncooperativelywithpolIIelongationfactorsanditskinaseactivityaffects enhancerRNA(eRNA)transcriptiondirectedbystimulusͲspecificTFs.Takentogether,theseresults establish new mechanistic roles for Mediator in transcription regulation and provide a more comprehensiveunderstandingofMediatorkinasefunction.
24 O101
FuzzyFreeͲforͲAll:RetentionofFuzzyBindingintheMultiͲDomain InteractionsofTranscriptionActivatorGcn4andMediatorSubunit Med15
LisaM.Tuttle1,2,DerekPacheco1,LindaWarfield1,JieLuo3,JeffRanish3,RachelE.Klevit2andSteven Hahn1
1FredHutchinsonCancerResearchCenter,Seattle,WA,USA2DepartmentofBiochemistry,UniversityofWashington, Seattle,WA,USA,3InstituteforSystemsBiology,Seattle,WA,USA
Transcriptional regulation is often mediated by interactions between intrinsically disordered activation domains (ADs) and transcription coactivators. These interactions often involve multiple activationregionsandmultiplebindingsitesasisthecaseforthetandemADsofyeasttranscription factor Gcn4 and the four activator binding domains (ABDs) of Med15, a subunit of the Mediator transcription coactivator complex. While interaction of multiple ADs and ABDs appears to be a commonthemeintranscriptionactivation,themechanismofhowthesemultidomaininteractions contributetotheaffinityandspecificityofADfunctionwasnotknown.
To address these questions, we used a combination of protein crosslinking, transcription activity assays, biochemistry, and NMR structural analysis to characterize the interactions between the tandemADsofGcn4andthefouractivatorbindingdomainsofMed15,individuallyandaspartofthe larger Gcn4ͲMed15 complex. Our data support a model where, instead of a specific higher order structurebeingformed,thereisafuzzyfreeͲforͲallinvolvingthehydrophobicpatchesofthetandem ADswiththefourstructureddomainsintheactivatorͲbindingregionofMed15.Ourresultsprovidea general model for the mechanism of many activators and demonstrate how multiple weak fuzzy interactionscancombinetogenerateabiologicallysignificantandspecificinteraction.
25 O111
Ccr4ͲNotisatthecoreofthegeneexpressioncircuitry
OlesyaPanasenko1,ZoltanVillanyi1,IshaanGupta2,ChristopherHughes3,LarsM.Steinmetz2,4,Peña ChouCohue5,SyamSomasekharan5,VikramPanse6,MartineA.Collart1
1DepartmentofMicrobiologyandMolecularMedicine,UniversityofGeneva,FacultyofMedicine,2EuropeanMolecular BiologyLaboratory3BritishColumbiaCancerResearchAgency4StanfordUniversity5ETHZurich6VancouverProstate Centre
The Ccr4ͲNot complex is a conserved multiͲprotein complex that regulates gene expression at all stages,fromproductionofthemRNAinthenucleustoitsdegradationinthecytoplasm.Challenging the general understanding of gene expression that considers transcription and translation to be independentprocesses,werecentlydemonstratedthattranslationefficiencyisdeterminedduring transcription elongation through imprinting of ribosomal protein mRNAs with Not1, the central scaffoldofCcr4ͲNot.WealsodeterminedthatNot5ͲdependentNot1associationwithspecificmRNAs wasimportantduringtranslationforassemblyofproteincomplexessuchasRNApolymeraseIIand theSAGAhistoneacetyltransferase.Thisregulationoftranscriptionduringtranslationandtranslation duringtranscriptionplacestheCcr4ͲNotcomplexatthecoreofthegeneexpressioncircuitry.More recentlywehavefocusedonunderstandingthemechanismsbywhichcoͲtranslationalassemblyof protein complexes is regulated and involves Not1. Our results indicate the existence of Not1Ͳ containingassemblysomes.
26 O121
StructuralbiologyofRNApolymeraseIandIIItranscription
ChristophW.Müller
EuropeanMolecularBiologyLaboratory(EMBL),Heidelberg,Germany
RNA polymerase I (Pol I) and RNA polymerase III (Pol III) mainly synthesize nonͲcoding RNA componentsrequiredforribosomeassemblyandproteinsynthesisineukaryotes.PolIsynthesizes precursor ribosomal RNA, whereas Pol III produces small RNAs including tRNAs, 5S RNA and U6 snRNA.PolIandPolIIItranscriptioniscarefullyregulatedinhealthycells,whilemisregulationofPolI andPolIIItranscriptionhasbeenobservedinavarietyofcancers.
Duringthelastyears,wedeterminedmolecularstructuresof14ͲsubunitPolIand17ͲsubunitPolIII unboundandincomplexwithdifferenttranscriptionscaffoldsbyXͲraycrystallographyandcryoͲEM that revealed the dynamics of both transcription machineries as well as their specific adaptions allowingthemtofulfilltheirdistincttranscriptiontasks.ThePolIandPolIIIstructuresalsoservedas starting points for assembling the Pol I and Pol III preͲinitiation complexes (PIC) followed by their cryoͲEManalysis.Inyeast,theminimalPolIPICincludesPolI,thetranscriptionfactorRrn3andCore Factor (CF) composed of subunits Rrn6, Rrn7 and Rrn11, while Pol III minimally requires threeͲ subunittranscriptionfactorTFIIIBwithsubunitsTPB,Brf1(BͲrelatedfactor1)andBdp1.
The Pol I PIC structure reveals an unusual architecture where TFIIBͲrelated factor Rrn7 binds promoterDNAconsiderablymoreupstreamcomparedtoTFIIBandBrf1inthePolIIandPolIIIPICs, respectively.InthePolIIIPIC,TFIIIBparticipatesinanintricateinteractionnetworkwiththePolIIIͲ specificheterotrimerthattrapspromoterDNAabovethePolIIIcleftandleadstoDNAmelting.These andotherdifferencesandsimilaritiesbetweenthePolI,PolIIandPolIIIPICswillbediscussed.
27 O131 iCLIPͲbasedmodelinguncovers3’splicesitedefinition:howU2AF65 specificityreliesonregulationbycoͲfactors
FXReymondSutandy1,StefanieEbersberger1,LuHuang1,AnkeBusch1,MaximilianBach1,HyunSeo Kang2,3,JörgFallmann4,DanielMaticzka5,RolfBackofen5,7,PeterF.Stadler5,KathiZarnack6,Michael Sattler2,3,StefanLegewie1,JulianKönig1
1InstituteofMolecularBiology(IMB)gGmbH,Ackermannweg4,55128Mainz,Germany2InstituteofStructuralBiology, HelmholtzZentrumMünchen,IngolstädterLandstr.1,85764Neuherberg,Germany3BiomolecularNMRandCenterfor IntegratedProteinScienceMunichatDepartmentofChemistry,TechnicalUniversityofMunich,Lichtenbergstr.4,85747 Garching,Germany4BioinformaticsGroup,DepartmentofComputerScienceandInterdisciplinaryCenterfor Bioinformatics,UniversityofLeipzig,Härtelstraße16Ͳ18,04107Leipzig,Germany5BioinformaticsGroup,Departmentof ComputerScience,UniversityofFreiburg,Freiburg,Germany6BuchmannInstituteforMolecularLifeSciences(BMLS),MaxͲ vonͲLaueͲStr.15,60438Frankfurta.M.,Germany7CentreforBiologicalSignallingStudies(BIOSS),UniversityofFreiburg, Freiburg,Germany
Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes.TheRNAͲbindingprotein(RBP)U2AF65iscentraltosplicingdecisions,asitrecognizes3' splice sites and recruits the spliceosome. We establish 'in vitro iCLIP' experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF65 recognizes RNA sequences and how this is modulated by transͲacting RBPs. We measure U2AF65 affinities at hundreds of binding sites, and compare in vitro and in vivo binding landscapes by mathematical modeling. We find that transͲacting RBPs extensively regulate U2AF65 binding in vivo, including enhancedrecruitmentto3'splicesitesandclearanceofintrons.Usingmachinelearning,weidentify and experimentally validate novel transͲacting RBPs (including FUBP1, BRUNOL6 and PCBP1) that modulate U2AF65bindingandaffectsplicingoutcomes.OurstudyoffersablueprintforthehighͲ throughputcharacterizationofinvitromRNPassemblyandinvivosplicingregulation.
28 O141
N6ͲmethyladenosinedemethylaseFTOtargetspreͲmRNAsand regulatesalternativesplicingand3’endprocessing
MarekBartosovic1,HelenaCoveloͲMolares1,PavlinaGregorova1,DominikaHrossova1,Grzegorz Kudla2andŠtĢpánkaVaŸáēová1
1CEITEC–CentralEuropeanInstituteofTechnology,MasarykUniversity,Brno,62500,CzechRepublic.2MRCHumanGenetics UnitMRCIGMM,UniversityofEdinburghWesternGeneralHospital,CreweRoad,EdinburghEH42XU,UnitedKingdom
N6Ͳmethyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs).Thereversibilityanddynamicsofm6AinmRNAshasbeenlinkedtoanumberofregulatory stepsinthemRNAmetabolism.Themajorfocusofmanyworkswasontheroleofthemethylase complexesandthemechanismsandfunctionofdemethylationremainesratherunexplored.There are at least two mRNA m6A demethylases in mammals, ALKBH5 and FTO. We have performed comprehensivetranscriptomeͲwideanalysesofFTOtargetsinhumancells.OurresultsrevealFTOas apotentregulatorofnuclearmRNAprocessingeventssuchasalternativesplicingand3഻endmRNA processing. Using crossͲlinking and immunoprecipitation coupled to highͲthroughput sequencing (CLIPͲseq)werevealthatFTObindspreferentiallytopreͲmRNAsinintronicregions,intheproximity ofalternativelyspliced(AS)exonsandpoly(A)sites.DepletionofFTObygeneknockoutresultsin substantialchangesinpreͲmRNAsplicingwithprevalenceofexonskippingevents.FTObindingwas highlyenrichedintheskippedexons,indicatingitsdirectroleinASregulation.TheASphenotype couldbereproducedfromaheterologousreportersystemandrescuedwithepisomalexpressionof WT,butnotmutantFTO.Deletionsofintronicregioncontainingm6AͲlinkedDRACHmotifresultedin partialdiminutionofFTOKOASphenotype.Insummary,theseexperimentsdemonstrated,thatthe splicing effects are dependent on the FTO catalytic activity in vivo and are mediated by m6A. Our resultsrevealforthefirsttimethedynamicconnectionbetweenFTORNAbindinganddemethylation activitythatinfluencesseveralmRNAprocessingevents.
29 O151
CellulardifferentiationstatemodulatesthemRNAexportand retentionactivitiesofSRproteins
FrançoisMcNicoll,FlorianM.Richter,AnfisaSolovyeva,MariusWegener,OliverD.Schwich,Ilka Wittig,KathiZarnack,MichaelaMüllerͲMcNicoll
GoetheUniversityFrankfurt
SRproteinsfunctioninnuclearpreͲmRNAprocessing,mRNAexportandtranslation.Toinvestigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleoͲ cytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining iCLIP and mass spectrometry, we show that elevated arginine methylationandlowerphosphorylationofcoͲboundSRSF2enhanceshuttlingofSRSF5inP19cellsby modulatingproteinͲproteinandproteinͲRNAinteractions.Moreover,SRSF5isboundtopluripotencyͲ specifictranscriptssuchasLin28aandPou5f1/Oct4inthecytoplasm.SRSF5depletionreducesand overexpressionincreasestheircytoplasmicmRNAlevels,suggestingthatenhancedmRNAexportby SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19cellsleadstodramaticallyreducedSRSF5shuttling.OurfindingsindicatethatpostͲtranslational modificationofSRproteinsunderliestheregulationoftheirmRNAexportactivitiesanddistinguishes pluripotentfromdifferentiatedcells.
30 O161
InsightsintospecificRNArecognitionmediatedbyhumanStaufen1 fromthecrystalstructureofitsminimalRNAͲbindingregionin complexwithaphysiologicaldsRNAtarget
DanielaLazzaretti,LinaBandholz,ChristianeEmmerichandFulviaBono
MaxPlanckInstituteforDevelopmentalBiology,Tuebingen,Germany
Members of the Staufen (Stau) protein family have conserved functions at several steps of postͲ transcriptional gene regulation. In Drosophila, Stau is involved in the intracellular localization of mRNAsrequiredforanteriorͲposterioraxisdeterminationduringoogenesis,neuroblastasymmetric cell division in the developing brain, and long term memory (LTM) formation in the adult brain. Mammals express two Stau paralogs, which mediate mRNA transport in neurons, and function in neuronalmorphogenesisandplasticity.Inaddition,mammalianStauproteinsareinvolvedinaform of translationͲdependent mRNA degradation known as StauͲmediated decay (SMD). All these functionsrequireStautotargetspecificmRNAs.
Stau family members contain several copies of the dsRNAͲbinding domain (dsRBD), although only two of these (dsRBDs 3 and 4) are required for RNA binding. dsRBDs primarily recognize RNA secondarystructuresinasequenceͲindependentmanner,raisingthequestionofhowStauproteins areabletorecognizetheirtargets.
Here we present the crystal structure of human Stau1 (hStau1) minimal RNAͲbinding region in complexwithadoublestrandedRNA(dsRNA)fromaphysiologicaltarget.Thestructureshowshow hStau1 dsRBDs sense dsRNA structure through contacts with the sugarͲphosphate backbone, and reveals positions at which baseͲspecific contacts are formed to achieve target specificity. RNAͲ dependent dimerization of hStau1, as well as dsRBD flexibility, could further modulate substrate recognition.
31 O171
DissectingthefunctionsofSMG5,SMG7andPNRC2innonsenseͲ mediatedmRNAdecayofhumancells
PamelaNicholson,AsiminaGkratsou,ChristophJosi,MartinoColomboandOliverMühlemann
DepartmentofChemistryandBiochemistryUniversityofBernSwitzerland
Theterm“nonsenseͲmediatedmRNAdecay”(NMD)originallydescribedthedegradationofmRNAs withprematuretranslationͲterminationcodons(PTCs),butitsmeaninghasrecentlybeenextended tobeatranslationͲdependentpostͲtranscriptionalregulatorofgeneexpressionaffecting3Ͳ10%of allmRNAs.ThedegradationofNMDtargetmRNAsinvolvesbothexonucleolyticandendonucleolytic pathwaysinmammaliancells.WhilethelatterismediatedbytheendonucleaseSMG6,theformer pathwayhasbeenreportedtorequireacomplexofSMG5ͲSMG7orSMG5ͲPNRC2bindingtoUPF1. However, the existence, dominance and mechanistic details of these exonucleolytic pathways are divisive.Therefore,wehaveinvestigatedthepossibleexonucleolyticmodesofmRNAdecayinNMD byexaminingtherolesofUPF1,SMG5,SMG7andPNRC2usingacombinationoffunctionalassays andinteractionmapping.Confirmingpreviouswork,wedetectedaninteractionbetweenSMG5and SMG7andalsoafunctionalneedforthiscomplexinNMD.Incontrast,wefoundnoevidenceforthe existenceofaphysicalorfunctionalinteractionbetweenSMG5andPNRC2.Instead,weshowthat UPF1interactswithPNRC2andthatittriggers5’Ͳ3’exonucleolyticdecayofreportertranscriptsin tetheringassays.PNRC2interactsmainlywithdecappingfactorsanditsknockdowndoesnotaffect theRNAlevelsofNMDreporters,indicatingthatPNRC2isanimportantmRNAdecappingfactorbut itisnotrequiredforNMD.
32 O181
TheRrp4ͲexosomecomplexrecruitsandchannelssubstrateRNAby auniquemechanism
CvetkovicMA,WurmJP,AudinMJ,SchützS,SprangersR
DepartmentofBiophysicsI,UniversityofRegensburg,93053Regensburg,Germany
The exosome is a large molecular machine involved in RNA degradation and processing. Here we address how the trimeric Rrp4 cap enhances the activity of the archaeal enzyme complex. Using methylͲTROSYNMRmethodsweidentifieda50ͲÅlongRNAbindingpathoneachRrp4protomer.We showthattheRrp4capcanthussimultaneouslyrecruitthreesubstrates,oneofwhichisdegradedin thecorewhiletheothersarepositionedforsubsequentdegradationrounds.Thelocalinteraction energybetweenthesubstrateandtheRrp4Ͳexosomeincreasesfromtheperipheryofthecomplex towardtheactivesites.Notably,theintrinsicinteractionstrengthbetweenthecapandthesubstrate is weakened as soon as substrates enter the catalytic barrel, which provides a means to reduce friction during substrate movements toward the active sites. Our data thus reveal a sophisticated exosomeͲsubstrateinteractionmechanismthatenablesefficientRNAdegradation.
33 O191
DecodingregulatoryproteinͲRNAinteractionsingeneregulation usingintegratedstructuralbiology
MichaelSattler
HelmholtzZentrumMünchen/TechnicalUniversityofMunich
RNAplaysessentialrolesinvirtuallyallaspectsofgeneregulation,whichinvolvetherecognitionof cis regulatory RNA sequences by RNA binding proteins (RBPs). Most eukaryotic RBPs are multiͲ domain proteins that comprise multiple structural domains to mediate proteinͲRNA or proteinͲ proteininteractions.Weemployintegratedstructuralbiologytounravelthemolecularmechanisms involving these regulatory RNP (ribonucleoprotein) complexes. For these studies, solution NMRͲ spectroscopy and SAXS/SANS provide unique information on functionally important dynamics and are combined with XͲray crystallography and electron microscopy to elucidate the structural mechanismsanddynamicsofregulatoryRNPs.Exampleswillbepresentedthathighlighttheroleof conformational dynamics in RNA recognition and proteinͲprotein interactions in the regulation of eukaryoticpreͲmRNAsplicing,translationandRNAstability.
34 O201
Mechanismofribosomebiogenesisanditslinkto nucleocytoplasmictransport
EdHurt
UniversityofHeidelberg,BiochemieͲZentrumHeidelberg(BZH),ImNeuenheimerFeld328,69120Heidelberg
RibosomesconsistingofribosomalRNAandribosomalproteinsarethemachinesthatsynthesizethe proteins of the cell. In eukaryotes, the two ribosomal subunits (60S and 40S subunit) are first assembled in the nucleolus before export to the cytoplasm. Ribosome biogenesis is not only complicatedbutthemostenergyconsumingprocessingrowingcells,andthusrequiresextensive regulation and coordination. Eukaryotic ribosome synthesis is initiated by transcription of a large rRNA precursor, which is subsequently modified and processed to 25S, 5.8S and 18S rRNA with a concomitant assembly of the ribosomal proteins. At the beginning of ribosome synthesis, a huge (90S)precursorparticleisformedthatissubsequentlysplittoinducetheformationofthepreͲ60S and preͲ40S particles, which each follow separate biogenesis and export routes. During ribosome synthesis about 200 nonͲribosomal factors and 100 small nonͲcoding RNAs (snoRNAs) transiently workontheevolvingribosomalsubunitstofacilitatetheirassembly,maturationandtransport.We study ribosome formation in vivo in the yeast Saccharomyces cerevisiae and exploit a eukaryotic thermophile, Chaetomium thermophilum, for structural studies. In my talk I will summarize our recentfindingsonthemechanismofribosomebiogenesisanditslinktonucleocytoplasmictransport, whichwereobtainedfrominvitroassayscombinedwithgeneticinvestigationsandstructuralstudies includingelectronmicroscopyandxͲraycrystallography.
35 O211
Coordinationofdistant40Sribosomalsubunitmaturationevents
ValentinMitterer,HajrijaOmanic,SarahWeigl,DieterKresslerandBrigittePertschy
KarlͲFranzensͲUniversitätGraz,InstitutfürMolekulareBiowissenschaften,Humboldtstrasse50/EG,8010Graz
Thesynthesisofeukaryoticribosomesisanintricateprocessinvolvingacomplexmaturationcascade withamultitudeofintermediateswhichtravelfromthesiteofinitialassemblyofribosomalproteins with ribosomal RNA in the nucleolus through the nucleoplasm and are then exported into the cytoplasm.Onthatpath,theyaretransientlyboundbymorethan200ribosomeassemblyfactors which participate in various aspects of their maturation, including rRNA processing, assembly of ribosomalproteins,restructuringeventsandreleaseofassemblyfactors.
Wearestudyingthematurationoffreshlyexportedprecursorparticlesofthesmall40Sribosomal subunit,whichstillcontain~10ribosomeassemblyfactorsboundatdifferentimportantfunctional sites of the 40S. Among them is Rio2, an ATPase positioned on the intersubunit side of the 40S subunit in the so called "head region". Rio2 hydrolyses ATP and is believed to thereby trigger structural reͲarrangements. Additionally, ATP hydrolysis results in Rio2 dissociation from preͲ40S particles.Anotherassemblyfactor,Ltv1isalsopositionedinthe40Shead,albeitdistantlyfromRio2 on the solvent exposed side. Its release involves phosphorylation by a protein kinase Hrr25 and allows for the stable incorporation of ribosomal protein Rps3 into 40S subunits. Intriguingly,despitethedistantpositioningofRio2andLtv1onopposingsidesofthe40Shead,we showthattheirreleaseismutuallylinked.ATPhydrolysisbyRio2isnotonlyapreͲrequisiteforRio2 dissociation, but is also required for Ltv1 release. Vice versa, Ltv1 phosphorylation triggers its liberation from preͲ40S particles, but is also necessary for Rio2 release. These results raise the question how such tight communication between physically distant maturation events is possible. Notably,aribosomalprotein,Rps20,ispositionedonthesolventsideofthe40Ssubunit,whereit interactswithLtv1andRps3.AdditionallyRps20hasanextensiondivingdeepintothe40Ssubunit andreachingalmostthroughthe40Shead,therebymakingcontactswith18SrRNAhelix31,which was previously shown to be also bound by Rio2. In this study, we provide evidence that Rps20 is involvedinRio2andLtv1releaseevents,andmighttherebycoordinatethesespatiallydistantsteps inpreͲ40Smaturation.
36 O221
Highthroughputdiscoveryofnovelregulatorsofhumanribosome biogenesis
KatherineI.FarleyͲBarnes1,KathleenMcCann1,LisaOgawa1,JanieMerkel2,YuliaSurovtseva2,Susan J.Baserga1
1YaleUniversityandtheYaleSchoolofMedicine,NewHaven,CT06520,2YaleWestCampus,Orange,CT06477
Itiswellknownthatincanceranincreasedsizeandnumberofnucleolicorrelateswithincreased malignant potential. Nucleoli make ribosomes, the essential protein synthesizers of the cell. Previouslywehaveshown(PLoSGenetics2012)thatdisruptionofnucleolarfunctioninmammalian cellsbydepletionoftheribosomebiogenesisfactorsUTP4orNOL11causesachangeinthenumber ofnucleolipercellfrom2Ͳ3toonly1.Takingadvantageofthisrelationshipbetweennumberand function,weexploitedthisassaytodiscovernewregulatorsofnucleolarfunctioninhumancellsviaa highͲcontent, highͲthroughput, genomeͲwide siRNA screen. This screen successfully identified approximately200proteinswhosedepletioncausedachangeinthenumberofnucleolipercellfrom 2Ͳ3toonly1.Thefunctionofasubsetofthesehitsinribosomebiogenesishasbeenvalidatedby oligonucleotidedeconvolution.OfthesehighͲconfidence,validatedhits,20werechosenforfurther study,includingbothnucleolarandnonͲnucleolarproteins.Whilethenucleolarproteinsaredirectly involvedinribosomebiogenesis,thenonͲnucleolarproteinslikelyreflectnewpathwaysofnucleolar regulationinmammaliancells.Outofthe20testedhits,wehaveidentifiednewrolesfor17hitsin regulatingeithertRNApolymeraseItranscriptionoftheribosomalDNA(7/20),theprocessingofthe preͲribosomal RNA (16/22) and/or global protein synthesis (14/20). This genomeͲwide analysis exploitstherelationshipbetweennucleolarnumberandfunctiontodiscoverunexpectedanddiverse cellularpathwaysthatregulatethemakingofribosomesandpavesthewayforfurtherexplorationof thelinksbetweenribosomebiogenesisandhumandisease.
SupportedbyNIHR01GM0115710andbytheYaleCancerCenter.
37 O231
AnoveldsRNAͲbindingmodulefromanuclearprolylisomerase
DavidDilworth,SantoshKumarUpadhyay,PierreBonnafous,AmiirahBibiEdoo,FrancyPesekͲJardim, GeoffGudavicius,ChristopherNelson,CameronMackereth
InsermU1212,Univ.Bordeaux,InstitutEuropeendeChimieetBiologie
ProlylisomerasesaredefinedbyacatalyticdomainthatfacilitatesthecisͲtransinterconversionof proline residues. In most cases, additional domains are also present in these enzymes that add importantbiologicalfunctions.Whileseveralisomeraseshavebeenlocatedinthenucleus,verylittle is known about the range of native substrates and the cellular function of nuclear FK506Ͳbinding proteins (FKBPs). In order to gain insight into the biological role of the nuclear prolyl isomerase FKBP25,wehaveperformedproteomicassaystoidentifypartnersofFKBP25andhaveidentifiedthat these interactions occur via an RNAͲdependent mechanism based on a previously unidentified dsRNAͲbindingdomain.Theproximitydependentbiotinidentification(BioID)approachprovidesan historical snapshot of neighboring protein interactions to FKBP25 fused to the E. coli biotin ligase BirA.BiotinylatedproteinsintheFKBP25ͲBirAsamplebelongtoRNAͲbindingproteinsandribosome biogenesis factors, including 22 ribosomal proteins and several proteins involved in the early maturationeventsofribosomeproduction.TheseFKBP25Ͳassociatedproteinswerefurthervalidated by using a complementary coͲimmunopurification assay. Strikingly, the addition of RNase A in a followͲupassaystronglyreducedtheinteractingpartnersofFKBP25,implicatingaroleforRNAin mediating the contacts. Furthermore, we find that RNA is necessary for the localization and recruitmentofFKBP25tonucleolarsitesofribosomebiogenesis,withFKBP25coͲlocalizingwiththe rRNA transcription factor UBF in an RNAͲdependent manner. We therefore predicted a direct interaction with RNA to explain these findings, and a series of in vitro experiments were used to elucidate the nature of the RNA association by FKBP25. We find that FKBP25 in fact harbours a module that binds specifically to doubleͲstranded RNA (dsRNA) and is selective for dsRNA over dsDNA,ssRNAandssDNA.AminimaldsRNAlengthofeightbaseͲpairsisrequiredfortheinteraction, and in the context of ribosome biogenesis this interaction also occurs with nucleolinͲbound rRNA stemloop structures. The atomic details were used to design a minimal mutation to abrogate the interaction with RNA in vitro, and this mutant also prevents interaction with RNA in vivo. We hypothesizethattherepertoireofsubstratesforFKBP25isdefinedbycellularlocalization,grantedin thiscasebyselectivebindingtodsRNA.
38 O241
The5SRNPprovidesthelinkbetweenribosomebiogenesisandthe tumoursuppressorp53
AndriaPelava,LorenGibson,JustineLee,DennisNagl,ZhaoZhao,ClaudiaSchneiderandNicholas Watkins
InstituteforCellandMolecularBiosciences,NewcastleUniversity,NewcastleuponTyne
The 5S RNP (RPL5, RPL11 and the 5S rRNA) is an essential assembly intermediate of the large ribosomalsubunitthatisalsoakeysignallingfactorimportantforthecellularreactiontostalledor reduced levels of ribosome production. Perturbations in ribosome production lead to the accumulationofthe5SRNP,whichbindsto,andinhibitsMDM2,themainregulatorofthetumour suppressorp53,resultinginp53activation.Theactivationofp53,atranscriptionregulator,leadsto antiͲproliferative responses such as cell cycle arrest, apoptosis or senescence. The 5S RNPͲMDM2 pathwayhasbeenshowntobeinducedinseveralgeneticdiseasescausedbydefectsinribosome biogenesis (ribosomopathies) and is central to the cell cancer defence systems. Indeed, the ribosomalproteinRPL5,whichismutatedinthemoresevereformsofDiamondBlackfananaemia, hasbeendescribedasa“cancergene”inseveraltumourtypes.
Wehaveshownthatdefectsineitherlargeorsmallribosomesubunitproduction,evencytoplasmic steps, lead to p53 activation through the 5S RNPͲMDM2 pathway. Indeed, this pathway monitors ribosomebiogenesisandp53activationoccurspriortochangesinribosomalsubunitlevels.Ourdata alsoshownthatablockinribosomebiogenesisresultsintherapidaccumulationofmillionsofcopies of the 5S RNP that sequester the cellular MDM2 pool. Interestingly, the 5S RNPͲMDM2 complex remainsboundtop53suggestingthatthe5SRNPcandirectlymodulatep53activity.Wehaveshown thatRPL5,infree(nonͲribosomal)5SRNP,isphosphorylatedandthatthismodificationcontrolsboth ribosomebiogenesisandcellularsignalling.Finally,wehavefoundthatthesplicingfactorSRSF1is required for efficient 5S RNP recruitment into the ribosome as well as the repression of MDM2 activity.Takentogether,ourdataindicatethatp53signallingandribosomeproductionareintimately linkedviathe5SRNPandthatthislinkiscriticaltoregularcellulargrowthandfunction.
39 O251
InteractionofhnRNPKandrpS19anditsfunctioninerythroid maturation
IsabelS.NaarmannͲdeVries1,BodoMoritz2,HenningUrlaub3,4,RobertaSenatore1,DierkNiessing5,6, AntjeOstareckͲLederer1andDirkH.Ostareck1
1DepartmentofIntensiveCareandIntermediateCare,ExperimentalResearchUnit,UniversityHospital,RWTHAachen University,AachenGermany;2InstituteofPharmacy,FacultyofNaturalSciences,MartinͲLutherͲUniversityHalleͲ Wittenberg,Halle(Saale),Germany;3MaxͲPlanckͲInstituteforBiophysicalChemistry,BioanalyticalMassSpectrometry Group,Göttingen;4DepartmentofClinicalChemistry,UniversityMedicalCenter,Göttingen,Germany;5DepartmentofCell Biology,BiomedicalCenteroftheLMUMunich,Martinsried;6InstituteofStructuralBiology,HelmholtzCenterMunich, Neuherberg,Germany
In erythropoiesis posttranscriptional control is essential to safeguard structural and metabolic transformationduringenucleatedreticulocytetoerythrocytestransition.Regulationofreticulocyte 15Ͳlipoxygenase(r15ͲLOX)mRNAtranslationbyhnRNPK,whichconstitutesasilencingcomplexat the 3’UTR DICE1Ͳ3 secures initiation of timely mitochondria degradation by newly synthesized r15Ͳ LOX.
To elucidate how hnRNP K interferes with 80S ribosome formation to suppress r15ͲLOX mRNA translation initiation, we applied three independent interaction screens: 1] DICE RNA affinity chromatography from cytoplasmic extracts of K562 cells, which represent a premature erythroid state was combined with hnRNPKͲimmunoprecipitation3. 2] HnRNP K interacting proteins were enriched from extracts of K562 cells induced for erythroid maturation and nonͲinduced cells2. 3] ConsideringalterationsinhnRNPKargininedimethylationduringerythroidmaturation2,4,methylated andnonͲmethylatedHisͲhnRNPK5wereemployedtopurifyinteractorsfromRNaseAͲtreatedK562 extracts.
All strategies identified the 40S ribosomal subunit protein rpS19. Mutations in rpS19 have been functionally linked to impaired erythropoiesis in Diamond Blackfan Anemia (DBA)6. Interestingly, hnRNPKͲrpS19interactiondeclinesduringerythroidmaturationofK562cellsinvitroandinvivo. WestudiedtheimpactofdifferentiallydimethylatedarginineresiduesinhnRNPKonrpS19binding inthermalshiftassays.FurthermoreweanalyzedthedisturbedhnRNPKinteractionwithDBArelated rpS19variants.Theirfunctioninr15ͲLOXmRNAsilencinganderythropoiesiswasexaminedininvitro translationassayswithrecombinantrpS19variantsandbydepletionfromK562cells.
1OstareckDetal.(1997)Cell 2NaarmannISetal.(2008)JBC 3NaarmannISetal.(2010)RNA 4OstareckͲLedererAetal.(2006)JBC 5MoritzBetal.(2014)Biol.Chem. 6DraptchinskaiaNetal.(1999)Nat.Gen.
40 O261
RibosomeͲtargetingantibioticsandmechanismsofbacterial resistance
DanielNWilson
InstitutfürBiochemieundMolekularbiologie,MartinͲLutherͲKingͲPlatz6,20146Hamburg
The ribosome is one of the main antibiotic targets in the bacterial cell. Structures of naturally producedantibioticsandtheirsemiͲsyntheticderivativesboundtoribosomalparticleshaveprovided unparalleledinsightintotheirmechanismsofaction,andtheyarealsofacilitatingthedesignofmore effectiveantibioticsfortargetingmultidrugͲresistantbacteria.Inthispresentation,Iwilldiscussthe recent structural insights into the mechanism of action of ribosomeͲtargeting antibiotics and the molecularmechanismsofbacterialresistance,inadditiontotheapproachesthatarebeingpursued fortheproductionofimproveddrugsthatinhibitbacterialproteinsynthesis.
REFERENCES
Arenz S, Wilson DN. (2016) Blast from the Past: Reassessing Forgotten Translation Inhibitors, Antibiotic Selectivity,andResistanceMechanismstoAidDrugDevelopment.MolCell.61(1):3Ͳ14. WilsonDN.(2014)RibosomeͲtargetingantibioticsandmechanismsofbacterialresistance.NatRevMicrobiol. 2014,12(1):35Ͳ48. SohmenD,HarmsJM,SchlunzenF,WilsonDN(2009)SnapShot:AntibioticinhibitionofproteinsynthesisI.Cell 138:1248e1241.
41 O271
DiscoveryandcharacterizationofribosomeͲarrestingpeptidesby HighͲthroughputInverseToeprinting
BrittaSeip,GuénaëlSacheau,DenisDupuy,AxelInnis
InstitutEuropéendeChimieetBiologie,UniversityofBordeaux,InsermU1212,CNRSUMR5320,Pessac33607,France
During translation by the ribosome, nascent peptides sometimes block their own synthesis by interactingwiththeexittunnelofthelargeribosomalsubunit.Althoughthisprocessmainlydepends upontheaminoacidsequenceoftheribosomeͲarrestingpeptide,itcanalsorelyuponthesensingof asmallmoleculebytheribosomenascentchaincomplex,explainingitsusefordrugormetaboliteͲ dependent gene regulation in bacteria and in eukaryotes. While numerous biochemical and structuralstudieshavesought todissectthemoleculardetailsofthearrestprocess,theabilityof ribosomeͲarresting peptides to sense different types of small molecules and their impact as regulatorsofgeneexpressioninnaturearestilllargelyunexplored.
Here, I will present our ongoing efforts to decipher the arrest code governing nascent chainͲ mediated translational arrest in bacteria. Specifically, I will describe HighͲthroughput iNverse Toeprinting (HiNT), a method for the systematic analysis of nascent chainͲassociated ribosomal stallingevents,andIwilldiscussitsapplicationtothestudyofdrugͲdependenttranslationalarrest. ByperformingHiNTonanmRNAlibraryencoding>1011randompeptidesequencestranslatedinvitro in the presence or absence of the macrolide antibiotic erythromycin, we could establish the completesequenceͲspecificarrestprofileofthisdrugandcomparethestrengthofdrugͲdependent pauses with those induced by short intrinsic arrest motifs. Importantly, successive cycles of HiNT shouldmakeitpossibletoevolveandenrichlonger,lessabundantarrestsequencespresentwithin theinputlibrary.WewillthereforeuseHiNTtodiscoversequencesthatcausetranslationalarrestin responsetodifferentsmallmolecules.Giventhatthesitesofactionofarrestpeptidescoincidewith thoseofknownantibioticsandnaturalantimicrobialpeptides,addressingthefunctionaldiversityand molecular basis of the arrest process will not only be key to understanding a central aspect of ribosomebiology,butcouldalsoprovideahandlefordesigningnextͲgenerationantibiotics.
42 O281
Extensions,extrafactorsandextremecomplexity:Ribosomal structuresprovideinsightsintoeukaryotictranslation
NenadBan
ETHZurich,MolecularBiologyandBiophysics,OttoSternWeg5,8093Zurich,Switzerland
We are investigating bacterial and eukaryotic ribosomes and their functional complexes to obtain insightsinto the process of protein synthesis. Eukaryotic ribosomes are much more complex than theirbacterialcounterparts,requirealargenumberofassemblyandmaturationfactorsduringtheir biogenesis, usenumerous initiation factors, and are subjected to extensive regulation. We have investigated the structuresof eukaryotic ribosomes and their complexes involved in initiation and maturationandcomplexesinvolvedinregulationofproteinsynthesis(1,2,3,4).Theseresultsprovide insightsintothearchitectureoftheeukaryoticribosomeandintovariouseukaryoticͲspecificaspects of protein synthesis. Recently, usingelectron microscopy, we determined the complete molecular structureofthe55Smammalianmitoribosome.Themapsthatwecalculatedbetween3.4and3.6A resolution allowed deͲnovo tracing of a large numberof mitochondrial specific ribosomal proteins and visualization of interactions between tRNA and mRNA inthe decoding centre, the peptidyl transferasecenter,andthepathofthenascentpolypeptidethroughtheidiosyncratictunnelofthe mammalian mitoribosome.Furthermore, the structure suggested a mechanismof how mitochondrial ribosomes, specialized for the synthesis of membrane proteins, are attached to membranes(5,6). 1.RablJ,LeibundgutM,SandroF.AtaideSF,HaagAandBanN.(2010)Nature331(6018):730Ͳ6. 2.KlingeS,VoigtsͲHoffmannF,LeibundgutM,ArpagausS,BanN.(2011)Science,334,941Ͳ8. 3.ErzbergerJPetal.(2014)Cell158(5):1123Ͳ35. 4.AylettCHetal.(2016)Science351(6268):48Ͳ52 5.GreberBJ,BoehringerDetal.(2014)Nature.505(7484):515Ͳ9. 6.GreberBJ,BieriP,etal.(2015)Science.348(6232):303Ͳ8.
43 O291
ThesmallribosomalproteinRPS3anditsfunctions
KristynaPoncova,MyrteEsmeraldaJansen,LeosShivayaValasek
InstituteofMicrobiology,CzechAcademyofSciences
Eukaryotictranslationalinitiationrequiresinteractionofmanyproteinfactorswiththe40Sribosomal subunit.Thesefactorsarecalledeukaryoticinitiationfactors(eIFs)andnumerousstudieshavebeen publishedovertheyearsdescribingtheirspecificrolesnotonlyintranslationalinitiation,butalsoin terminationandribosomalrecycling.SmallribosomalproteinsarekeyplayersinanchoringtheeIFs totheribosomalsurfaceandperhapseveninmodulatingtheirfunction,buttheirexactrolesremain largelyunexplored.Inthisstudy,wefocusedonsmallribosomalproteinRPS3,whichliesnearthe mRNA entry channel and is known to be a part of the latch mechanism modulating mRNA recruitmentandmovementoftheribosomealongmRNA.Inaddition,RPS3interactswiththeTIF32 andTIF35subunitsofeIF3,recentlyimplicatedincontrollingtranslationterminationandpromoting programmed stop codon readthrough. Indeed, our experiments identified two RPS3 variants with altered readthrough levels. Interestingly, both mutants seem to have an opposite effect on the efficiencyofreadthrough(increasevs.decrease),whichmostlikelyreflectstheirspecificorientation towardsotherconstituentsofthelatchmechanism.
44 O301
EfficientPreventionofNeurodegenerativeDiseasesbyDepletionof StarvationResponseFactorAtaxinͲ2
AuburgerG1,SenNE2,MeierhoferD3,BaƔakAN4,GitlerAD5
1DepartmentofNeurology,GoetheUniversityMedicalSchool,60590FrankfurtamMain,Germany.Electronicaddress: [email protected]Ͳfrankfurt.de.2DepartmentofNeurology,GoetheUniversityMedicalSchool,60590FrankfurtamMain, Germany.3MaxPlanckInstituteforMolecularGenetics,Ihnestraɴe63Ͳ73,14195Berlin,Germany.4Sunaand7nanKŦraç Foundation,NeurodegenerationResearchLaboratory(NDAL),BoŒaziçiUniversity,34342Istanbul,Turkey.5Departmentof Genetics,StanfordUniversitySchoolofMedicine,Stanford,CA94305,USA
AtaxinͲ2 (ATXN2) homologs exist in all eukaryotic organisms and may have contributed to their origin. Apart from a role in endocytosis, they are known for global effects on mRNA repair and ribosomal translation. Cell size, protein synthesis, and fat and glycogen storage are repressed by ATXN2 via mTORC1 signaling. However, specific liver mitochondrial matrix enzymes and the mitochondrialrepairfactorPINK1requireATXN2abundance.Duringperiodsofstarvation,ATXN2is transcriptionally induced and localized to cytosolic stress granules, where nuclear factors dock to compensateRNApathology.Thesephysiologicalactionswerenowrevealedtobecrucialforhuman neurodegenerativediseases,giventhatATXN2depletionissurprisinglyefficientinpreventingmotor neuronandcerebellaratrophy,asdemonstratedinmousemodels,flies,andyeast.
45 O311
MutantTDPͲ43selectivelyaffectstranslationofspecificmRNAsin culturedcorticalneuronsandmotorneuronͲlikecells
NagammalNeelagandan,StefanDang,GiorgioGonnella,MalikAlawi,PhillipChristophJaniesch, KatrinKüchler,KatharineK.Miller,DanielaIndenbirken,AdamGrundhoff,StefanKurtz,KentDuncan
CenterforMolecularNeurobiology(ZMNH),UniversityMedicalCenterHamburgͲEppendorf,Hamburg,Germany
TDPͲ43isanRNAͲbindingproteinimplicatedinseveralneurodegenerativediseases,particularlyALS andFTLD.Inpatients,numerousmutationshavebeenidentifiedinTARDBP,thegenethatcodesfor TDPͲ43suggestingacausallinkbetweenalteredTDPͲ43functionanddisease.TDPͲ43isprimarilya nuclearprotein,butlevelsinthecytoplasmincreaseindisease,whereitisthemajorcomponentof aggregatesinmostformsofALS.CellandanimalmodelsindicatethatTDPͲ43’sRNAͲbindingactivity isessentialfortoxicityandexpressionofmutantTDPͲ43inmotorneuronsissufficientfordisease phenotypes.Thus,alteredregulationofcellularRNAsbyTDPͲ43inmotorneuronspresumablydrives disease.PreviousstudieshaverevealedTDPͲ43’sdirectRNAtargetsandfunctioninmanyaspectsof mRNA metabolism. However, it remains unresolved which effects on gene expression are the key drivers of disease. We report here that mutant TDPͲ43 can selectively modulate translation of specificmRNAsinneuronalcelllinesandculturedprimaryneurons.
To examine possible effects of TDPͲ43 on translation of specific mRNAs, we applied the sensitive genomeͲwide ribosome footprint profiling method to mouse motor neuronͲlike cells and primary cortical neurons expressing either wild type TDPͲ43 or patient mutant protein. In each case we performed deep sequencing of ribosome “footprints” and corresponding mRNA samples from at least two independent replicates. Bioinformatic analysis provided a short list of candidate mRNAs thatappeartobedifferentiallyregulatedatthetranslationallevelincellsexpressingthemutated protein.WeanalyzedasubsetofthesemRNAsviaanindependentapproach,classicalpolyribosome profiling,andwereabletovalidateselectiveeffectsofmutantTDPͲ43ontheirtranslation.Strikingly, some of these mRNAs encode proteins that have already been implicated in neurodegenerative diseases. We also detected a subset of overlapping mRNAs in data sets from both models, highlighting a potential “common signature” for altered translation in the two different cell populations affected by disease (cortical and spinal motor neurons). Taken together, our data indicate that mutant alleles of TDPͲ43 from ALS patients can selectively modulate translation of specificmRNAsinmammaliancells.
46 O321
RibosomequalitycontrolofprematurepolyadenylatedmRNAsbya uniqueE3ubiquitinligaseandRNAͲbindingprotein
SeyedMehdiJafarnejad,AitorGarzia,CindyMeyer,ClémentChapat,TasosGogakos,PavelMorozov, MehdiAmiri,MaayanShapiro,HenrikMolina,ThomasTuschl,andNahumSonenberg
McGillUniversityMontrealCanada
Crypticpolyadenylationwithincodingsequencesorincompletelyremovedintronsproducesaberrant transcripts that lack inͲframe stop codons and are subject to ribosomeͲassociated quality control (RQC). Premature polyadenylated mRNAs trigger ribosome stalling and activation of the RQC pathway, leading to degradation of the aberrant mRNA and nascent polypeptide, ribosome disassemblyandrecyclingofitssubunits.Whileribosomalsubunitdissociationandnascentpeptide degradationarewellͲunderstood,themolecularsensorsofaberrantmRNAsandtheirmechanismof action, especially in mammalian cells, remain largely unknown. We show that the unique RNAͲ bindingproteinandE3ubiquitinligase,ZincFingerProtein598(ZNF598)mayfunctionasasensorfor detectionoftheprematurepolyadenylatedmRNAs.PARͲCLIPassayrevealedthatZNF598crosslinks totRNAs,mRNAs,andrRNAs,therebyplacingtheproteinontranslatingribosomes.Crosslinkedreads originatingfromAAAͲdecodingtRNALys(UUU)were10Ͳfoldenrichedoveritscellularabundance,and polyͲlysineencodedbypoly(AAA),butnotpoly(AAG),inducedRQCinaZNF598Ͳdependentmanner. SensingofprematurepolyAtailsbyZNF598triggeredubiquitinationoftheribosomalproteinsRPS3A, RPS10,andRPS20requiringtheE2ubiquitinligaseUBE2D3andtherebyinitiatingRQC.Considering thathumancodingsequencesaredevoidof>4repeatedAAAcodons,sensingofprematurepolyAby a specialized RNAͲbinding protein is a novel nucleicͲacidͲbased surveillance mechanism for contributingtoRQC.
47 O331
MadaboutU:regulatingthelet7preͲmiRNA
ChrisFaehnle1,JackWalleshauser1,2andLeemorJoshuaͲTor1,2
1W.M.KeckStructuralBiologyLaboratory,HowardHughesMedicalInstitute,2WatsonSchoolofBiologicalSciences,Cold SpringHarborLaboratory,1BungtownRoad,ColdSpringHarbor,NY11724,USA
ThepluripotencyfactorLin28inhibitsthebiogenesisoftheletͲ7familyofmammalianmicroRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Alternatively, Lin28 overexpression is correlated with the onset of numerous cancers, while letͲ7, a tumor suppressor, silences several human oncogenes. Lin28 binds to precursor letͲ7 (preͲletͲ7) hairpins, triggering the 3’ oligouridylationactivityofTUT4/7.TheoligoUtailaddedtopreͲletͲ7servesasadecaysignal,asitis rapidly degraded by the exonuclease Dis3L2. Disruption of this pathway has been associated with pediatrickidneycancerandothercancersaswell.Insomaticcells,intheabsenceofLin28,TUT4/7 promotesletͲ7biogenesisbycatalyzingsingleuridineadditiontoasubsetofpreͲletͲ7miRNAs.Iwill discuss our studies toward the molecular basis and mechanism of Lin28 mediated recruitment of TUT4/7 to preͲletͲ7, and its effect on the uridylation activity of TUT4/7, switching it from a monouridylationactivitytoanoligouridylase,andthesubsequentdegradationofpreͲletͲ7byDis3L2.
48 O341
Frombenchtoclinicaltrial:microRNA122asanantiviraltargetfor hepatitisCvirus
PeterSarnow
DepartmentofMicrobiologyandImmunology,StanfordUniversitySchoolofMedicine,Stanford,CA94305,USA
LiverͲspecificmicroRNAmiRͲ122isknowntobindattwoadjacentsitesthatareclosetothe5'endof thehepatitisCvirus(HCV)RNAgenome,resultinginupregulationofviralRNAabundance.Extensive analyses have shown that miRͲ122 regulates the turnover of HCV RNA. Specifically, cellular 5’Ͳ3’ exonucleasesXRN1andXRN2havebeenshowntodegradetheviral,5’triphosphateͲcontainingRNA intheabsenceofmiRͲ122.ThemainfunctionofmiRͲ122intheliverinvolvestheupregulationof cholesterol biosynthesis by a mechanism that involves the downregulation of an Insig1 mRNA isoformthatencodesaninhibitorofcholesterolbiosynthesis.Thisfindingpromptedstudiesinwhich miRͲ122wassequesteredintheliverafterinjectionofmodifiedantisenseoligonucleotides(miRͲ122 antagomir).PhaseIandphaseIIclinicaltrialsshowedthatsinglesubcutaneousinjectionsresultedin loss of HCV in infected patients (Regulus Therapeutics (Carlsbad, CA). Curiously, HCV rebound in someofthepatients.Analysisofthisvirus,isolatedfromserumofpatients,revealedthattheviral RNAgenomeharboredasingleC3UchangethatispredictedtochangetheinteractionbetweenmiRͲ 122andHCVRNAfromaGͲCtoaGͲUbasepairintheir3'endpairing.TheC3Urevertantvirusdoes growinliverDroshaͲ/Ͳcells,albeitverypoorly,wheremiRͲ122isabsent.Thisdatasuggeststhatthe C3UHCVRNAcanpersistinpatientserum,whichlacksmiRͲ122,byfoldingintoanalternateRNA structureorbyformationofspecificRNAͲproteincomplexesthatstabilizetheviralRNA.Indeed,in vivoproximityligationassayshaveidentifiedcellularfactorsthatbindspecifictothe5'endofthe viralC3Ugenome.ThesefindingsarguethataspecificHCVgenotypecanpersistinanextrahepatic reservoir.
49 O351
TheroleofpiRNAsinplanarianregeneration
IanaKim1,BethDuncan2,EricRoss2,AlejandroSánchezAlvarado2andClausͲD.Kuhn1
1BIOmacResearchCenter,UniversityofBayreuth,Bayreuth,Germany2StowersInstituteforMedicalResearch,KansasCity, MO,USA
Planarianflatwormspossessfantasticregenerativeabilitiesthatcanbeattributedtothepresenceof stemcellsallacrosstheirbodies.Intriguingly,PIWIproteinsandPIWIͲinteractingRNAs(piRNAs)were foundessentialforproperstemcellfunctionandhenceregenerationinplanarians.However,how thethreeplanarianPIWIhomologs(SMEDWIͲ1/2/3)contributetoregenerationisnotunderstood.
Thus, employing biochemical techniques paired with nextͲgeneration sequencing, we set out to unraveltheroleofplanarianpiRNAsduringregeneration.WeshowthatSMEDWIͲ1andSMEDWIͲ3 are cytoplasmic proteins and that they direct the postͲtranscriptional degradation of active transposons. In contrast, SMEDWIͲ2 is mostly nuclear and regulates transposons on the transcriptional level. Further analysis revealed that only piRNAs bound by SMEDWIͲ1 andͲ3 are derived from mRNAs in planarians. This raises the intriguing possibility that piRNAs in planarians directlyregulateproteinͲcodingmRNAsthatareimportantforregeneration.
UsingHITSͲCLIPonplanarianPIWIproteinswearecurrentlyexploringtheroleofthepiRNApathway inthepostͲtranscriptionalregulationofplanarianmRNAs.Inaddition,weareemployingChIPͲseqon heterochromatinmarkstouncoverthemechanismoftranscriptionalregulationbySMEDWIͲ2.Our resultswillhelptoestablishhowsmallRNAscontributetothepluripotencyofstemcells.Inaddition, understanding the role of piRNAs during planarian regeneration will facilitate attempts to grow humanorgansfrompluripotentstemcells.
50 O361
AnickelͲregulatedsmallRNArepressesexpressionofmultiple majorvirulencefactorsinHelicobacterpylori
SaraK.Eisenbart1,MonaAlzheimer1,#,SandyR.Pernitzsch1,#,StephanieStahl1,RainerHaas2,Cynthia M.Sharma1,*
1ResearchCenterofInfectiousDiseases(ZINF),UniversityofWürzburg,97080Würzburg,Germany2MaxvonPettenkoferͲ InstituteforHygieneandMedicalMicrobiology,LudwigͲMaximiliansͲUniversity,80336Munich,Germany#Equally contributingauthors*CorrespondingAuthor:cynthia.sharma@uniͲwuerzburg.de
TheGramͲnegativeEpsilonproteobacteriumHelicobacterpyloricolonizesthestomachsofabout50% oftheworld’spopulationandtherebyleadstogastritis,ulcers,andgastriccancer.Severalvirulence factorsincludingsecretedeffectors,exotoxinsandoutermembraneproteinshavebeendescribedin H.pylorithatallowthisspiralͲshapedbacteriumtosurviveintheacidicenvironmentofthehuman stomachandtointeractwithhostcells.Formanyofthesefactorstheircontributiontopathogenicity hasbeenwellstudied.However,howtheirexpressionisregulatedislessunderstood.Whereasmany genesimportantforpathogenicityareregulatedatthetranscriptionallevel,e.g.inresponsetolow pH or the availability of metal ions, almost nothing is known about their regulation at the postͲ transcriptionallevel.
BasedonadifferentialRNAͲseqapproachwehadidentifiedanunexpectednumberofmorethan60 smallRNA(sRNA)candidatesinH.pyloristrain26695(1).Whiletheirfunctionsandtargetsremained largely unknown, we now report the characterization of an abundant and conserved sRNA, NikS (NikRͲdependent sRNA), and show that it directly represses expression of several major virulence factorsatthepostͲtranscriptionallevel.Usinginvitroandinvivoexperiments,wedemonstratethat NikSsRNAfoldsintoastemͲloopstructurewithanextendedloopregionanddirectlybindstothe mRNAsofmultiplevirulencegeneswithdifferentsingleͲstrandedregionswithinitsextendedloop. Moreover,wedemonstratethatexpressionofNikSitselfistranscriptionallyrepressedinresponseto nickelstressthroughthetranscriptionalregulatorNikR.Inturn,themajorvirulencefactorsarepostͲ transcriptionallyrepressedthroughNikSsRNAdependentonnickelavailability.Invitrocellculture infection assays revealed that deletion and overexpression of nikS impacts host cell interaction, indicating that fineͲtuning and coordinated regulation of virulence gene expression by NikS is important for pathogenesis. Overall, NikS represents the first potential virulence regulating sRNA fromH.pylori.
Reference:
1.Sharma,C.M.,Hoffmann,S.,Darfeuille,F.,Reignier,J.,Findeiss,S.,Sittka,A.,Chabas,S.,Reiche,K., Hackermuller, J., Reinhardt, R. et al. The primary transcriptome of the major human pathogen Helicobacterpylori.Nature,2010,464,250Ͳ255.
51 O371
SelfͲtargetingandgenerepressionwiththeCRISPRͲCassystemof Haloferaxvolcanii
ArisͲEddaStachler1,EllaShtifmanSegal2,ThorstenAllers3,LisaͲKatharinaMaier1,UriGophna2and AnitaMarchfelder1
1BiologyII,UniversityofUlm,Germany;2DepartmentofMolecularMicrobiologyandBiotechnology,GeorgeS.Wise FacultyofLifeSciences,TelAvivUniversity,Israel;3SchoolofLifeSciences,UniversityofNottingham,UK
CRISPRͲCassystemsareprevalentamongstarchaeaandwellknownforprovidingheritableimmunity byincorporationofforeignDNA.ThesequenceinformationacquiredistranscribedtocrRNAsand incorporated into protein complexes, which can thus reͲrecognize the foreign DNA and trigger degradation.OnthedownͲside,liketheeukaryoticimmunesystem,CRISPRͲCassystemsareproneto autoͲimmunityifselfͲtargetingspacersareacquiredthatleadtocleavageofhostDNA.
Here,weinvestigatedwhetherarchaeacantolerateautoͲimmunityandelucidatedtheeffectsofselfͲ targetingoncellularfitnessinthehalophilicarchaeonHaloferaxvolcanii.Toinvestigatethedamage causedbyCRISPRͲCasautoͲimmunityinHaloferax,wereͲprogrammedtheendogenousCRISPRͲCas systemtotargetanonͲessentialgeneinvolvedincarotenoidbiosynthesis.Uponlossormutationof theORFforphytoenedehydrogenase(crtL)thephenotypeofH.volcaniicolonieschangestowhite ratherthanred(wildtype).Thusdamagestothisgenecanbeeasilyobserved.Wedesignedthree crRNAstotargetthechromosomalcrtLgeneandinvestigatedtheeffectoftargetingindetail.
Wecouldshowthatincontrasttotheobservationsinbacteria,selfͲtargetingisneitherlethalnor even strongly deleterious in Haloferax. Furthermore, our results imply that either efficient repair mechanisms,thehighlypolyploidgenome,orthecombinationofbothpreventlethalitybyCRISPRͲ CasselfͲtargeting.
52 O381
METTL16isaN6Ͳmethyladenosine(m6A)methyltransferasethat targetspreͲmRNAs,lncRNAsandtheU6snRNA
AhmedS.Warda,JensKretschmer,PhilippHackert,ClaudiaHöbartner,KatherineE.Sloan,MarkusT. Bohnsack
DepartmentofMolecularBiology,UniversityMedicalCenterGöttingen,Göttingen,Germany
N6Ͳmethyladenosine(m6A)isahighlydynamicRNAmodificationthathasrecentlyemergedasakey regulatorofgeneexpression.Whilemanym6AmodificationsareinstalledbytheMETTL3ͲMETTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A “writer” protein METTL16 binds to the U6 snRNA, as well as numerouslncRNAsandpreͲmRNAs.WedemonstratethatMETTL16isresponsibleforN6Ͳmethylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16Ͳinteraction partners. Interestingly, A43 lies withinanessentialACAGAGAsequenceofU6thatbasepairswith5’splicesitesofpreͲmRNAsduring thecatalyticstepofthesplicingreaction.ThissuggeststhatMETTL16Ͳmediatedmodificationofthis site fineͲtunes U6 snRNAͲpreͲmRNA interactions, thereby regulating 5’ splice site recognition or spliceosomeassemblyonitssubstratepreͲmRNAs.TheidentificationofMETTL16asanactivem6A methyltransferaseinhumancellsexpandsourunderstandingofthemechanismsbywhichthem6A landscapeisinstalledoncellularRNAs.
53 O391
lncRNA&RibonucleoproteinsinCancer
SvenDiederichs
DivisionofCancerResearch,DepartmentofThoracicSurgery,UniversityHospitalFreiburg,79106Freiburg,Germany; GermanCancerResearchCenter(DKFZ),DivisionofRNABiology&Cancer,69120Heidelberg,Germany
Long nonͲcoding RNAs (lncRNAs) and their protein interaction partners (RBPs) can play important rolesinmalignantdiseases.ThelncRNAMALAT1isamarkerandessentialfactorinthemetastasisof lung cancer. It controls cell migration epigenetically and could serve as a therapeutic target. Numerous additional lncRNAs execute important functions in cancer initiation and progression. Many lncRNAs as well as all other classes often interact with proteins altering their confirmation, localization, function or activity. Hence, the elucidation of the composition and functional consequencesofribonucleoproteincomplexesinhealthanddiseaseisofgreatimportance.
54 O401
MechanisticdissectionofUsnRNPbiogenesisanditsroleindisease
ArchanaPrusty,RajiMeduri,AravindanViswanathan,JyotishmanVeepaschit,ClemensGrimmand UtzFischer
DepartmentofBiochemistryBiocentreUniversityofWuerzburgGermany
Theformationofmacromolecularcomplexeswithinthecrowdedenvironmentofcellsoftenrequires aidfromassemblychaperones.AnelaboratesystemofassemblyfactorsunitedinPRMT5ͲandSMNͲ complexesmediatesformationofthecommoncorestructureofthepreͲmRNAprocessingUsnRNPs composed of seven Sm/Lsm proteins bound to snRNA. Core formation is initiated by the PRMT5Ͳ complex subunit pICln, which preͲarranges Sm/Lsm proteins into spatial positions occupied in the assembled snRNP. The SMNͲcomplex then catalyzes snRNP formation by accepting these Sm/Lsm proteinsanduniting themwithsnRNA.Wehaveinvestigated hownewlysynthesizedproteinsare channeledintotheassemblymachinerytoevadeaggregationand/ormisͲassembly.Weshowthat Sm/Lsmproteinsinitiallyremainboundtotheribosomenearthepolypeptideexittunnel.Release fromtheribosomeisdependentonpICln,whichworksinremarkableanalogytofoldingchaperones. The PRMT5Ͳcomplex then ensures the formation of cognate Sm/Lsm heterooligomers and their guidanceintothelateassemblystagemediatedbytheSMNͲcomplex.SMN,onekeycomponentof the SMNͲcomplexisreducedinthe neuromusculardisorderSpinal MuscularAtrophy (SMA). Iwill discusshowreducedactivityofthisproteinmayleadtothisdevastatingdisorder.
55 O411
GenomeͲwidestudyoftranscriptioncomplexityandribosome dynamics
VicentPelechano
SciLifeLab,DepartmentofMicrobiology,TumorandCellBiology.KarolinskaInstitutet,PͲBox1031.17121Solna,Sweden
One of the biggest challenges in biology is to understand how apparently identical cells respond differently to the same stimulus. During the last decade, thanks to the development of genomics tools,researchhasuncoveredextensivevariabilityintheRNAmoleculespresentwithinthecells.
In the past we have developed a diversity of novel genomeͲwide approaches to study eukaryotic geneexpressionusingbothbuddingyeastandmammaliancells.Bysimultaneouslysequencingboth the5’and3’endsofeachRNAmolecule(TIFͲSeq),weshowedthatthecomplexityofoverlapping transcriptisoformshadbeengreatlyunderestimatedeveninageneticallyhomogeneouspopulation of cells. More recently, we have shown how the existence of widespread coͲtranslational mRNA degradation allows studying ribosome dynamics by sequencing mRNA degradation intermediates (5PͲSeq).
I will discuss our current efforts to study transcriptome complexity and its implications for gene expressionregulation.
56 O421
GradͲseqdiscoversthethirddomainofsmallRNAͲmediatedgene regulationinbacteria
JörgVogel
HelmholtzInstituteforRNAͲbasedInfectionResearch;InstituteforMolecularInfectionBiology,UniversityofWürzburg, JosefͲSchneiderͲStraße2/D15,DͲ97080Würzburg,Germany
RNAͲseqcanrapidlyprofiletheexpressionoftheoreticallyallRNAmoleculesinanygivenorganism buttheprimarysequenceofthesetranscriptsisapoorpredictorofcellularfunction.Thishasbeen particularlyevidentfortheregulatorysmallRNAsofbacteriawhichdramaticallyvaryinlengthand sequencewithinandbetweenorganisms.
Wehaveestablishedanewmethod(gradientprofilingbysequencing;GradͲseq)topartitionthefull ensembleofcellularRNAsbasedontheirbiochemicalbehavior.Ourapproachenabledustodrawan RNA landscape of the model pathogen Salmonella Typhimurium, identifying clusters functionally related noncoding RNAs irrespective of their primary sequence. The map revealed a previously unnoticed class of transcripts that commonly interact with the osmoregulatory protein ProQ in Salmonellaenterica.WeshowthatProQisaconservedabundantglobalRNAͲbindingproteinwitha widerangeoftargets,includinganewclassofProQͲassociatedsmallRNAsthatarehighlystructured, andmRNAsfrommanycellularpathways.OurfunctionalcharacterizationofthesesmallRNAsthat suggests that they constitute a previously unrecognized third domain of RNAͲmediated control in bacteriawhichrivalsthescopeofthewellͲestablishedregulonsofthesmallRNAͲbindingproteins, HfqandCsrA.
By its ability to describe a functional RNA landscape based on expressed cellular transcripts irrespectiveoftheirprimarysequence,ourgenericgradientprofilingapproachpromisestoaidthe discoveryofmajorfunctionalRNAclassesandRNAͲbindingproteinsinmanyorganisms.
SmirnovA,FörstnerKU,HolmqvistE,OttoA,GünsterR,BecherD,ReinhardtR,VogelJ(2016);GradͲseqguides thediscoveryofProQasamajorsmallRNAbindingprotein;PNAS113(41):11591Ͳ6 SmirnovA,WangC,DrewryLL,VogelJ(2017);MolecularmechanismofmRNArepressionintransbyaProQͲ dependentsmallRNA;EMBOJournal36(8):1029Ͳ1045 GonzalezGM,HardwickSW,MaslenSL,SkehelJM,HolmqvistE,VogelJ,BatemanA,LuisiBF,BroadhurstRW (2017);StructureoftheEscherichiacoliProQRNAbindingprotein;RNA23(5):696Ͳ711 ChaoY,LiL,GirodatD,FörstnerKU,SaidN,CorcoranC,_migaM,PapenfortK,ReinhardtR,WiedenHJ,LuisiBF, VogelJ(2017);InvivocleavagemapilluminatesthecentralroleofRNaseEincodingandnoncodingRNA pathways;MolecularCell65(1):39Ͳ51 WestermannAJ,FörstnerKU,AmmanF,BarquistL,ChaoY,SchulteLN,MüllerL,ReinhardtR,StadlerPF,Vogel J(2016)DualRNAͲsequnveilsnoncodingRNAfunctionsinhostͲpathogeninteractions.Nature529:496Ͳ 501
57 O431
FunctionalcharacterizationofhypoxiaͲregulatedlncRNAsin endothelialcells
NicolasJaé1,PhilippNeumann1,YoussefFouani1,DavidJohn1,IlkaWittig2,OliverRossbach3,Albrecht Bindereif3,MarcusKrüger4,andStefanieDimmeler1
1InstituteforCardiovascularRegeneration,UniversityofFrankfurt,Germany2FunctionalProteomicsCoreUnit,Facultyof Medicine,UniversityofFrankfurt,Germany3InstituteofBiochemistry,JustusLiebigUniversityofGiessen,Germany4CECAD ResearchCenter,UniversityofCologne,Germany
Withtheriseofnextgenerationsequencingtechnologiesitbecameevidentthatthehumangenomeisalmost pervasivelytranscribed,however,onlyaminorpartofthegenomeactuallyaccountsforproteincodingexons. Consequently, the majorityof all transcripts is nonͲcoding. Accordingto their size,nonͲcoding RNAs canbe divided into small (<200nt) and long nonͲcoding RNAs (lncRNAs; >200nt). Whereas the role of distinct nonͲ codingRNAs,e.g.microRNAs,iswellunderstood,littleisknownaboutthedetailedmolecularmechanismsof lncRNAs. Recent studies suggest a broad functional spectrum including sponging of miRNAs, modulation of mRNAsplicingandstability,orrecruitmentofchromatinmodifyingenzymes.Regardingendothelialcells(ECs), the response to hypoxia and the regulation of angiogenic activity are key events in the context of several diseases,however,theinvolvementofregulatorylncRNAsandtheirmolecularfunctionisnotwellunderstood.
To analyze the influence of hypoxia on lncRNA expression, we performed RNA deep sequencing in hypoxic
(24h;0.2%O2)andnormoxicECsandidentified71and78lncRNAstobesignificantlyupͲordownͲregulated, respectively.Fromthesetranscripts,wechosetworobustlyupͲregulatedanduncharacterizedlncRNAsͲnamed GATA6ͲAS and NTRASͲ for a detailed investigation. First functional assays revealed that silencing of both candidate lncRNAs using LNA GapmeRs significantly alters the angiogenic activity of ECs, underlining their involvementinendothelialbiology.Moreover,cellularfractionationshowedapredominantnuclearlocalization ofGATA6ͲASandNTRASandanalysisoftherespectivegenelociruledoutcisͲregulatorymechanisms.Thus,in ordertogetdetailedinsightsintothemolecularfunctionsofthesetranscripts,wedeployedantisenseaffinity purificationfollowedbymassspectrometrytoenrichforandanalyzetherespectiveendogenousRNAͲproteinͲ complexes. 1) For GATA6ͲAS, this uncovered the chromatin modifying enzyme LOXL2, which catalyzes the oxidativedeaminationoftrimethylatedH3K4residues,asaboundproteinfactor.Strikingly,geneexpression profilingidentified~71%ofGATA6ͲASͲregulatedgenestobeinverselyregulatedbyLOXL2silencing,including numerous angiogenesisͲrelated genes, like PTGS2 and POSTN.Moreover, silencingof GATA6ͲAS significantly decreasedH4K3me3ChIPefficienciesonPTGS2andPOSTNpromotersandinparallelreducedglobalH3K4me3 levels. Taken together, these results strongly argue for GATA6ͲAS acting as negative regulator of LOXL2 function.2)ForNTRAS,affinitypurificationandmassspectrometryidentifiedthesplicingregulatorhnRNPLto be strongly associated with the lncRNA, which is in line with a bona fide hnRNPL binding motif within the NTRAS sequence. Using RNA immunoprecipitation and RTͲqPCR, we were able to confirm the observed interaction.Basedonthesefindings,weassayedforafunctionalinvolvementofNTRASinalternativesplicing regulationbymicroarrays.Strikingly,silencingofNTRASchangedthesplicingpatternofmorethan400genes, among those the wellͲknown hnRNPL target TJP1. In detail, NTRAS was found to activate TJP1 exon 20 inclusion,whichisrepressedbyhnRNPL.Onafunctionallevel,TJP1isrequiredfortightjunctionassembly,and wecurrentlyinvestigatetheroleofthe“ɲͲmotif”encodedbyexon20inthecontextofangiogenicintegrityand permeability. In conclusion, our data demonstrates that the hypoxiaͲregulated lncRNA NTRAS controls endothelialcellfunctionsputativelyviamodulatinghnRNPLͲdependentalternativesplicing. 58 O441
HighͲthroughputscreeningforsplicingregulatoryelements
SimonBraun1,SamarthThontaSetty2,MihaelaEnculescu1,MarielaCortesLopez1,MarkusSeiler2, AnkeBusch1,StefanieEbersberger1,StefanLegewie1,JulianKönig1,andKathiZarnack2
1InstituteofMolecularBiologygGmbH(IMB),Ackermannweg4,55128Mainz,Germany2BuchmannInstituteforMolecular LifeSciences(BMLS),MaxͲvonͲLaueͲStr.15,60438Frankfurt,Germany
Alternativesplicingconstitutesamajorstepineukaryoticgeneexpressionandrequirestightcontrol of transͲacting factors that recognise cisͲregulatory elements in the RNA sequence. However, the majority of cisͲregulatory elements are poorly defined, and the impact of intronic and exonic sequencevariantsonthesplicingoutcomeremainselusive.
Here,weestablishahighͲthroughputscreentocomprehensivelyidentifyallcisͲregulatoryelements that determine a particular splicing decision. As a prototype example, we screen a minigene harbouring the cancerͲrelevant alternative exon of the RON receptor kinase gene. A library of thousandsofrandomlymutagenisedminigenevariantsistransfectedasapoolintohumanHEK293T cells, and the splicing products are subsequently analysed via RNA sequencing. In parallel, DNA sequencing enables reliable point mutation discovery which are assigned to the corresponding splicingproductsviaauniquebarcodesequence.InadditiontoknowncisͲregulatoryelements,we identifynumerouspreviouslyunknownregulatorysites.Thisapproachprovesparticularlypowerful whencombinedwithknockdownexperimentswhichallowstoconnecttransͲactingfactorswiththeir correspondingcisͲregulatoryelements.Insummary,thisnovelscreeningapproachintroducesatool to study the relationship of cisͲregulatory elements, sequence variants and their impact on the splicing outcome, offering new insights into alternative splicing regulation and the implication of mutationsinhumandisease.
59 O451
RegulationofgeneexpressionbyproteinsboundtoLINEͲderived RNAelements
JanAttig1,2,FedericoAgostini1,NejcHaberman1,2,IgorMozos1,2,AartiSingh2,ChrisSmith3,Nicolas Luscombe1andJernejUle1,2
1TheFrancisCrickInstitute,1MidlandRoad,London,NW11AT,UK;2UCLInstituteofNeurology,QueenSquare,London, WC1N3BG,UK;3DepartmentofBiochemistry,UniversityofCambridge,TennisCourtRoad,Cambridge,CB21QW,UK
Thehumangenomecontainsover1.5milliondegenerateLINEͲderivedsequences,manyofwhichare transcribedaspartoflongergenes.WeanalysediCLIP,eCLIPandRNAseqdatatofind thatmany RNAͲbinding proteins (RBPs) bind LINEͲderived RNA sequences, and thereby regulate gene expression.MATR3andPTBP1areparticularlyenriched,andbothproteinsrepresssplicingaswellas 3’endprocessingwithinornearbytheboundLINEs,whichaffectsbothannotatedandcrypticexons.
MATR3isanRNAͲandDNAͲbindingproteinrecentlyimplicatedinmotorneurondisease,whichhas beenreportedtoplaymultiplepresumablydistinctfunctionsinenhanceractivationandregulationof alternatively splicing. We show that LINEs are associated with both functions. MATR3 can repress splicesitesinasfaras2kbdistancetoLINEs,whichwedemonstratebymutagenesisexperiments. Moreover,MATR3andPTBP1bindnonͲcodingRNAscontainingLINEsequencesandarerequiredfor theirexpression.
We also analysed data from PTBP2Ͳ/Ͳ mouse brain, which confirms that PTBP proteins regulate tissueͲspecificsplicingofLINEͲderivedexons,whicharefrequentlyspeciesͲspecific.Weconcludethat RBPs are employed to proofread RNA processing at thousands of LINEs. We propose that the invasionofvertebrategenomebyLINEandotherretrotransposonshasledtotheircoͲevolutionwith RNAͲbindingproteins,whichiscrucialfortheevolutionaryprocessesthatshapethespeciesͲspecific geneexpressionpatterns.
60 O461 iCLIPdeterminesthetargetrepertoireandbindinglandscapeofthe clockͲregulatedRNAͲbindingproteinAtGRP7
KatjaMeyer,TinoKöster,ChristineNolte,MartinLewinski,ClausWeinholdt,IvoGrosse,Dorothee Staiger
RNABiologyandMolecularPhysiolgyFacultyofBiologyBielefeldUnivesityBielefeld,Germany
A key function for RNAͲbinding proteins in orchestrating plant development and environmental responses is well established. However, the lack of a genomeͲwide view on their in vivo binding targetsandbindinglandscapesrepresentsagapinunderstandingthemodeofactionofplantRNAͲ bindingproteins.Here,weadaptindividualnucleotideresolutioncrosslinkingimmunoprecipitation (iCLIP) for genomeͲwide determining the binding repertoire of the circadian clockͲregulated ArabidopsisthalianaglycineͲrichRNAͲbindingproteinAtGRP7.iCLIPidentified858transcriptswith significantlyenrichedcrosslinksitesinplantsexpressingAtGRP7ͲGFPandabsentinplantsexpressing an RNAͲbindingͲdead AtGRP7 variant or GFP alone. To independently validate the targets, we performed RNA immunoprecipitation (RIP)Ͳsequencing of AtGRP7ͲGFP plants subjected to formaldehydefixation.452oftheiCLIPtargetswerealsoidentifiedbyRIPͲseq,thusrepresentinga set of highͲconfidence binders. AtGRP7 can bind to all transcript regions with a preference for 3´untranslatedregions.Inthevicinityofcrosslinksites,UCͲrichmotifswereoverrepresented.CrossͲ referencing the targets against transcriptome changes in AtGRP7 lossͲofͲfunction mutants or AtGRP7ͲoverexpressingplantsrevealedapredominantlynegativeeffectofAtGRP7onitstargets.In particular,elevatedAtGRP7levelsleadtodampingofcircadianoscillationsoftranscriptsincluding DORMANCY/AUXINASSOCIATEDFAMILYPROTEIN2andCCRͲLIKE.Furthermore,severaltargetsshow changesinalternativesplicingorpolyadenylationinresponsetoalteredAtGRP7levels.Establishing iCLIPforplantstoidentifytargettranscriptsoftheRNAͲbindingproteinAtGRP7pavesthewayto investigatethedynamicsofposttranscriptionalnetworksinresponsetoexogenousandendogenous cues.
61 O471
FunctionalcharacterizationoftheCRISPRͲCas9systemofthe foodborneͲpathogenCampylobacterjejuni
GauravDugar1,RyanLeenay2,SaraEisenbart1,ThorstenBischler1,BelindaAul1,ChaseBeisel2,Cynthia M.Sharma1
1InstituteofMolecularInfectionBiology,MolecularInfectionBiologyII,UniversityofWürzburg,Germany2NorthCarolina StateUniversity,DepartmentofChemicalandBiomolecularEngineering,Raleigh,NC27695,UnitedStates
BasedonacomparativeRNAͲseqanalysisofmultiplestrainsofthefoodͲbornebacterialpathogen Campylobacterjejuni,wehavedetectedstrainͲspecifictranscriptionaloutputandsmallRNA(sRNA) repertoires(1).OurC.jejuniglobaltranscriptomemapsalsorevealedaminimalvariantoftheRNAͲ basedCRISPRͲCasimmunesystemofthetypeͲIIsubtype,whichreliesonthehostfactorRNaseIIIand atransͲencodedsRNA(tracrRNA)formaturationofcrRNAs.ThisminimaltypeͲIICsystemofC.jejuni employs a unique maturation pathway, where the CRISPR RNAs (crRNAs) are transcribed from individual promoters in the CRISPR repeats and thereby minimize the requirements for the maturationmachinery.TypeIICRISPRͲCassystemsandtheirCas9nucleasesnaturallyutilizecrRNAs andtracrRNAtobindandcleavecomplementaryinvadingDNAsequences.Besidesapotentialrolein phage defense, Cas9 in the bacterial pathogen Francisella novicida, was reported to use an alternativescaRNAtorepressanendogenoustranscriptandtherebyaffectsvirulence(2).
TostudyRNAͲproteincomplexesinCampylobacter,wehavesetͲupseveralapproaches,suchascoͲ immunoprecipitationcombinedwithRNAͲseq(RIPͲseq)toidentifythedirectRNAsubstratesofan RNAbindingprotein(3).OurRIPͲseqanalysisofC.jejuniCas9revealedthatnotonlythecrRNAsand tracrRNA are abundantly bound by Cas9, but also uncovered several endogenous mRNAs that coͲ purifywithCas9.Usinginvivoandinvitroapproaches,weshowthatC.jejuniCas9mediatescrRNAͲ andtracrRNAͲdependentmRNAcleavageintheabsenceofascaRNA.Moreover,weshowthatRNA cleavagebyCas9canbereprogrammedinvitrobyasingleguideRNA.Thesefindingssuggestthat the C. jejuni Cas9 is a promiscuous nucleic acid nuclease and could serve in applications of programmableRNAbindingorcleavage.
1.Dugar,G.,Herbig,A.,Förstner,K.U.,Heidrich,N.,Reinhardt,R.,Nieselt,K.andSharma,C.M.(2013)HighͲ Resolution Transcriptome Maps Reveal StrainͲSpecific Regulatory Features of Multiple Campylobacter jejuni Isolates.PLoSGenet,9,e1003495. 2.Sampson,T.R.,Saroj,S.D.,Llewellyn,A.C.,Tzeng,Y.L.andWeiss,D.S.(2013)ACRISPR/Cassystemmediates bacterialinnateimmuneevasionandvirulence.Nature,497,254Ͳ257. 3.Dugar,G.,Svensson,S.L.,Bischler,T.,Waldchen,S.,Reinhardt,R.,Sauer,M.andSharma,C.M.(2016)The CsrAͲFliW network controls polar localization of the dualͲfunction flagellin mRNA in Campylobacter jejuni. Naturecommunications,7,11667.
62
POSTER ABSTRACTS (A-Z)
63 P1 1
FunctionalcharacterizationofHMGN5,aspecificRNAor nucleosomebindingproteinopeninghigherorderstructuresof chromatin
IngridAraya,JuliaWimmer,UweSchwartz,IvoCarrasco,GernotLängst
DepartmentofBiochemistryIII,UniversityofRegensburg,Regensburg,Germany.
Packaging of DNA into higher order structure of chromatin has profound implications in the regulation of nuclear processes, like transcription, replication, recombination or DNA repair. The reversible opening and closing of chromatin domains is mediated by architectural proteins of the “highmobilitygroupnucleosomalbindingdomain”(HMGN)family,whichareknowntoreorganize chromatin structure and to regulate gene expression. However, the mechanisms underlying this processarestillunclear.HereweusethehumanHMGN5asamodeltostudytheopeningofhigher orderstructureofchromatinusinggenomeͲwideanalysismethods.WerevealthatHMGN5mainly associateswithactivepromotersandregulatoryregionslikeCpGislands,maintainingtheiraccessible state. HMGN5 binding overlaps with PolII and CTCF binding sites and localizes to DNase I hypersensitivesites(DHSs).Activelyregulatedtargetgenesbelongtothegroupofgenesinvolvedin RNAmetabolicprocesses.Surprisingly,weidentifiedaspecificRNAbindingdomainoverlappingwith the nucleosomal binding domain of HMGN5. Our data suggest a bimodal state or a switch that enablesHMGN5bindingeitherchromatinorRNAsincetheHMGN5ͲboundRNAshavenofunctional relationshipwiththechromatinfunctionofHMGN5.WesuggestthatHMGN5hasadualfunction eitherregulatingchromatinstructureorRNAmetabolism.
64 P2 1
Assembly,localizationandtranslationalcontrolofWUSͲ/WOXͲ containingmRNPsinA.thaliana
AnastasiiaBazhenova,AndreaBleckmann,ThomasDresselhaus
DepartmentofPlantBiochemistryandCellBiology,UniversityofRegensburg,Universitaetsstr.31,93053Regensburg, Germany
Plantreproduction,embryogenesisanddevelopmentareregulatedbyacomplexsignalingnetwork. WUSCHELͲrelatedhomeobox(WOX)superfamilymembersarekeyregulatorsofsuchdevelopmental processes.ThisgenefamilyisfoundedbyWUSCHEL(WUS)andconsistsof15members,whichare essentialforstemcellmaintenanceinshootapicalmeristem.OthermemberssuchasWOX2,WOX8 and WOX9 are essential for proͲembryo development. And WOX5 is essential for root meristem formation.
TemporalandspatialcontrolofmRNAtranslationisaprecisemechanismtocontrolthepresenceof gene products. This mechanism is found in all kingdoms of life and appears to be essential for asymmetric cell division, cell fate determination and many other developmental processes. In contrast to animals and fungi, to date little is known about mRNPs localization and translational controlinplants.UsingfluorescentwholeͲmountinsituhybridization(FͲWISH)wecouldshowthat WOX2/5/8 and WUS mRNAs accumulate in globular structures. These globular structures indicate specific mRNPs formation, which might be essential for localization and translational control. I currently try to identify response elements for mRNP association using deletion and synthetic derivatives in FͲWISH experiments. To characterize mRNP composition I perform RNA pull down experimentsusingspecificRNAͲtagorlabeledantisenseprobes.
65 P3 1
AuxiliaryFactorsoftheRNAhelicasePrp43inribosomebiogenesis
PriyaBhutada,StefanUnterweger,LisaKofler,ValentinMittererandBrigittePertschy
UniversityofGraz,InstituteforMolecularBiosciences
Ribosomebiogenesisisacomplex,tightlyregulatedandhighlyenergydemandingprocess.Itneeds participation of RNAͲpolymerases, 82 ribosomal proteins, 66 (small nucleolar RNA) snoRNAs and morethan200nonribosomalproteinswhichcarryoutmolecularrearrangements,modificationsand rRNA processing steps within ribosomal precursors for maturation and functional assembly of ribosome.Theseincludeassemblyfactors,endo–andexonucleases,ATPases,transportfactors,RNA helicases. AmongtheRNAhelicases,Prp43DEAH/RHAhelicaseplaysmultiplerolesatdifferentstagesinboth 40S and 60S ribosomal subunit biogenesis and also participates in mRNA splicing. As Prp43 is responsibleforessentialprocesses,itnecessitatestheengagementofmany coordinating auxiliary factorsfortheaccomplishmentofitsfunctions.ThesecoͲfactorsofPrp43includeagroupofproteins sharingaglycineͲrichdomain,termedGͲpatchdomain.GͲpatchproteinsNtr1,Pxr1,Pfa1andCmg1 areknowntostimulateATPaseandRNAhelicaseactivityofPrp43.InmRNAsplicingGͲpatchprotein Ntr1isresponsibleforPrp43helicaseactivation,whilethenonGͲpatchauxiliaryfactorNtr2functions asadaptorfortherecruitmentofPrp43tothespliceosome.Pxr1andPfa1areinvolvedinribosome biogenesis. HereweshowthatPrp43isacomponentofribosomeprecursorparticlesfrom90Stoboth60Sand 40Ssubunitbiogenesis.Pxr1andPfa1activatePrp43inribosomebiogenesis,withPxr1beingmainly involvedatthelevelof90Sparticles,andPfa1presumablyfunctioningatlaterstagesofboth40Sand 60S maturation. This finding indicates distinct roles of Pxr1 and Pfa1 during ribosome biogenesis. AlongwithPrp43coͲfactorswealsoidentifiedaGͲpatchproteinAssociatedFactornamedGaf1.Gaf1 isanovelribosomebiogenesisfactorinvolvedatthelevelof90Sparticles,aswellasatearlystages of60Smaturation.ItisinterestingtoinvestigatethefunctionalroleofGaf1inribosomebiogenesisin associationwithPrp43.
66 P4 1
mRNPsassemblyandlocalizationduringearlyembryogenesisin Arabidopsisthaliana
AndreaBleckmann,AnastasiiaBazhenova,ThomasDresselhaus
CellBiologyandPlantBiochemistry,UniversityofRegensburg,93053Regensburg,Germany
ThespatialͲtemporalcontrolofmRNAtranslationisaprecisemechanismtoregulatethepresenceof geneproducts,whichinvolvestheformationofspecificmRNA/proteincomplexessoͲcalledmRNPs. SuchmRNPsarewelldescribedinotherorganismsandappeartobeessentialforasymmetriccell division, cell fate determination and cell differentiation. Using fluorescent whole mount in situ hybridization (FͲWISH), we could show that the mRNA of severla homeobox transcription factors accumulate in globular structures and localize at specific sites of the cell during embryogenesis. These globular structures indicate the formation of specific mRNPs, which might be essential for mRNA localization and translational control. We currently focusing on the mechanism of mRNP formationanditsimpactonplantdevelopmentalprocesses.
67 P5 1
HighͲthroughputrandommutagenesisscreeningunveilsextensive regulationofRONalternativesplicingbyhnRNPH
SimonBraun1,SamarthT.Setty2,MihaelaEnculescu1,MarielaCortesLopez1,AnkeBusch1,Marion Scheibe1,ReymondSutandy1,JeanͲLouisMergny3,FalkButter1,StefanLegewie1,JulianKönig1,Kathi Zarnack2
1InstituteofMolecularBiology(IMB),55128Mainz,Germany2BuchmannInstituteforMolecularLifeSciences(BMLS), 60438Frankfurt,Germany3InstitutEuropéendeChimieetBiologie,33607Pessac,France
Alternativesplicingincreasesthecodingcapacityofmosthumangenes,andnumerousdiseasescan arise from defects in this process. Control of alternative splicing is realized by cisͲregulatory elements, such as RNA sequence and structure, which recruit transͲacting RNAͲbinding proteins. Although several of those interactions are already described in detail, we lack a comprehensive understandingoftheregulatorycodethatunderliesaspecificsplicingdecision.Here,weestablished ahighͲthroughputrandommutagenesisscreentocomprehensivelystudyallcisͲregulatoryelements that control a selected splicing decision in the protoͲoncogene RON. To this end, we generated a library of thousands of randomly mutagenized minigene variants and characterized the mutations presentinthelibraryusingnextͲgenerationsequencing.Next,wetransfectedthislibraryasapool intohumancells,andsubsequentlyquantifiedthesplicedisoformsbyRNAsequencing.Importantly, we used a barcode sequence to tag the minigene variants and thereby linked mutations to their corresponding spliced products. We identify numerous sites involved in the regulation of the alternativeexonthatspreadacrosstheentiresequencespace,includingthesurroundingintronsand neighbouringconstitutiveexons.Furthermore,wefindthatthesplicingfactorhnRNPHactsasan extensiveregulatorofRONsplicingviamultiplecisͲregulatoryelements.Overall,ourresultsunveilan unprecedentedviewonthecomplexityofsplicingregulationatasingleexon.
68 P6 1
StructuralstudiesonnovelMLLEͲPAM2Linteractionindynamic endosomemediatedmRNAtransport
SenthilKumarDevan,ThomasPohlmann,SebastianBaumannandMichaelFeldbrügge
HeinrichHeineUniversityDüsseldorf,InstituteforMicrobiology,40225Düsseldorf,Germany
LongͲdistance transport of macromolecules is essential for highly polarized cells such as fungal hyphae.IntheplantpathogenicfungusUstilagomaydisactivemRNAtransportiscarriedoutbyearly endosomesalongmicrotubules.
During the transport, mRNAs are bound to the key RNAͲbinding protein Rrm4 on the cytoplasmic surfaceoftheendosome.PolyͲAbindingprotein(Pab1)isotherpredominantlyfoundmRNAbinding protein on endosomes. Rrm4 has three RNA recognition motifs (RRM) in its NͲterminal and two Mademoiselle(MLLE)domainsinitsCͲterminal.Incontrast,PolyͲAbindingprotein(Pab1)contains onlyoneMLLEdomain.However,both Rrm4andPab1donothavealipidbindingdomainhence cannotbedirectlylinkedtoendosomes.MLLEdomainsareknowntointeractwithproteinswhich containpolyAbindingproteininteractingmotif(PAM2).Inthepast,weidentifiedanadapterprotein, with a PAM2 domain and a FYVE domain for lipid binding, it was named Upa1. Interestingly, in additiontoPAM2motifUpa1alsohasanovelPAM2Ͳlike(PAM2L)motifwhichspecificallyinteracts withMLLEdomainfromRrm4butnotPab1.
TheobjectiveofourstudyistodecipherthestructuralbasisforthespecificityofMLLEPab1ͲPAM2Upa1 andMLLERrm4–PAM2LUpa1interactionsbyusingbiochemical,biophysicalcharacterizationandXray crystallography.
69 P7 1
IdentificationofProteinsthatInteractwithEndogenouslyProduced tRNAFragments
AleksejDrino,MatthiasSchaefer
MedicalUniversityofVienna,CenterforAnatomyandCellBiology,Schwarzspanierstrasse17,1090Vienna,Austria
Transfer RNAs (tRNAs) have been widely appreciated as fundamental components of the translationalmachinery,actingasadaptormoleculesthatareessentialformRNAdecodinginto proteins.Inaddition,tRNAmoleculescanbeendonucleolyticallycleavedunderawidevarietyof stressconditionsthroughtheactivityofendonucleases,resultingintheproductionofdistinct tRNA fragments. Although various tRNA fragments have been shown to modulate a range of biological processes, namely proliferation, stress responses and apoptosis, the full scope and mechanisticdetailsoftheirbiologicalimpactarestillpoorlyunderstood. TobetterdefinethebiologicalfunctionoftRNAfragments,wehavesetouttoidentifyhuman proteins, which interact with specific and endogenously produced tRNA fragments during the stress response. To that end, we employ a combination of biochemical approaches. Specific tRNAfragmentsarepurifiedfromhumancellsbychromatographicmethodsafterexposureto stress or after transient expression of a tRNA endonuclease. RNA affinity capture is being performedusingproteinextractsobtainedfromstressͲexposedcellsallowingtheidentification of tRNA fragmentͲinteracting proteins using mass spectrometry. This poster discusses the progress,bottlenecksandfuturedirectionsoftheproject.
70 P8 1
StructuralinsightsintoribosomalantiterminationofEscherichiacoli
BenjaminDudenhöffer,PaulRösch,StefanH.Knauer
LehrstuhlBiopolymereundForschungszentrumfürBioͲMakromoleküle,UniversitätBayreuth,Universitätsstraße30,95447 Bayreuth,Germany
Transcriptionisthefirststepingeneexpressionandconsistsofthreesteps(initiation,elongation, termination)withRNApolymerase(RNAP)beingthecoremachineryresponsibleforRNAsynthesis. Eachstepofthetranscriptioncycleishighlyregulatedbyavarietyoftranscriptionfactors.During antitermination RNAP is modified to suppress termination signals, allowing the expressing of downstreamgenes.InEscherichiacoli(E.coli)theexpressionoftheribosomal(rrn)operonsrelieson antitermination.Processiverrnantiterminationrequirestheassemblyofamulticomponentcomplex andNͲutilizationsubstance(Nus)factorsA,B,E,andGaswellasribosomalproteinS4aresuggested tobepartofit.Recently,SuhB,aninositolmonophosphatase,wasidentifiedtobealsoessentialfor robustexpressionoftherrnoperon1.Thestructuralbasisofrrnantiterminationhassofarremained elusive and in particular the roles of S4 and SuhB are not understood. Here we present first molecular insights into rrn antitermination based on a combinatorial approach of analytical size exclusion chromatography and solution state NMR spectroscopy. First experiments indicated that NusA,NusB,NusEandNusGtogetherwithS4andSuhBcanformastablecomplexonrrnGRNAand inthepresenceofelongatingRNAP.WethenshowedthatthesixdomainproteinNusAmightplaya central role in rrn antitermination as it directly interacts with S4 and SuhB. Whereas the NusA domain that binds to S4 remains to be determined, we identified SuhB to bind specifically to the acidic repeat (AR) 2 domain of NusA. We determined the SuhB interaction surface on NusAͲAR2, which comprises the CͲterminal helix of NusAͲAR2 and overlaps with the binding site for the CͲ terminaldomainoftheɲͲsubunitofRNAPaswellaswiththebindingsitefortheNͲterminaldomain ofNusG2,3.Duringelongation,andmostprobablyduringantitermination,NusAistetheredtoRNAP via its NTD. Thus we propose that NusAͲAR2 serves as general interaction platform for various transcriptionfactorsandmaythusberesponsiblefortherecruitmentofSuhBduringtheassemblyof therrnantiterminationcomplex.
[1]Singh,N.;Bubunenko,M.;Smith,C.;Abbott,D.M.;Stringer,A.M.;Shi,R.;Court,D.L.;WadeJ.T.mBio2016, 7(2),e00114Ͳ16. [2]Schweimer,K.;Prasch,S.;Sujatha,P.S.;Bubunenko,M.;Gottesman,M.E.;Rösch,P.Structure2011,19(7), 945–954. [3]Strauss,M.;Vitiello,C.;Schweimer,K.;Gottesman,M.E.;Rösch,P.;Knauer,S.H.NAR2016,44(12),S.5971– 5982
71 P9 1
UPF1inthecellnucleus
AndreaB.EberleandOliverMühlemann
UniversityofBernDepartmentofChemistryandBiochemistryFreiestrasse33012Bern,Switzerland
UPF1 binds RNA and is an ATPͲdependent RNA helicase belonging to the SF1 superfamily. Functionally,UPF1isbestknownforitsessentialroleinnonsenseͲmediatedmRNAdecay(NMD),a postͲtranscriptional quality control mechanism that recognizes transcripts with premature stop codons and efficiently degrades them. In addition to NMD, UPF1 is implicated in several nuclear processes including telomere homeostasis, S phase progression, DNA repair, and DNA replication. However,itsexactrolesintheseprocessesarepoorlyunderstoodanddespiteitsshuttlingcapacity, the majority of UPF1 is found in the cytoplasm of unperturbed cells. We aim at a better understanding of UPF1’s function(s) in the nucleus and its recruitment to RNA. RNA immunoprecipitation(RIP)showedthatUPF1associateswithadiversesetofRNAsincludingboth, codingandnonͲcodingtranscripts.Interestingly,nuclearlongnonͲcodingRNAslikeANRIL,GAS5and FIRRE appear to be extensively bound with UPF1, pointing towards a recruitment of UPF1 to transcripts already in the nucleus and independent of their coding potential. We currently try to understand why some transcripts are highly enriched in UPF1 IPs while others lack UPF1 almost completely. So far, the molecular basis for this differential association of UPF1 with RNA is not understood.FurtherinvestigationoftheproposednuclearfunctionsofUPF1wouldgreatlybenefit fromhavingaUPF1mutantwithadefectivenuclearlocalizationsignal(NLS)thatlocalizesexclusively tothecytoplasm.Tothisend,wetrytofunctionallymaptheNLSofUPF1bytestingdifferentUPF1 mutantsincellstreatedwithleptomycinB,whichblocksthenuclearexportpathway.
72 P101
ALYRNAͲbindingproteinsarerequiredfornucleoͲcytosolicmRNA transportandmodulateplantgrowthanddevelopmentin Arabidopsis
HansF.Ehrnsberger,ChristinaPfaff,MaríaFloresͲTornero,BrianB.Sørensen,ThomasSchubert, GernotLängst,StefanieSprunck,MarionGrasserandKlausD.Grasser
CellBiologyandPlantBiochemistryͲUniversityofRegensburg
The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step in the expressionofproteinͲcodinggenes,linkingtranscriptsynthesisandprocessingwithtranslation.Still onlyfewplantfactorsofthemRNAexportpathwayhavebeenfunctionallycharacterised.Flowering plant genomes encode several members of the ALY protein family that in other organisms act as mRNA export factors. Here we show that the four nuclear Arabidopsis ALY proteins (ALY1Ͳ4) are commonlydetectedinrootandleafcells,buttheyaredifferentiallyexpressedinreproductivetissue. ThesubnucleardistributionofALY1/2differsfromthatofALY3/4.ALY1bindswithhigheraffinityto ssRNArelativeto dsRNA andssDNAanditinteractspreferentiallywith5Ͳmethylcytosine modified ssRNA.ComparedtothefullͲlengthALY1itsindividualRNArecognitionmotifbindsRNAonlyweakly. The ALY proteins interact with the RNA helicase UAP56, suggesting a link to the mRNA export machinery.Whileindividualalymutantshavewildtypeappearance,simultaneousinactivationofall fourALYgenesinthe4xalyplantsresultsinvegetativeandreproductivedefectsincludingstrongly reducedgrowth,alteredflowermorphologyandpartiallyabnormalovulesandfemalegametophytes thatcausereducedseedproduction.Comparedtocontrolcells,in4xalycellspolyadenylatedmRNAs accumulate in the nuclei, supporting the role of Arabidopsis ALY proteins as plant mRNA export factors. The four ALY proteins might act partly redundantly in mRNA export, but differences in expressionandsubnuclearlocalisationsuggestalsodistinctfunctions.
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SmallRNApathwaycomponentsinthefemalegametophyteof Arabidopsisthaliana
MariaEnglhart1,MarcUrban1,AndreaBleckmann1,NingXia1,NicolasStrieder2,ThomasDresselhaus1, JuliaC.Engelmann2#,StefanieSprunck1
1CellBiologyandPlantBiochemistry,BiochemieͲZentrumRegensburg,UniversityofRegensburg,Germany,2Statistical Bioinformatics,UniversityofRegensburg,Germany,#currentaddress:DepartmentofMarineMicrobiologyand Biogeochemistry(MMB),NIOZRoyalNetherlandsInstituteforSeaResearch,Netherlands
Thereproductivelineageinitiateswhenthemeiocyteprecursorcellsspecifyandundergoatransition fromamitotictoameioticcellfate.Unlikeanimals,wherethegermlineisestablishedduringearly embryogenesis, the plant male and female reproductive lineages arise late during development, when the flowers are formed. Notably, the haploid products of meiosis (the spores) do not immediately differentiate to form the gametes but undergo further mitotic divisions in a process termedgametogenesis.Asaresultthehaploid“gametophytic”generationisformed,comprisingthe gametesandaccessorycellswhicharenecessaryforsuccessfulreproduction.
SpecificArgonauteproteinsandtheirsmallRNAtargetsareimportantforbothanimalandflowering plant germline development. The role of Argonautes and sRNAs during male and female sporogenesishasbeenintensivelystudiedinriceandmaize,demonstratingthatepigeneticfactors participateinintercellularinteractionsleadingtotheestablishmentofreproductivecellnumberand fateinplants.
However, it is not known whether small RNA pathway components are also important for female gametogenesis. The composition of small RNAs in the cells of the female gametophyte remains elusiveandtheirgametogenesisortransgenerationalrole(s)areunexplored.Reasonsforthislackof knowledgearetheirsmallsizeandinaccessibility,astheyaredeeplyembeddedinthesporophytic tissuesoftheovule,andtheovary.Here,wewillpresentourmethodologicalapproachestoidentify small RNAs expressed in the cells of the female gametophyte as well as AGO effector ribonucleoproteincomplexespresentintheArabidopsiseggcell.
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TheArabidopsisTHO/TREXcomponentTEX1functionallyinteracts withMOS11andmodulatesmRNAexportandalternativesplicing events
SilviaEsposito,BrianB.Sørensen,HansF.Ehrnsberger,AstridBruckmann,GunterMeister,Rainer Merkl,ThomasSchubert,GernotLängst,MarionGrasserandKlausD.Grasser
CellBiologyandPlantBiochemistryͲUniversityofRegensburg
TREX(transcriptionͲexport)isamultiproteincomplexthatplaysacentralroleinthecoordinationof synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicaseUAP56andthemRNAexportfactorsALY2Ͳ4andMOS11wedetectedinteractionswiththe mRNAexportcomplexTREXͲ2andmultiplespliceosomalcomponents.PlantsdefectiveintheTHO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 doubleͲmutant plants show marked defects in vegetative and reproductivedevelopment.Intex1plants,thelevelsoftasiRNAsarereduced,whilemiR173levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed,whilenomRNAexportdefectwasdetectedwithtex1cells.Nevertheless,intex1mos11 doubleͲmutants,themRNAexportdefectwasclearlyenhancedrelativetomos11.Thesubnuclear distributionofTEX1substantiallyoverlapswiththatofsplicingͲrelatedSRproteinsandintex1plants theratioofcertainalternativesplicingeventsisaltered.OurresultsdemonstratethatArabidopsis TEX1andMOS11areinvolvedindistinctstepsofthebiogenesisofmRNAsandsmallRNAs,andthat theyinteractregardingsomeaspects,butactindependentlyinothers.
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NewinsightsintoprevalenceandfunctionsofsigmaͲdependent pausinginbacteria
DariaEsyunina,IvanPetushkov,DanilPupov,AndreyKulbachinskiy
InstituteofMolecularGenetics,RussianAcademyofSciences,Moscow123182,Russia
TheʍsubunitassociateswithbacterialRNAͲpolymerase(RNAP)toassistitinpromoterrecognition andtranscriptioninitiation.Mostbacteriacontain oneprincipalandseveralalternativeʍsubunits that have different promoter specificities and are responsible for gene expression under varying conditions.TranscriptionelongationisperformedbythecoreenzymeofRNAPfollowingpromoter escapeandʍsubunitdissociation.However,theprincipals70subunitinE.colicanstayboundtothe transcription elongation complex and recognize promoterͲlike sequences in initially transcribed regions, resulting in transcriptional pausing and increased s retention at later steps of elongation. Thisphenomenonwasproposedtobelimitedtotheprincipalʍsubunitonly.WeshowedthatʍͲ dependentpausingcanbeobservedwithRNAPsfromvariousbacteria.Furthermore,wefoundthat alternativessubunitscanalsoinducepausing.Inparticular,ʍ38andʍ24subunitsofE.coliinduced efficientpausingatpromoterͲlikesitesinthetranscribedDNA.Inbothcases,wedetectedformation of backtracked complexes that could be reactivated by GreͲfactors, similarly toʍ70Ͳdependent pausing. To understand the role of sͲdependent pausing in gene expression, we designed several modelsystemsbasedontheuseofluciferaseandlacZreporterscontainingnaturalandartificially designedʍͲpausesequences.WefoundthatthepresenceofpromoterͲlikesequencesintheearly transcribed regions significantly modulates the level of gene expression. In conclusion, our data suggestthatʍͲdependentpausingisawidespreadphenomenonthatcanbeinducedbyvarioustypes ofʍsubunitsandmodulategeneexpression.
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AgeneralmethodforrapidandcostͲefficientlargeͲscaleproduction of5'cappedRNA
AnnaͲLisaFuchs,AncillaNeuandRemcoSprangers
UniversityofRegensburg;MaxPlanckInstituteforDevelopmentalBiology,Tuebingen
The eukaryotic mRNA 5഻ cap structure is indispensible for preͲmRNA processing, mRNA export, translationinitiation,andmRNAstability.Despitethisimportance,structuralandbiophysicalstudies thatinvolvecappedRNAarechallengingandrareduetothelackofageneralmethodtoprepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce cappedRNAin theamountsthatarerequiredforlargeͲscalestructuralstudies. Wehave thereforedesignedanefficientexpressionandpurificationprotocolforthevacciniacappingenzyme. Usingthisapproach,thereactionscalecanbeincreasedinacostͲefficientmanner,wheretheyields ofthecappedRNAsolelydependontheamountofavailableuncappedRNAtarget.Usingalarge number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highͲresolution NMR spectroscopy.Incombinationwith 13CͲmethylͲlabeledSͲadenosylmethionine,themethylgroupsin theRNAcanbelabeledformethylTROSYNMRspectroscopy.Finally,weshowthatourapproachcan producebothcapͲ0andcapͲ1RNAinhighamounts.Insummary,wehereintroduceageneraland straightforwardmethodthatopensnewmeansforstructuralandfunctionalstudiesofproteinsand enzymesincomplexwithcappedRNA.
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DepletionofthelongnonͲcodingRNASINCR1inhibitscell proliferationandinducessenescenceinlivercancercells
MinakshiGandhi1,2,MatthiasGroß1,MathiasMunschauer3,EricLander3,4,5,SvenDiederichs1,6
1DivisionofRNABiology&Cancer(B150),GermanCancerResearchCenter(DKFZ),ImNeuenheimerFeld280,69120 Heidelberg,Germany2DeutscherAkademischerAustauschdient(DAAD),Kennedyallee50,53175Bonn3BroadInstituteof MITandHarvard,Cambridge,MA02142,USA.4DepartmentofBiology,MIT,Cambridge,MA02139,USA5Departmentof SystemsBiology,HarvardMedicalSchool,Boston,MA02115,USA.6DivisionofCancerResearch,Dept.ofThoracicSurgery, MedicalCenterͲUniversityofFreiburg,BreisacherStr.115,79106Freiburg,Germany
Hepatocellularcarcinoma(HCC)isthesixthmostcommoncancerinthepopulationworldwidewitha poor 5Ͳyear overall survival of less than 15% illustrating the need for deeper investigation into hepatocarcinogenesis. Long nonͲcoding RNAs (lncRNAs) have emerged as critical players for regulating various pathological and physiological processes including HCC. InͲspite of the clinical relevance, their functional role and molecular mechanisms remain poorly characterized. Here, we usedagenomeͲwidescreeningapproachtoidentifySINCR1(SenescenceInducingNonCodingRNA1) ͲanlncRNAinducedby5.57foldinHCC.ThedepletionofSINCR1inmultipleHCCcelllinesevokesa strongproliferationdefect.SINCR1depletedcellsarearrestedinG1/SresultinginasenescenceͲlike phenotype.ThisstrongphenotypeissupportedbyproteomicsprofilesofSINCR1Ͳdepletedcellsusing tripleͲlabelSILACexperiments.DepletionofSINCR1ledtodownregulationofgenessuchasCCNB1, TOP2A,CDK2,KIF11,MKiͲ67andTYMSconfirmingthelinktocellcycleprocesses.AninvivoRNA Affinity Purification approach identified direct interaction partners of SINCR1 with known roles in regulating G1/S transition and promoting Senescence. Further validation of these interaction partnerswillunravelitsmolecularmechanisminfurtherdetail.OurfindingshighlightSINCR1asa crucialregulatorofcancercellproliferationandsenescenceinHCC
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MolecularmechanismsofBdp1inTFIIIBassemblyandRNA polymeraseIIItranscriptioninitiation
JeromeGouge1,NicolasGuthertz1,KevinKramm2,GuillermoAbascalͲPalacios1,KarishmaSatia1,Dina Grohmann2&AlessandroVannini1
1DepartmentofBiochemistry,GeneticsandMicrobiology,InstituteofMicrobiology,UniversityofRegensburg,93053 Regensburg,Germany.2TheInstituteofCancerResearch,LondonSW73RP,UnitedKingdom
Eukaryotes encode at least three distinct RNA polymerases that transcribe the genome into functionalandregulatoryRNAs.Amongthem,RNApolymeraseIII(PolIII)synthesizesadiversepool ofabundantRNAsincludingtRNAsandtheU6spliceosomalRNA.InitiationoftranscriptionbyPolIII requires the activity of the initiation factor TFIIIB, a complex formed by Brf1 (or Brf2, BͲrelated factor),TBP(TATAͲbindingprotein),andBdp1.TFIIIBrecruitsPolIIItothepromoterandsupportsthe transitionfromaclosedtoanopenpreͲinitiationcomplex,aprocessdependentontheactivityofthe Bdp1subunit.WhilesometranscriptionfactorsaresharedbetweenPolI,IIandIII,Bdp1isuniqueto the Pol III system. Structural data showed that Bdp1 utilizes similar binding surfaces as the Pol II transcription factors TFIIA and TFIIF that promote the stability of the complex and suggest an auxiliaryroleinpromoteropening.WhileBdp1doesshowDNAinteractionssimilartobacterialsigma factorsitdoesnotinduceDNAmeltingalone,supportingtheideaofaconcertedinteractionwithPol IIIsubunits.SingleͲmoleculeFRETmeasurementsonimmobilizedandfreelydiffusingmoleculescan provideinformationbeyondthestaticimageofthecrystalstructure.Here,weusedtimeresolved singleͲmoleculeFRETmeasurementstogaininsightsintothearchitectureanddynamicassemblyof TFIIIBonaU6promoter[1].OurmeasurementsrevealedthatTBPonlytransientlybindsandbends the U6 promoter (complex lifetime of 0.3 s). This is reminiscent of the dynamic archaeal TBPͲ promoter DNA interaction [2]. In contrast, the interaction of TBP with Pol II promoters lasts for minutes to hours. The transient TBPͲDNA interaction is stabilized by Brf2 (complex lifetime of 6.5min),lockingthepromoterinasinglebentconformation. Subsequent bindingofBdp1further stabilizesthecomplextoalifetimeof9minbutdoesnotaltertheDNAconformation.Thesedata suggestthatadditionalinitiationfactorslikeBrf1/2andBdp1evolvedtostabilizethepreͲinitiation complexandtoensureefficienttranscriptionfromasubsetofpromoters.
[1]J.Gouge,N.Guthertz,K.Kramm,O.Dergai,G.AbascalͲPalacios,K.Satia,P.Cousin,N.Hernandez,D.Grohmann,andA. Vannini,‘MolecularmechanismsofBdp1inTFIIIBassemblyandRNApolymeraseIIItranscriptioninitiation’,Nat.Commun., vol.8,no.1,p.130,2017.
[2]A.Gietl,P.Holzmeister,F.Blombach,S.Schulz,L.V.VonVoithenberg,D.C.Lamb,F.Werner,P.Tinnefeld, and D. Grohmann, ‘Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways’,NucleicAcidsRes.,vol.42,no.10,pp.6219–6231,2014
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GlobaltranscriptionalstartsitemappinginPyrococcusfuriosus: Transcriptionalnoiseorpartofarchaealgeneregulation?
FelixGrünberger,RobertReichelt,MartinFenk,WinfriedHausner
UniversityofRegensburg,Microbiology,Regensburg,Germany
Occupying a key position in the tree of life, archaeal organisms and their complex transcriptomes sharemolecularcharacteristicswithEukaryaandBacteria.WeuseddifferentialRNAͲseq(dRNAͲseq)1 to map transcriptional start sites (TSS) in a newly annotated genome of the hyperthermophilic archaeon Pyrococcus furiosus (Pfu). To reach maximum transcriptome coverage, sequencing cDNA librariesweregeneratedfromRNAisolatedfromcellsunder9differentgrowthconditions.Mapping resultswerecomplementedwithasingleͲnucleotideresolutionmapofthewholetranscriptomewith a6000foldsequencecoverage.Inaddition,TSSofgeneswithvarying5`Ͳuntranslatedregion(5`ͲUTR) lengthswereconfirmedusingprimerextensionanalysis.UsingTSSpredator2,morethan5800TSSfor about 2000 annotated genes have been identified and categorized as primary (pTSS), secondary, internalorantisensetranscriptsaccordingtotheirrelativegeneposition.Only968pTSS(50%ofall genes) have been detected with a median 5`ͲUTR length of 27 nt. Archaeal promoter motifs, consistingofaBREͲelementandaTATAͲbox,werepresentforall4categoriesofTSS.Geneswith strongpromoterstendtobeleaderless.Overall,extensiveRNAͲbasedregulationisconsistentwith resultsofotherarchaealmodelorganisms.DifferentialRNAͲseqfurtherrevealedthepresenceofa biͲdirectionalpromotermotifupstreamofalmost30%ofallpTSS.Sofar,thishasnotbeenreported inarchaea,whileitiswidespreadineukaryoticspecies3,4.Aquestionthatremainsis,towhatextent Pfu´s densely packed genome and low GC content (41%) result in these promoters, leading to transcriptionalnoiseorifitisanenergyefficientwayfortranscriptionalregulation5.
[1]Sharma,C.M.,&Vogel,J.(2014).DifferentialRNAͲseq:Theapproachbehindandthebiologicalinsightgained.Current OpinioninMicrobiology [2]Dugar,G.,Herbig,A.,Förstner,K.U.,Heidrich,N.,Reinhardt,R.,Nieselt,K.,&Sharma,C.M.(2013).HighͲResolution TranscriptomeMapsRevealStrainͲSpecificRegulatoryFeaturesofMultipleCampylobacterjejuniIsolates.PLoSGenetics [3]Xu,Z.,Wei, W.,Gagneur,J.,Perocchi,F.,ClauderͲMünster,S.,Camblong,J.,…Steinmetz,L.M.(2009).Bidirectional promotersgeneratepervasivetranscriptioninyeast.Nature [4]Jin,Y.,Eser,U.,Struhl,K.,&Churchman,L.S.(2017).TheGroundStateandEvolutionofPromoterRegionDirectionality. Cell [5]LlorénsͲRico,V.,Cano,J.,Kamminga,T.,Gil,R.,Latorre,A.,Chen,W.ͲH.,…LluchͲSenar,M.(2016).Bacterialantisense RNAsaremainlytheproductoftranscriptionalnoise.ScienceAdvances
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NMRanalysisconformationaldynamicsduringUsnRNPassembly
NishthaGulati1,2;HyunͲSeoKang1,2;SamAsami1,2;MichaelSattler1,2
1InstituteofStructuralBiology,HelmholtzZentrumMünchen,85764Neuherberg,Germany2BiomolecularNMRandCenter forIntegratedProteinScienceMunich(CIPSM),DepartmentChemie,TechnischeUniversitätMünchen,85747Garching, Germany
pIClnisahighlyconservedchaperone,whichregulatestheassemblyofspliceosomalsmallnuclear ribonucleoproteins(snRNPs).Thesearethebuildingblocksofthespliceosomeandtherebyplaysa crucialroleinsplicingofcellularpreͲmRNAs.AhallmarkofthesnRNPsisasevenͲmemberedringof SmproteinsthatbindstoauridineͲrichRNAsequenceintheUsnRNAs.ThebiogenesisofsnRNPsis tightlycontrolledandinvolvesadditionalfactorsthatensurefidelityofcorrectsnRNPassembly.One oftheseproteins,pIClnassemblessubcomplexesofSmproteinsinthecytosolandmediatescorrect assemblyofthecompleteSmringbytheSMN(survivalmotorneuron)complex.Wearestudyingthe role of conformational dynamics and flexibility within intermediate complexes involving pICln, a subset of Sm proteins and various components of the SMN complex using NMRͲspectroscopy. Furthermore,wewouldliketounderstandtheroleoftheSMNTudordomainanditsinteractionwith thedimethylatedargininesinthe8ScomplexandUsnRNAbinding.Preliminaryresultsshowthat isotopeͲlabelled pICln can be expressed, purified and incorporated into Sm protein complexes. To betterunderstandthestructuraldynamicsofpIClnintheseprocesses,weareusingsolutionNMR relaxation experiments and small angle scattering experiments to study the role of pICln in the complexassembly
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SLAMͲFRET:surveyingsiteͲspecificallylabelledeukaryotic biomolecularcomplexesonthesinglemoleculelevel
AlexanderGust1,LeonhardJakob1,DanielaM.Zeitler2,KevinKramm1,SarahWillkomm1,Philip Tinnefeld3,GunterMeister2andDinaGrohmann1
1DepartmentofBiochemistry,GeneticsandMicrobiology,InstituteofMicrobiology,SingleͲMoleculeBiochemistryLab, UniversityofRegensburg,Universitätsstraße31,93053Regensburg,Germany2DepartmentforBiochemistryI,Biochemistry CentreUniversityofRegensburg,Universitätsstraße31,93053Regensburg,Germany3InstituteofPhysicalandTheoretical Chemistry,andBraunschweigIntegratedCentreofSystemsBiology(BRICS),andLaboratoryforEmergingNanometrology (LENA),TechnischeUniversitätBraunschweig,Rebenring56,38106Braunschweig,Germany
In the cell, biomolecules constantly form new interactions or dissociate from bindings partners to fulfillthemyriadoftasksrequiredforthesurvivalofacell.Misregulationoftheseprocessesoften causes diseases. Oftentimes, conformational changes in a biomolecule initiate complex formation, dissociationorfunctionaltransitions.Inordertoexploittheseprocessesforbiotechnicalapplications or to target these reactions with suitable inhibitors, it is necessary to understand the detailed molecular mechanisms. In this context, singleͲmolecule measurements, like single molecule FRET (smFRET), allow the precise and timeͲresolved interrogation of biological interactions and conformational changes on the single molecule level.In order to carry out fluorescenceͲbased singleͲmolecules studies, it is necessary to equip them with an organic fluorophore. SiteͲspecific labellingofbiologicalmoleculesisachallengingtask,especiallyifhumanproteinsaretobestudied. Oftenitisnotpossibletoproduceeukaryoticproteinsinarecombinantmannerduetotheircomplex posttranslational modifications essential for protein function preventing traditional fluorophore engineeringapproaches.Toovercometheseproblems,wehaveestablishedthesiteͲspecificlabelling ofendogenousmammalianproteinsforsingleͲmoleculeFRETmeasurements,amethodwetermed SLAMͲFRET(siteͲspecificlabelingofmammalianproteinsforsingleͲmoleculeFRETmeasurements). HereweshowthattheSLAMͲFRETmethodcanbeappliedtohumanproteinsandproteincomplexto carryoutsmFRETmeasurementsandthattheFRETͲderiveddistancesareinverygoodagreement with structural studies. Using this technique, we succeeded to carry out singleͲmolecule FRET measurements using fluorescently labeled human Argonaute 2 (hAgo2). hAgo2 could not be producedinafluorescentlylabelledformyetandconsequently,singleͲmoleculestudiesthatfollow the conformational changes of this protein throughout its activity cycle are lacking. Labelling two amino acids in hAgo2, it was possible to measure intramolecular FRET, which reports on an interdomaindistanceinhAgo2.Furthermore,wemeasuredintermolecularFRETbetweenhAgo2and an interaction partner, in this case a fluorescent labeled guide RNA. Notably, the modified hAgo2 variantswerefullyposttranslationallymodifiedandactive.Wealsoextendedourmethodtosurveya humanproteincomplexviaFRET,namelytheRNApolymeraseIIsubunitsRPB4/7
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TheCͲterminaldomainofS.cerevisiaeNet1isapotentactivatorof RNApolymeraseItranscriptioninvivoandinvitro
KatharinaHannig,UlrikeStöckl,AndreasMaier,MichaelPilsl,ShuangLi,VirginiaBabl,Philipp Milkereit,HerbertTschochner,WolfgangSeufert,JoachimGriesenbeck
UniversitätRegensburgInstitutfürBiochemie,GenetikundMikrobiologie93053Regensburg,Germany
RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) producing a precursor of the large ribosomalRNAs(rRNAs)inalleukaryotes.rRNAtranscriptionisanessentialprocessandaccountsfor morethanhalfoftotalcellulartranscriptioninproliferatingcells.Toachievethishightranscriptional output,aspecificsetofproteinfactorshasevolvedtosupportPolItranscriptioninitiation.Whereas someofthesefactorsarehighlyconservedfromyeasttohuman,othersareconservedratheratthe functionalandstructurallevel.InS.cerevisiae(hereaftercalledyeast)aminimalsetoftranscription factorscomposedofRrn3,whichassociateswithPolItoformtheinitiationcompetentpolymerase, and a three subunit complex called core factor support promoter dependent transcription in vitro. Anothersixsubunitcomplexcalledupstreamactivatingfactor,whichformsacomplexwith TATAbindingprotein,isrequiredforPolItranscriptioninvivo.Previously,ithasbeenreportedthata largeprotein,Net1,stimulatescellgrowthcorrelatingwithitsabilitytoactivatePolItranscription. Net1isamultifunctionalprotein,whichfulfillsadditionalimportantrolesinregulatingthecellcycle andsilencingoftranscriptionbyRNApolymeraseIIattherDNAlocus.ThemechanismbywhichNet1 stimulatesPolItranscriptionisunknown.
Weshow,thatthePolIstimulatingfunctionofNet1canbeattributedtoanindividualdomainwithin theprotein.Thus,theveryCͲterminaldomain(CTD)ofNet1,comprisedofonly139aminoacidsis requiredfornormalcellgrowthandsufficienttopartiallyrescuethegrowthdefectobservedinthe absenceoffullͲlengthNet1.Ingoodcorrelation,theCTDenhancesPolIloadingonrRNAgenesin vivoandpromoterͲdependenttranscriptioninaminimaltranscriptionsysteminvitro.Interestingly, phosphorylationoftheCTDappearstomodulateitspotentialtostimulatePolItranscription.The data suggests, that the Net1ͲCTD might be an important regulator of Pol I transcription and cell proliferation.PossiblemechanismshowtheCTDmayinfluencePolItranscriptionarediscussed.
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AnovelfunctionoftheLaͲrelatedproteinLARP7inthemodification ofU6snRNA
DanieleHasler,RajyalakshmiMeduri,GerhardLehmann,UtzFischer,GunterMeister
BiochemistryCenterRegensburg(BZR),LaboratoryforRNABiology,UniversityofRegensburg,Regensburg(Germany)
TheLupusautoantigenLaandtheLaͲrelatedproteins(LARPs)areRNAͲbindingproteins(RBPs)which exerttheirfunctionswithinamultitudeofpathwaysinRNAbiology.Ofparticularrelevanceisthe interactionoftheLaproteinwiththe3’ͲendofallnascentRNApolymeraseIII(PolIII)transcripts, whichhasnotmerelyaprotectiverolebutalsosupportsthecorrectfoldingoftheseRNAs.
Intermsofproteindomainorganization,aswellassubstratebindingcharacteristics,genuineLais mostcloselyrelatedtotheLARP7protein.Sofar,LARP7hasbeenmainlydescribedasacomponent of the 7SK small nuclear ribonucleoprotein (snRNP) complex which negatively regulates RNA polymeraseII(PolII)bysequesteringthepositivetranscriptionelongationfactorb(PͲTEFb).Within the 7SK snRNP, LARP7 stably associates with the 3’Ͳend of the 7SK RNA, an approximately 330 nucleotideslongnon codingRNAtranscribedbyPolIII. Inourstudieswe discoveredanovel,7SK snRNPͲindependent function of LARP7. We show that LARP7 specifically interacts with the spliceosomal U6 snRNA as well as with the small nucleolar RNAs (snoRNAs) which direct the 2’OͲ methylationofU6.Importantly,ourinvestigationsindicatethatintheabsenceofLARP7significantly less 2’OͲmethylations are deposited on the U6 snRNA. We finally analyze the impact of LARP7 depletiononthespliceosome.
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CircularRNAZNF609promotestheprogressionofcolorectalcancer
HungHoͲXuan,PetarGlazar,MarvinAnders,ClaudiaLatini,IvanoLegnini,GerhardLehmann,Norbert Eichner,IreneBozzoni,NikolausRajewsky,ChristinaHacklandGunterMeister
BiochemistryCenterRegensburg(BZR),LaboratoryforRNABiology,UniversityofRegensburg,Regensburg
NonͲcoding RNAs can function as potent gene regulators and consequently, such RNA molecules have been implicated in many diseases including different types of cancer. Using an intrasplenic tumormodelforcolorectalcancer,wehaveidentifiedseverallongnoncodingRNAs(lncRNAs)and circularRNAs(circRNAs)differentiallyexpressedinprimarytumorsandlivermetastasessuggestinga roleoftheseRNAsintumorprogression.OfthecircRNAcandidates,wehaveidentifiedcircularRNA ZNF609 (circZNF609), which is upͲregulated in primary tumors as well as liver metastases in our mousemodel.Wedevelopeda methodforknockingdownand overexpressionofcircZNF609and established inducible stable cell lines for in vivo analyses. In a xenograft mouse model, overexpressionofcircZNF609promotescolorectalcancerprogression.Interestingly,wefoundthat circZNF609encodesforsmallpeptides,whichcouldpotentiallyhavefunctionintumordevelopment. UsingFlagreporterconstructsandmutagenesis,wefoundthatseveralATGs(evenexogenousATGs) canbeusedtoinitiatetranslationfromcircZNF609.Moreover,circZNF609proteinlocalizestothe cytoplasminseveraltestedcelllineswhiletheparentalZNF609proteinexclusivelylocalizestothe nucleus. Mechanistically, mass spec analysis identified several interaction partners which bind to both circZNF609 and ZNF609 proteins suggesting an interesting mode of circZNF609 functions. Currently, we are developing methods for rescuing the phenotype of circZNF609 depletion and investigatingfurthercircZNF609mechanisms.
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CHD3andCHD4formdistinctNuRDcomplexeswithdifferentyet overlappingfunctionality
HelenHoffmeister,AndreasFuchs,FabianErdel,SophiaPinz,ReginaGroebnerͲFerreira,Astrid Bruckmann,RainerDeutzmann,UweSchwartz,RodrigoMaldonado,ClaudiaHuber,AnneͲSarah Dendorfer,KarstenRippeandGernotLaengst
InstituteofBiochemistry,GeneticsandMicrobiology,UniversityofRegensburg,93053Regensburg
CHD3andCHD4(ChromodomainHelicaseDNAbindingprotein),twohighlysimilarrepresentativesof theMiͲ2subfamilyofSF2helicases,arecoexpressedinmanycelllinesandtissuesandhavebeen reportedtoactasthemotorsubunitoftheNuRDcomplex(nucleosomeremodelinganddeacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3,arguingforaroleasatranscriptionalrepressor.However,thesubunitcompositionvaries among cellͲ and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4coexistinthesameNuRDcomplexorwhethertheyformdistinctNuRDcomplexeswithspecific functions.WemappedtheCHDcompositionofNuRDcomplexesinmammaliancellsanddiscovered thattheyareisoformͲspecific,containingeitherthemonomericCHD3orCHD4ATPase.Bothtypesof complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UVͲ induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbedcells,revealingasubsetofspecifictargetgenes.Furthermore,CHD3andCHD4differin theirnucleosomeremodelingandpositioningbehaviourinvitro.TheproteinsformdistinctCHD3Ͳ andCHD4ͲNuRDcomplexesthatdonotonlyrepress,butcanjustaswellactivategenetranscription ofoverlappingandspecifictargetgenes.
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CompositionoftheArabidopsisRNApolymeraseIItranscript elongationcomplexrevealsinterplayofelongationandmRNA processingfactors
PhilippHolzinger,WojciechAntosz,AlexanderPfab,HansF.Ehrnsberger,AstridBruckmann,Thomas Schubert,GernotLängst,JoachimGriesenbeck,VeitSchubert,MarionGrasserandKlausD.Grasser
CellBiologyandPlantBiochemistryͲUniversityofRegensburg
Transcriptelongationfactors(TEFs)areaheterogeneousgroupofproteinsthatcontroltheefficiency oftranscriptelongationbyRNApolymeraseII(RNAPII)ofsubsetsofgenesinthechromatincontext. Using reciprocal tagging in combination with affinityͲpurification and mass spectrometry, we demonstratethattheTEFsSPT4/SPT5,SPT6,FACT,PAF1ͲCandTFIIScoͲpurifiedwitheachotherand elongating RNAPII, while PͲTEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATPͲdependent chromatin remodelling factors and some histone modifying enzymes includingElongatorwererepeatedlyfoundassociatedwithTEFs.AnalysisofdoubleͲmutantplants defectivein differentcombinationsofTEFsrevealedgeneticinteractions betweengenesencoding subunits of PAF1ͲC, FACT and TFIIS, resulting in synergistic/epistatic effects on plant growth/development. The analysis of subͲnuclear localisation, gene expression and chromatinͲ associationdidnotprovideevidenceforaninvolvementoftheTEFsinthetranscriptionbyRNAPI (and RNAPIII). The proteomics analyses showed also multiple interactions between the transcript elongation complex and factors involved in mRNA splicing and polyadenylation including an associationofPAF1ͲCwiththepolyadenylationfactorCstF.OurdataestablishthattheArabidopsis RNAPIItranscriptelongationcomplexrepresentsaplatformforinteractionsamongdifferentTEFsas wellasforcoordinatingongoingtranscriptionwithmRNAprocessing.
87 P251
Translationalregulationofpollendevelopmentandpollentube growth;implicationsformaleͲfemalecommunication
DavidHonys,SaidHafidh,DavidPotĢšil,KatarínaKulichová,PavelBokvaj,ZbynĢkZdráhal
LaboratoryofPollenBiology,InstituteofExperimentalBotanyASCR,Prague,CzechRepublic;CentralEuropeanInstituteof Technology,MasarykUniversity,Brno,CzechRepublic;NationalCentreforBiomolecularResearch,MasarykUniversity, Brno,CzechRepublic
The male gametophyte, highly organized haploid flower organ, offers an unique opportunity to analyzedevelopmentanddifferentiationofsinglehaploidcells,cellͲcellinteractionsandrecognition, cellularpolarityandpollentubetipgrowth.Transcriptionalandposttranscriptionalcontrolofgene expressionplayavitalroleduringtobaccopollenmaturationandtubegrowth.Theneedforahigh rateoftranslationduringpollentubegrowthsuggestsademandforarobuststoragesystemthat couldwithstandalongͲtermstorageandtransport,ongoingcellularmorphogenesis,andyetdeliver the message efficiently accompanied with instant translation. A number of pollen genes showed apparentexpressiondiscrepancyatmRNAandproteinlevelsandtheirrespectivetranscriptswere shown to be associated with longͲterm stored ribonucleoprotein particles. Similarly to the role played in growing mammalian neurons, these particles represent preͲloaded complex machinery devoted to mRNA processing, transport, subcellular localization and protein synthesis. Here, we present functional, transcriptomic and proteomic characterisation of pollen storage ribonucleoproteinparticles.Inparticular,weaimedtointegrateourknowledgeonthecategorization oftranslationallyregulatedtranscriptsindevelopingpollenandtoidentifythemodeofactionofthe translational repression and derepression of mRNAs stored in developing pollen and which have graduallyactivatedduringtheprogamicphase.Wealsodiscusstheselectiveactivationofparticular transcriptsfollowingmaleͲfemaleinteractioninsemiͲinvivoconditions.
Acknowledgement:TheauthorsgratefullyacknowledgethefinancialsupportfromCzechScienceFoundation (projects13Ͳ06943S,15Ͳ16050S,17Ͳ23183S).
88 P261
DiscoveryofRNAͲproteincomplexesbyGradͲseq
JensHör1,KonradU.Förstner1,2,JörgVogel1,2
1InstituteforMolecularInfectionBiology,UniversityofWürzburg,Germany2CoreUnitSystemsMedicine,Universityof Würzburg,Germany2HelmholtzInstituteforRNAͲbasedInfectionResearch(HIRI),Würzburg,Germany
RNAͲbindingproteins(RBPs)areimportantfactorsintheregulationofgeneexpression.Inbacteria, threemajorfamiliesofregulatoryRBPsareknown:Hfq,ProQandCsrA,allofwhichactbyregulating geneexpressiononmRNAlevel.WhileHfqandProQfacilitatebindingofsmallRNAs(sRNAs)totheir targetmRNAsandtherebyleadtoregulation,CsrAbindsdirectlytomRNAs,alteringtheirtranslation andstability.sRNAsarealsoakeyfactorinCsrAregulation,astheyareabletosequesterCsrAaway fromitsmRNAtargets.Furthermore,RBPscanbeessentialfactorsforbacterialvirulenceasitwas shown for Hfq in the major human pathogen Salmonella Typhimurium. However, many bacterial species lack one or more of these three wellͲstudied RBPs even though they express regulatory sRNAs,suggestingtheexistenceofcurrentlyunknownRBPs.Usinggradientprofilingbysequencing (GradͲseq), we are investigating the existence of overlooked RBPs. For this, whole bacterial cell lysatesarerunonalinearglycerolgradient,leadingtopartitioningofallsolublecontent.SinceGradͲ seq is performed under native conditions, RNAͲprotein complexes stay intact and sediment as a whole according to their biochemical properties. These interactions can then be investigated by fractionationofthegradientfollowedbyRNAͲseqandmassspectrometryofeachofthosefractions. ThecombinedanalysisoftheresultingdatasetsallowsustodrawconclusionswhichRNAsmight interactwithwhichproteins.
89 P271
TranslationalrepressionofnanosmRNAinearlyflyoogenesis
AndreasHorn1,DanielaStrauss1,MonikaGaik2,SimonaPalusci3,SebastianGlatt2,JanMedenbach1
1InstituteofBiochemistryI,UniversityofRegensburg,Universitaetsstrasse31,93053Regensburg,Germany2MaxPlanck Laboratory,MaųopolskieCentrumBiotechnologii,Gronostajowa7A,30Ͳ387Krakow,Poland3BiomarkerResearch Laboratory,UniversityofHuddersfield,Queensgate,HD13DHHuddersfield,UnitedKingdom
Tissuehomeostasisrequiresafinebalancebetweengermcellmaintenanceanddifferentiation.In thefemalegermlineofD.melanogastertwotothreegermlinestemcells(GSCs)resideatthetipof the germarium. Signaling from the surrounding tissue defines a stem cell niche that prevents differentiation.AftermitoticdivisionofaGSC,thetwodaughtercellsadaptdifferentcellfates:one remains a GSC to replenish the pool of stem cells, the other undergoes four synchronous cell divisionswithincompletecytokinesistogenerateacystofsixteencells,oneofwhichdevelopsinto theoocyte.Toinitiatethisdifferentiationprocess,productionofthestemcellfactorNanos(nos)has to be transiently repressed. This requires the femaleͲspecific master regulator protein SexͲlethal (Sxl),whichbindstonosmRNAtorepressitstranslation.Failuretodosoresultsintheformationof germlinetumorsandsterility.Furthermore,theproteinsBagofmarbles(Bam),Benigngonialcell neoplasm (Bgcn) and MeiͲP26 have been implicated in SxlͲdependent regulation of nos, however, theirmolecularfunctionremainsunexplored.HerewereportthatbothBamandBgcncanrepress translation upon tethering to an RNA reporter, suggesting that they play an active role in nos repressionandtissuehomeostasisinthefemaleDrosophilagermline.
90 P281
ForceͲdependencyofCas9targetrecognitioninvestigatedwitha DNAorigamiͲbasednanoscopicforceclamp
LeonhardJakob,PhilippNickels,DongfangWang,PhilipTinnefeldandDinaGrohmann
UniversityofRegensburgAGGrohmannMikrobiologie
The discovery of prokaryotic adaptive immune systems consisting of the so called CRISPR–Cas (clustered regularly interspaced short palindromic repeats (CRISPR)Ͳassociated) proteins and their applicability for genome editing in eukaryotes is one of the most exciting biochemical advances withinthepastyears.AmongthemanyCRISPRsystemvariants,thetypeIICRISPRsystemismost suitableforgeneeditingapplications.Itcontainsasingleeffectorprotein,theendonucleaseCas9, whichassociateswithanRNAduplexconsistingofaCRISPRRNA(crRNA)andatransͲactingCRISPR RNA(tracrRNA)tospecificallycleavedoubleͲstrandedDNA(dsDNA)targetscomplementarytothe crRNA.Additionaltosequencecomplementarity,aprotospaceradjacentmotif(PAM)isrequiredfor efficient target recognition and cleavage. For genome editing applications, single guide RNAs (sgRNA),whicharechimerasofcrRNAandtracrRNAarecommonlyusedtoreducethecomplexityof heterologousexpressionineukaryoticcells.Foreditingpurposes,sgRNAͲassociatedCas9needstobe efficientlyimportedintothenucleustoencounteritstargetsequence.However,withinthenucleus, genomic DNA is seldom easily accessible but often tightly packed within chromatin regions. The wrappingandbendingofDNAinducestensionsonthedsDNAandthismightconstituteadecisive factorforCas9efficiencyandaccuracy.
We employed our recently developed DNA origamiͲbased nanoscopic force clamp (Nickels et al., 2016)toanalyzewhethertheextentofstrainonDNAinfluencesCas9binding.Thisapproachallows high throughput force measurements in the biological relevant piconewton range on the singleͲ moleculelevel.Usingthenanoscopicforceclamp,weexertedadefinedforceonthetargetDNAand monitoredCas9bindingonthesingleͲmoleculelevelusingafluorescenceresonanceenergytransfer (FRET)signalbetweenthedonorͲlabeledtargetDNAandtheacceptorͲlabeledCas9ͲboundcrRNAas readout.WecanshowthatthebindingbehavioroftheCas9ͲsgRNAtoitstargetDNAchangesina forceͲdependentmannerandthathighforcesabolishCas9ͲsgRNAbindingtoitstargetDNA.These resultssuggestthatCas9isaforceͲsensitiveenzymeandthatstrainedDNAregionsinthegenomic DNAwilllessefficientlyrecognizedbyCas9.
91 P291
VisualisingsequentialpreͲ60Smaturationbychemicalinhibition
VasileiosKargas,PabloCastroͲHartmann,FelixWeis,EmmanuelGiudice,GertrudeZisser,Paul Emsley*,GaribMurshadov*,HelmutBergler,AlanJ.Warren
CambridgeInstituteforMedicalResearchUniversityofCambridgeHillsRoadCambridgeCB20XY*MRCLaboratoryof MolecularBiologyCambridgeUK
Ribosomalsubunitassemblyisahighlydynamicprocessthatisinitiatedinthenucleusandinvolves theactionofmorethan200transͲactingfactors,someofwhichaccompanythepreͲribosomesinto the cytoplasm and must be recycled back into the nucleus. Diazaborine arrests 60S subunit maturation at an early maturation step by inhibiting the AAAͲATPase Drg1 and preventing the cytoplasmicreleaseandrecyclingofshuttlingpreͲ60Smaturationfactors.Wehavetakenadvantage of the rapid onset of diazaborine inhibition to investigate the structural changes occurring during export of the 60S preͲribosomal particle using single particle cryoͲelectron microscopy. We will present the structures of sequential late nuclear and early cytoplasmic preͲ60S intermediates, supported by a comprehensive series of immunopurification and laser scanning microscopy experiments. Our results highlight the power of combining chemical inhibition with cryoͲelectron microscopytointerrogatethemechanismsof60Ssubunitmaturationwithtemporalresolution
92 P301
InsightsintotheevolutionaryconservedregulationofRioATPase activity
RobertKnüppel1,RegitseH.Christensen2,FionaC.Gray2,DominikEsser3,DanielaStrauß4,Jan Medenbach4,BettinaSiebers3,StuartA.MacNeill2,5,NicoleLaRonde6,andSébastienFerreiraͲCerca1
1BiochemistryIII–InstituteforBiochemistry,GeneticsandMicrobiology,UniversityofRegensburg,Universitätsstraße31, 93053Regensburg,Germany.2DepartmentofBiology,UniversityofCopenhagen,CopenhagenBiocenter,Universityof Copenhagen,OleMaaløesVej5,2200CopenhagenN,Denmark.3MolecularEnzymeTechnologyandBiochemistry,Biofilm Centre,FacultyofChemistry,UniversityofDuisburgͲEssen,Universitätsstraße5,45141Essen,Germany.4BiochemistryI– InstituteforBiochemistry,GeneticsandMicrobiology,UniversityofRegensburg,Universitätsstraße31,93053Regensburg, Germany.5SchoolofBiology,UniversityofStAndrews,NorthHaugh,StAndrewsKY169ST,UK.6DepartmentofChemistry andBiochemistry,UniversityofMaryland,CollegePark,MD20742,USAUniversityofMarylandMarleneandStewart GreenebaumCancerCenter,Baltimore,MD21201,USA
Eukaryotic ribosome biogenesis is a complex and dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1,Rio2andRio3)belongtoanancientconservedatypicalproteinkinase/ATPasefamilyrequired forthematurationofthesmallribosomalsubunit(SSU).RecentstructureͲfunctionanalysissuggested anATPaseͲdependentroleoftheRioproteinstoregulatetheirdynamicassociationwiththenascent preͲSSU. However, the detailed molecular mechanism that allows the timely activation of Rio catalytic activity and consequent release of Rio proteins from the nascent ribosomal subunit remainedtobedetermined.Inaddition,theancientfunctionaloriginoftheRioproteinsinribosome biogenesis has not been characterized to date. In this work we provide functional evidence supportinganancientroleoftheRioproteinsinthesynthesisofthearchaealSSU.Moreover,we unravel a conserved ribosomal RNAͲmediated crossͲtalk regulatory mechanism enabling timely stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent preͲ40S particle.BasedonourfindingswesuggestaunifiedreleasemechanismoftheRioproteinsfromthe preͲSSU.
93 P311
ToolkitsfortheanalysisofRNAmetabolisminArchaea
R.Knüppel,C.Kuttenberger,M.Kern,N.Ostheimer,M.Weiß,M.Jüttner,S.FerreiraͲCerca
BiochemistryIII,ChairTschochner,GroupFerreiraͲCerca
ArchaeaarewidespreadorganismscolonizingalmosteveryhabitatonEarth.However,themolecular biology of archaea still remains relatively uncharacterized. RNA metabolism is a central cellular process,whichhasbeenextensivelyanalyzedinbothbacteriaandeukarya.Inarchaea,howeverRNA metabolismingeneralandribosomebiogenesisinparticulararestillpoorlycharacterized.Inorderto shed light on these fundamental processes, we are currently establishing and applying several methodologies allowing to analyze archaeal gene expression network dynamic in unprecedented detail.AsummaryofnonͲradioactivepulselabelingofRNA,usingthenucleotideanalog4Ͳthiouracil (4TU),aswellastheapplicationsofarDNAͲreportersystemwillbepresented.
94 P321
SynthesisTakesOnePause!Screeningfornovelinhibitorsof eukaryoticribosomebiogenesis.
LisaKofler,MelaniePertl,MirjamPennauer,DominikAwad,MathiasLoibl,BrigittePertschyand HelmutBergler
InstituteofMolecularBiosciences,UniversityofGraz,Austria
Ribosomesareoutstandingmolecularnanomachinesthatsynthesizeallcellularproteinsandhence form the basis for cellular growth. Eukaryotic ribosome biogenesis, an enormously intricate and highlydynamicprocess,requiresnotonly80ribosomalproteinsandfourribosomalRNAs(rRNAs) butmorethan200ribosomeassemblyfactors.Inthecourseofthisprocesstheseassemblyfactors performmolecularrearrangements,rRNAprocessingandmodificationsorareinvolvedinthenuclear exportofmaturingribosomalparticles.Duetothehighcomplexityandimmensespeedofribosome synthesis, the specific functions of most ribosome assembly factors remain still elusive. A valuable tool to gain deeper insights into these mechanisms is to block the rapidly ongoing ribosomeformationatacertainstep.Tothisend,wedevelopedascreentoidentifynovelinhibitors thattargeteitherthe40Sor60Sribosomalsubunitmaturation.Wediscoveredseveralcompounds blocking ribosome biogenesis leading to various rRNA processing defects. Treatment with these individual drugs might provide a wellͲequipped toolbox to study the process with high temporal resolution.
95 P331
IdentificationofCircularRNAdegradationpathways
ClaudiaLatiniandGunterMeister
RegensburgUniversity
CircularRNAs(circRNAs)arehighlyabundantandevolutionaryconservednonͲcodingRNAs.Several evidencesindicatethatcircRNAscanregulategeneexpressionbytitratingmicroRNAsorRNAbinding proteins,controllingtranscriptionandinterferingwithsplicing.AlthoughthebiogenesisofcircRNAs is considerably well understood, an intriguing question remains how circRNAs are ultimately degraded, as they are stable and resistant to exonucleolytic degradation. We aim to identify the cellular degradation pathways of circRNAs and the responsible endonucleases by a biochemical approach.Therefore,wefirstselectedcircRNAcandidatesandvalidatedtheirexpressionindifferent cell lines. Then, using an enzymatic ligation method, we performed in vitro synthesis of selected circRNAsandconfirmedtheircircularity.Finally,wemeasuredthehalfͲlivesofthesyntheticcircRNAs indifferentcelllysates.OurdataconfirmthehighstabilityofcircRNAsandsuggestthatcircRNAsare moresensitivitytodegradationincytoplasmicextracts.Altogether,ourstudieselucidateanewand fascinatingaspectofcircRNAbiology,whichhasnotbeenanalysedsofar.
96 P341
TriplexesonNucleosomes:basesforchromatinregulationoftriplex function
RodrigoMaldonado,UweSchwartz,ElisabethSilberhorn,GernotLängst
BiochemistryIII,UniversityofRegensburg
The epigenetic landscape of chromatin and its associated gene expression programs is modulated throughtheinteractionwithnonͲcodingRNAs(ncRNAs).However,themolecularmechanismsthat allow ncRNA targeting to chromatin, favouring its stable and sequence specific binding, are still uncleartodate.ThencRNAscanbetargetedtochromatinviasequencespecificbindingproteins,or theydodirectlyinteractwithDNAthroughsequencespecificcontacts,informofRNAͲdsDNAtriple helix structures. The RNA binds to the major groove, stabilised by Hoogsteen bonds between the bases.Ithasbeendescribedthattriplexsequencesareinvolvedintheregulationofgeneexpression, butexperimentalevidencefortheformationoftriplexesinvivoisstilllacking.RNAͲdsDNAtriplex structures, in comparison with dsDNA, are unstable due to the charge repulsion forces and it is believedthatthenucleosomesformamajorobstaclefortriplexformation.Inthepresentstudywe analysedtheeffectofchromatinonthestabilityandsequencespecificformationofaRNAͲdsDNA triplex.Interestingly,wediscoveredthatthetriplexformationisstabilisedbythenucleosomes.Our resultsshowthatthehistonetails,especiallyhistoneH3tail,dospecificallycontributetothestable formationoftriplexesinchromatin.Triplexesareformedinthelinkerregions,andevenintherealm of the nucleosome. GenomeͲwide analyses revealed functional arrangements of nucleosome positioningandputativetriplextargetingsites,correlatingwithspecificstatesofgeneactivityand epigeneticmodifications.
97 P351
AnalysisofRNAPolymeraseItranscribingnucleosomaltemplates
PhilippMerkl1,MichaelPilsl1,JochenGerber1,VirginiaBabl1,KatharinaFreitag1,ChristophGstöttner1, AstridBruckmann1,OlivierGadal2,GernotLängst1,JorgePerezͲFernandez1,PhilippMilkereit1, JoachimGriesenbeck1andHerbertTschochner1
1UniversitätRegensburg,InstitutfürBiochemie,GenetikundMikrobiologie,Universitätsstr.31,Regensburg,Germany 2UniversitéToulouse,LaboratoiredeBiologieMoléculaireEucaryote,UMR5099
RNApolymeraseI(PolI)isaspecializedRNApolymerasewhichtranscribesrRNAgenesineukaryotes synthesizingupto60%ofthetotalcellularRNAcontent.Thedetailedmolecularmechanismdriving Pol I transcription is still unknown. Pol IͲdriven transcription occurs on DNA templates with a particularchromatinstructure.WeanalyzedtheabilityofPolItotranscribethroughnucleosomesin directcomparisonwithRNApolymerasesIIandIII(PolIIandPolIII).PurifiedPolIandPolIIIfromS. cerevisiae transcribed in vitro assembled nucleosomal templates more efficiently than Pol II. Pol I processivity and speed were dependent on both, nucleotide concentration, and reaction temperature.UndersuboptimalassayconditionstheefficiencyofPolItoelongatethroughasingle nucleosomewassignificantlyreduced.ItwaspreviouslypublishedthatPolIsubunitsA49andA12 areinvolvedinPolIprocessivityand/orRNAcleavage,therebyaffectingtranscriptionelongation.We present data how depletion of either one of these subunits or some of their domains influence subunit composition, Pol I elongation speed, Pol I processivity and Pol I passage through a nucleosome.TheresultsoftheseinvitroexperimentsarecorrelatedwithinvivoanalysesofPolI andPolImutantslackingA49ͲorA12Ͳdomainswithregardtotheirabilitytosupportcellulargrowth orrDNAtranscription.
98 P361
DrosophilaSisterofSexͲlethalantagonizesSxlͲlethalͲdependent splicingtomaintainamaleͲspecificgeneexpressionpattern
RebeccaMoschall1,OliverRossbach2,GerhardLehmann1,LarsKullmann3,NorbertEichner1,Daniela Strauss1,NicholasStrieder4,JuliaC.Engelmann4,GunterMeister1,MichaelKrahn3,5,andJan Medenbach1
1InstituteofBiochemistryI,UniversityofRegensburg,Universitaetsstrasse31,DͲ93053Regensburg,Germany2Instituteof Biochemistry,JustusͲLiebigͲUniversityGiessen,HeinrichͲBuffͲRing17,DͲ35392Giessen,Germany3InstituteforMolecular andCellularAnatomy,UniversityofRegensburg,Universitaetsstrasse31,DͲ93053Regensburg,Germany4Departmentfor StatisticalBioinformatics,UniversityofRegensburg,AmBioPark9,DͲ93053Regensburg,Germany5MedicalClinicD, UniversityofMuenster,Domagkstrasse3,DͲ48149Muenster
InDrosophilafemaledevelopmentisgovernedbyasingleRNAͲbindingprotein,Sexlethal(Sxl),that controlstheexpressionofkeyfactorsinvolvedindosagecompensation,germlinehomeostasisand theestablishmentoffemalemorphologyandbehaviour.Sxlexpressioninfemalefliesismaintained by an autoͲregulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now,itremainedunclearhowmalescompletelyshutdowntheSxlexpressioncascadeandprotect themselvesagainstrunawayproteinproduction.
HereweidentifytheproteinSisterofsexlethal(Ssx)asanantagonistofSxlautoͲregulatorysplicing. Sxl and Ssx have a comparable RNAͲbinding specificity and compete for RNA regulatory elements presentintheSxltranscript.AblationofthessxgeneresultsinalowlevelofproductiveSxlmRNA splicinginmaleflies;andinculturedDrosophilacells,SxlͲinducedchangestoalternativesplicingcan berevertedbytheexpressionofSsx.Insum,thisdemonstratesthatSsxsafeguardsmaleanimals againstSxlproteinproductiontoestablishastable,maleͲspecificgeneexpressionpattern.
99 P371
RNAͲbindingproteinsinthemalegametophyte
AlenaNáprstková,DavidHonys
LaboratoryofPollenBiology,InstituteofExperimentalBotanyASCR,v.v.i.,Rozvojová263,16502Prague6,Czech Republic;[email protected]
Pollen, an extremely reduced biͲcellular or triͲcellular male reproductive structure of flowering plants, serves as a model for numerous studies covering a wide range of developmental and physiological processes. The pollen development and subsequent progamic phase represent two fragileandvitalphasesofplantontogenesis,andpollenwasamongthefirstsingularplanttissues thoroughlycharacterisedatthetranscriptomiclevel.Posttranscriptionalcontrolofgeneexpression includingRNAaccumulationandstorageplaysavitalroleduringtobaccopollenmaturationandtube growth. Storage ribonucleoprotein particles were described in pollen and pollen tubes of tobacco andwereshowntocompriseribosomalsubunits,translationallysilentmRNAsandRNAprocessing proteins. Following our previous transcriptomic studies, we identified several members of RNAͲ bindingproteinfamilyinvolvedinRNAprocessingandmetabolism.Analysedproteinswereshownto localise to male gametophyte vegetative or sperm cells and roots. Here we present functional characterisationandtissueͲspecificlocalisationoffourfromsixpollenͲexpressedhomologs(RNP1Ͳ6) ingenerativetissues.
TheauthorsthankfullyacknowledgementthefinancialsupportfromCzechScienceFoundation(grantsNo.13Ͳ 06943Sand15Ͳ16050S).
100 P381
eIF2assemblybyCdc123:mechanismandlinktocellproliferation control
LeaNeumannͲArnold,SvenVanselow,FranziskaWojciechandWolfgangSeufert
UniversityofRegensburgInstituteofBiochemistry,GeneticsandMicrobiology
eIF2 is a central player in the initiation of mRNA translation. In its active, GTPͲbound form eIF2 recruitstheinitiatormethionylͲtRNAtotheribosome,participatesinmRNAscanningandstartcodon recognition. eIF2 also serves an important regulatory function as part of the integrated stress response, where phosphorylation of eIF2 reduces global protein synthesis, but upͲregulates the mRNA translation of a stressͲresponsive transcription factor gene. eIF2 is a complex of 3 protein subunits.Surprisingly,however,eIF2heterotrimerformationdoesnotoccurautonomouslyinvivo, butrequiresadedicatedassemblyfactorcalledCdc123,whichbindstheunͲassembledeIF2Ͳgamma subunit.Cdc123isconservedamongeukaryoticorganismsandessentialforcellviabilityinyeastand human cells. As indicated by recent structural studies, Cdc123 resembles ATPͲGrasp enzymes and binds ATP. To further define the mechanism by which Cdc123 assembles the eIF2 complex, we studiedvariousmutantversionsofeIF2Ͳgamma.ThealphaͲandbetaͲsubunitsbindtoseparatesites oneIF2Ͳgamma.Mutationofeithersitedisruptedbindingoftherespectivesubunit,butleftbinding of the other subunit intact. In contrast, a mutation in the Cdc123 binding site located in the CͲ terminaldomainIIIofeIF2Ͳgammadisruptedthebindingofboth,thealphaͲandbetaͲsubunits,to eIF2Ͳgamma.TheseandfurtherdatasuggestthatCdc123mayreshapeeIF2Ͳgammabyanallosteric mechanismtoexposethealphaandbetabindingsites.Interestingly,amutationineIF2Ͳgammaclose totheputativeCdc123bindingsitecausesacomplexneurologicaldiseaseinhumans.Experiments are under way to check potential assembly defects of this mutated human eIF2Ͳgamma version. Cdc123wasfirstidentifiedinmammalsduetoitsroleinsupportingcellproliferation.Weconfirmed thatCdc123isneededforcellcycleentryinyeast.Moreover,depletionofeIF2subunitscausedaG1Ͳ specificcellcyclearrestinyeast.Thus,normalratesofproteinsynthesisandeIF2functionappearto beofparticularimportanceforcellcycleentry.TobetterunderstandtheeIF2Ͳcellcyclelinkage,we aimtodeterminehowreducedeIF2functionaffectsthetranslationofvariousmRNAsinyeastby RNAsequencingofpolysomeͲassociatedmRNAs.
101 P391
TheS.pombemRNAdecappingcomplexrecruitscofactorsandan Edc1Ͳlikeactivatorthroughasingledynamicsurface
OverbeckJH,WurmJP,SprangersR
UniversitätRegensburg
Theremovalofthe5'7ͲmethylguanosinemRNAcapstructure(decapping)isacentralstepinthe5'Ͳ 3'mRNAdegradationpathwayandisperformedbytheDcp1:Dcp2decappingcomplex.Theactivity of this complex is tightly regulated to prevent premature degradation of the transcript. Here, we establish that the aromatic groove of the EVH1 domain of Schizosaccharomyces pombe Dcp1 can interact with prolineͲrich sequences in the exonuclease Xrn1, the scaffolding protein Pat1, the helicaseDhh1,andtheCͲterminaldisorderedregionofDcp2.WeshowthatthisregionofDcp1can also recruit a previously unidentified enhancer of decapping protein (Edc1) and solved the crystal structureofthecomplex.NMRrelaxationdispersionexperimentsrevealthattheDcp1bindingsite can adopt multiple conformations, thus providing the plasticity that is required to accommodate different ligands. We show that the activator Edc1 makes additional contacts with the regulatory domainofDcp2andthatanactivationmotifinEdc1increasestheRNAaffinityofDcp1:Dcp2.Our datasupportamodelwhereEdc1stabilizes theRNAintheactivesite,whichresultsin enhanced decapping rates. In summary, we show that multiple decapping factors, including the Dcp2 CͲ terminalregion,competewithEdc1forDcp1binding.Ourdatathusrevealanetworkofinteractions thatcanfineͲtunethecatalyticactivityofthedecappingcomplex.
102 P401
EvolutionofRNAͲbindingproteinsacrossdifferentyeastspecies
MerveÖztürk&FalkButter
InstituteofMolecularBiology,Ackermannweg4,55128Mainz,Germany
Unlike prokaryotic cells, where translation is directly coupled to transcription; eukaryotic cells are compartmentalized.Thegeneticmaterialispreservedinsidethenucleus,andmRNAsaresubjectto severalpostͲtranscriptionalprocessesbeforebeingtranslatedintoproteinsinthecytoplasm.RNAͲ bindingproteins(RBPs)arekeyregulatorsofthesetightlycontrolledgeneregulatorynetwork.RBPs areabletorecognizeRNAͲrecognitionelements(RREs)ontheirtargetRNA,whichmayrangefrom sequence motifs to structured threeͲdimensional folds. Thereby RBPs influence gene expression postͲtranscriptionallybyregulatingthe“fate”oftheRNAatdifferentlevels:transcription,capping, splicing,polyadenylation,cellularlocalization,translation,andRNAturnover.Inthelastyearsnew methods were established to identify RBPs in vivo. We here apply the previously developed interactome capture approach to identify RBPs across a monophyletic group of 10 different yeast species covering 500 million years ofevolution. Because of their relatively low complex genomes, yeasthavebeenawidespreadmodelsysteminbiology.However,whileallspeciesoriginatefroma commonancestor,theyhavetakendifferentdirectionsfortheirpostͲtranscriptionalgeneregulation. For example, S. pombe preserved a more complex splicing machinery and alternative splicing patternsnotfoundinS.cerevisiae.Acomparativestudybasedoninteractomecapturedataallowsto characterize RNAͲbinding proteins by their ability to bind mRNA rather than just based on overall sequencehomology.Wehavepreviouslyshownbyphylointeractomicsthatsequencehomologydoes not necessarily equate to functional homology and a few amino acid exchanges can change a proteins’capabilitytobindtonucleicacids.Besidesidentificationofdivergentmolecularevolution, wealsoaimtouncovernewRBPsbasedonevolutionaryconservation,andtotracetheevolutionof RBPsandtheirRBDsacrossseveralspecies.
103 P411
TranscriptomeanalysisofmolecularsubtypesofGliomastemcells revealdifferentialexpressionofcircRNAsandlncRNAs
BalagopalPai,ArabelVollmann,GerhardLehmann,PeterHau,GunterMeister
FakultätfürBiologieundVorklinischeMedizin,UniversitätRegensburgKlinikundPoliklinikfürNeurologie,Klinikumder UniversitätRegensburg
WorldHealthOrganizationclassifiesGlioblastomamultiforme(GBM)asGradeIVastrocytoma,witha median survival rate of just 14 to 16 months. The solid tumours in GBM constitutes of a highly heterogeneous population of cells. Among these are a small group of cells termed brain tumour initiatingcells(BTICs)orcancerstemcells(CSCs).Theseareoftenthoughttobethecontributorsof resistancetowardsradiationandchemotherapyandhencerelapse.TheBTICsoftencontainahigher population of CD133 expressing cells; this can be used to enrich the stem cellͲlike cells. Recent studiesalsorevealedthatGBMfromvariouspatientscouldbeclassifiedintofourmolecularsubtypes basedonmolecularsignaturesandmutations.Thesemolecularsubtypeshavedifferentdegreesof response to therapy and hence varying degrees of survival rates among patients. Contribution of nonͲcodinggenometowardssuchacomplexdiseasestillrequiresinvestigation.Inthepresentstudy, patientsampleswereclassifiedintothetwomajorsubtypes,MesenchymalandProneural,basedon marker gene expression. With special, focus on long nonͲcoding RNAs (lncRNAs) and the circular RNAs (circRNAs) us performed RNA sequencing on cells raised from glioma patients. Primary cells raised in culture were sorted into CD133+ and CD133Ͳ cells or differentiated inͲvitro. A whole transcriptome analysis of these fractions revealed the presence of lncRNAs with differential expression among subtypes and fractions. Unlike lncRNAs only a very few circRNAs exhibited differential expression. We are currently performing additional validation in additional cells from patientsamplesandfunctionalcharacterisationonselectlncRNAsandcircRNAs
104 P421
ThededicatedchaperoneAcl4escortsRpl4toitsnuclearpreͲ60S assemblysite
BenjaminPillet,PatrickPausch,LaurentFalquet,GertBange,JesúsdelaCruzandDieterKressler
UniversityofFribourg
Ineukaryoticcells,mostribosomalproteinsneedtobetransportedfromthecytoplasmwherethey are synthesised to their ribosome assembly site in the nucleus. Due to the high abundance and difficultphysicochemicalpropertiesofrͲproteins,theircorrectfoldingandfailͲsafetargetingtothe assembly site depends largely on general, as well as highly specialized, chaperone and transport systems.ManyrͲproteinscontainuniversallyconservedoreukaryoteͲspecificinternalloopsand/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicatedchaperonesinagrowingnumberofcases.The60SrͲproteinRpl4isparticularlyinteresting since it harbours a conserved long internal loop and a prominent CͲterminal eukaryoteͲspecific extension. Here we show that both the long internal loop and the CͲterminal eukaryoteͲspecific extensionarestrictlyrequiredforthefunctionalityofRpl4.WhileRpl4containsatleastfivedistinct nuclear localization signals (NLS), the CͲterminal part of the long internal loop associates with a specificbindingpartner,termedAcl4.AbsenceofAcl4confersasevereslowͲgrowthphenotypeanda deficiencyintheproductionof60Ssubunits.Nevertheless,Acl4isnotessentialtoproduceribosomes andstrainslackingAcl4quicklyaccumulatemutationsthatcompletelysuppresstheirslowgrowth phenotype. By studying these mutations, we hope to uncover regulation pathways, protection mechanismsorassemblymechanismsofRpl4
105 P431
ReconstitutionofthePolIpreinitiationcomplexinS.cerevisiae
PilslMichael,BöhmMaria,GrafSaskia,SteinbauerRobert,GadalOlivier,GriesenbeckJoachim, MilkereitPhilipp,TschochnerHerbert
UniversitätRegensburg,BiochemieͲZentrumRegensburg(BZR),LehrstuhlBiochemieIII,93053Regensburg,Germany
Infastgrowingcells,upto60%oftotaltranscriptionisdevotedtoribosomalRNA(rRNA)synthesis.In eukaryotes a specialized enzyme, RNA polymerase I (Pol I), synthesizes a polycistronic precursor rRNAwhichisthe35SrRNAintheyeastS.cerevisiae.Thisprimarytranscript,isprocessedtothe mature18S,5.8Sand25SrRNAs.ThetranscriptioninitiationfactorCF(corefactor)togetherwithPol IandthePolIͲboundinitiationfactorRrn3supportPolIͲpromoterdependentinvitrotranscriptionat a basal level. Binding of UpstreamͲActivatingͲFactor (UAF) and TATAͲBindingͲProtein (TBP) to the upstreampromoterelement(UE)enhancePolItranscriptioninitiationinvitroandarerequiredfor Pol IͲdependent rRNA synthesis in vivo. Recent studies presented highͲresolution structures of a minimal Pol IͲRrn3ͲCF complex, leading to hypotheses about the mechanism of Pol I transcription initiation.Nevertheless,structuralinformationaboutthecompletepreͲinitiationcomplexislacking and the detailed mechanism how Pol I recognizes its promoter and how it achieves its high transcriptionaloutputremaintobefurtherinvestigated.ItwassuggestedthatUAF,TBPandCFbind thepromoterandformaplatformtowhichRrn3boundPolIisrecruited.Earlierstudiesassigned positionsͲ38to+8relativetothetranscriptionstartsiteascorepromoterelement(CE)responsible forCFbindingandstartsiteselection,andtheactivatingupstream(cis)ͲelementUEfromͲ155toͲ39. Wedevelopedareconstitutedinvitrotranscriptionsystemusinghighlypurifiedfactorssupporting promoterͲdependenttranscriptionandusegelshiftassaystobetterdefineDNAinteractionsitesand functionalcisͲelementsofthePolIpromoter.WewanttounderstandtheconcertedassemblyofUAF andTBPandtheirinteractionwithCF,Rrn3andPolItoformthefullPolIpreinitiationcomplexas wellastheirroleinstimulatingPolIactivity.WediscussfunctionalrequirementsofPolItoinitiate transcriptionconsideringthespecificroleofPolItranscriptionfactorUAFandTBP.
106 P441
WhenapreͲribosomehitsaroadblock:Usinginhibitorstostudythe maturationofpreͲribosomalparticles
MichaelPrattes,GertrudeZisser,ChristinaMauerhoferandHelmutBergler
InstituteofMolecularBiosciences,UniversityofGraz,8010Graz,Austria
Eukaryoticribosomebiogenesishasemergedasoneofthemostenergyconsumingandelaborate cellular pathways. More than 200 transͲacting factors are needed to guarantee the correct processingandassemblyoftheindividualRNAandproteincomponentsofamatureribosome.Allof thesefactorshavetoactinatimelyandspatiallystrictlyconcertedmannertobeabletoproducea functioningribosomeattheendoftheassemblyline.Oneofthemajorchallengesthatslowsdown theelucidationofthispathwayisitsfastpacesincethecompletematurationoftherRNA,themajor component of the ribosome, can be finished in minutes. Most available genetic and biochemical techniques only allow rather static pictures of individual steps representing only a glimpse of the dynamicsandtemporalorderofevents.Thereforewemakeuseoflowmolecularweightinhibitors to induce roadblocks at specific points in the maturation of the ribosomal subunits. This allows a focusedviewonthefinalprocessingstepsoftheparticlesdownstreamoftheblock.Amongothers thisenablesustogainnewinsightsintheformationofexportcompetentpreͲribosomes.
107 P451
Guardiansoftheribosomalproteins
IngridRössler,JuliaEmbacher,ValentinMitterer,JuliaUnterluggauer,BrigittePertschy
UniversityofGraz,Austria
Theribosome,anoutstandingmolecularmachine,synthesizesallcellularproteinsandisassembled aswellasmaturedinacomplexprocessinvolvingmorethan200ribosomebiogenesisfactors.Inthe course of this process, most ribosomal proteins have to be transported into the nucleus and incorporated into the preͲribosomal particles. Thereby, chaperones and importins protect the ribosomalproteinsfromaggregationandguardthemfromthecytoplasmthroughtheNuclearPore Complexintothenucleus.
Up to the present only a few chaperones are identified that show an interaction with ribosomal proteins,butitisstillunknownhowchaperonesescorttheseproteinsefficientlytotheirlocationof assembly.Inthisstudy,wearelookingforunknownspecificchaperones,importinsandothernonͲ ribosomalinteractionpartnersofribosomalproteinstogainfurtherinsightsintothemechanismof their assembly path. All ribosomal proteins of the 40S subunit were purified via Tandem Affinity Purificationandpotentialinteractionpartnerswereidentifiedbymassspectrometry.Subsequently, the most promising candidates, including importins and chaperones, are characterized using biochemical, genetic and cell biological methods to verify the interplay between the interaction partneranditsribosomalprotein.Here,wewanttoexemplarilypresentpotentialnewguardiansof ribosomalproteins.
108 P461
TranslationalreprogrammingunderERstress
StefanReich1,SoniHimanshu2,ChiNguyen3,SaschaSteltgens4,RobertAhrends3,ChristianeKnobbeͲ Thomsen4,BjörnTews2,GrischaTödt5,JanMedenbach1
1InstituteofBiochemistryI,UniversityofRegensburg,Universitaetsstrasse31,DͲ93053Regensburg,Germany2Department ofMolecularMechanismsofTumorInvasion,DeutschesKrebsforschungszentrum,ImNeuenheimerFeld581,69120 Heidelberg,Germany3InstituteofLipidomics,ISASe.V.,OttoͲHahnͲStraße6b,44227Dortmund,Germany4Institutefor Neuropathology,UniversitätsklinikumDüsseldorf,Moorenstrassse5,40225Düsseldorf,Germany5DepartmentofStructural andComputationalBiology,EMBLHeidelberg,Meyerhofstrasse1,69117Heidelberg,Germany
Endoplasmic reticulum (ER) stress triggers a signaling cascade to cope with the accumulation of misfoldedproteins:theunfoldedproteinresponse(UPR).Itaimstoreinstatecellularhomeostasisby adjustingtranscriptionandtranslation.Ifcellularhomeostasiscannotbereinstated,theUPRtriggers apoptosis.
Inrecentyears,theUPRhasgainedwidespreadattentionduetoitsinvolvementinmanydifferent diseases such as diabetes, Alzheimer’s, or various types of cancer. In most cancers, due to rapid growthandelevatedproteinsynthesisinnutrientlimitedandhypoxicenvironment,tumorcellsoften exhibithighlevelsofERstresswhichactivatestheUPR.TumorcellsexploittheproͲsurvivalsignalsof the UPR while avoiding apoptosis even under continuous ER stress conditions. This has also been linkedtoresistanceofchemotherapeutictreatment(e.g.ingliomas).
Despiteitsbroadclinicalimportance,qualitativeandquantitativemodelsthatdescribetheUPRin cancer cellsarestillmissing.Theaimofourconsortium (SystemsBiologyoftheUnfoldedProtein ResponseinGlioma,SUPRͲG)istoprovidethesemodelsbysystemͲwideanalysesoftheUPR.Inour projectwefocusonUPRͲdependenttranslationalreprogramminginvariousdifferentcelllinesand under different conditions. To this end we globally analyze translation by employing ribosomal profiling, aiming to establish a quantitative model of the UPR in glioma to reveal potential therapeuticcandidates.
109 P471
RNAmatchmakers:exploringtheRNAinteractomeofeuryarchaeal Lsmproteins
RobertReichelt,SarahWillkomm,SarahStöckl,WinfriedHausnerundDinaGrohman
LehrstuhlfürMikrobiologie&ArchaeenzentrumUniversitätRegensburg
SmandSmͲlike(Lsm)proteinsarekeyplayersinRNAmetabolismandarefoundinalldomainsoflife. WhereaseukaryoticandbacterialLsmhomologueshavebeenextensivelystudied,thefunctionof Lsmproteinsfromthearchaealdomainoflifeisnotwellunderstood.Ingeneral,Sm/Lsmproteins areknownasRNAbindingproteinsinvolvedinRNAbiogenesisandsmallRNAmediatedregulationof geneexpression.ToshedlightonthemechanismsthatenablesSm/Lsmproteinstoassociatewtih with multiple RNA interaction partners, we are studying members of the Lsm family from two hyperthermophilic archaeal organisms: Methanocaldococcus jannaschii (Mja) and Pyrococcus furiosus (Pfu). While the PfuLsm protein resembles a heptamer and is a typical representative of archaealSmͲlikeproteins,thehexamericMjaLsmproteinismorecloselyrelatedtobacterialHfq,a protein known as RNA chaperone that facilitates interactions between small RNAs (sRNAs) and messenger RNAs (mRNAs). Therefore, besides analysing the mechanism of interaction, we aim to identifyRNA interactionpartnersthat associatewiththearchaealPfuLsmand MjaLsmvariantsto gaininsightsintodifferencesandsimilaritiesaswellasintothebiologicalfunctionofLsmproteinsof thesecloselyrelatedeuryarchaealorganisms.Tothisend,werecombinantlyexpressedPfuLsmand MjaHfqinE.coliandpurifiedthemultimericproteinsviaastrepͲtag.Thisallowedustoi)carryouta biochemical analysis of the RNA binding and RNA chaperon functions and ii) to coͲpurify RNA interactionpartnersbyincubatingtheproteinswithtotalcellularRNAisolatedfromtherespective organismsfollowedbyhighͲthroughputsequencing.Additionally,wefollowaexperimentalstrategy thatexploitsthefactthatP.furiosus(unlikeM.jannaschii)isoneofthefewarchaealorganimsthat canbegeneticallymanipulated.WecreatedamutantP.furiosusstrainthatconstitutivelyexpresses strepͲtaggedPfuLsm.ThisallowsustocoͲpurifyandsequenceRNAsboundtoPfuLsminthecellular context and to compare this dataset to the RNA interactome data of the recombinant PfuLsm variant.
110 P481
DifferentialexpressionofpromoterandenhancerͲassociatedlong nonͲcodingRNAsandtheirneighboringproteinͲcodinggenesin diabeticnephropathy
SimoneReicheltͲWurm,DominikChittka,LauraLennartz,KathrinEidenschink,SebastianBeck, BernhardBanas,MiriamCBanas
UniversityHospitalofRegensburg,DepartmentofNephrology
The discovery of prokaryotic adaptive immune systems consisting of the so called CRISPR–Cas (clustered regularly interspaced short palindromic repeats (CRISPR)Ͳassociated) proteins and their applicability for genome editing in eukaryotes is one of the most exciting biochemical advances withinthepastyears.AmongthemanyCRISPRsystemvariants,thetypeIICRISPRsystemismost suitableforgeneeditingapplications.Itcontainsasingleeffectorprotein,theendonucleaseCas9, whichassociateswithanRNAduplexconsistingofaCRISPRRNA(crRNA)andatransͲactingCRISPR RNA(tracrRNA)tospecificallycleavedoubleͲstrandedDNA(dsDNA)targetscomplementarytothe crRNA.Additionaltosequencecomplementarity,aprotospaceradjacentmotif(PAM)isrequiredfor efficient target recognition and cleavage. For genome editing applications, single guide RNAs (sgRNA),whicharechimerasofcrRNAandtracrRNAarecommonlyusedtoreducethecomplexityof heterologousexpressionineukaryoticcells.Foreditingpurposes,sgRNAͲassociatedCas9needstobe efficientlyimportedintothenucleustoencounteritstargetsequence.However,withinthenucleus, genomic DNA is seldom easily accessible but often tightly packed within chromatin regions. The wrappingandbendingofDNAinducestensionsonthedsDNAandthismightconstituteadecisive factorforCas9efficiencyandaccuracy.
We employed our recently developed DNA origamiͲbased nanoscopic force clamp (Nickels et al., 2016)toanalyzewhethertheextentofstrainonDNAinfluencesCas9binding.Thisapproachallows high throughput force measurements in the biological relevant piconewton range on the singleͲ moleculelevel.Usingthenanoscopicforceclamp,weexertedadefinedforceonthetargetDNAand monitoredCas9bindingonthesingleͲmoleculelevelusingafluorescenceresonanceenergytransfer (FRET)signalbetweenthedonorͲlabeledtargetDNAandtheacceptorͲlabeledCas9ͲboundcrRNAas readout.WecanshowthatthebindingbehavioroftheCas9ͲsgRNAtoitstargetDNAchangesina forceͲdependentmannerandthathighforcesabolishCas9ͲsgRNAbindingtoitstargetDNA.These resultssuggestthatCas9isaforceͲsensitiveenzymeandthatstrainedDNAregionsinthegenomic DNAwilllessefficientlyrecognizedbyCas9.
111 P491
Reversibleproteinaggregationisaprotectivemechanismtoensure cellcyclerestartafterstress
ShadySaad,GeaCereghetti,YuehanFeng,PaolaPicotti,MatthiasPeter,ReinhardDechant
InstituteofBiochemistry,DepartmentofBiology,ETHZürichandLifeScienceZürich,PhDProgramforMolecularLife Sciences
Protein aggregation is usually considered as an irreversible process responsible for several pathologies. However, recent evidence suggests that the aggregation of an increasing number of proteins is a reversible, highlyͲregulated physiological mechanism used by cells to adapt to stress conditions. Despite these findings, the molecular mechanisms underlying reversible protein aggregationanditsrelationtoirreversibleaggregationhaveremainedelusive.Here,wecharacterize themechanismscontrollingreversibleaggregationofyeastpyruvatekinaseCdc19uponstress.We showthataggregationofCdc19isregulatedbyoligomerizationandbindingtoallostericregulators. Moreover,weidentifyaregionoflowcompositionalcomplexity(LCR)withinCdc19thatisnecessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while deͲ phosphorylationoftheLCRisnecessaryforreversibleaggregationuponstress.Importantly,wealso demonstratethatCdc19aggregationtriggersitslocalizationtostressgranules,andmodulatestheir formation and dissolution. Moreover, recruitment to stress granules protects Cdc19 from stressͲ induced degradation, suggesting that stress granules act as storage compartments for proteins necessary to restart the cell cycle after stress relief. As LCRs are also responsible for irreversible aggregation of proteins causing several diseases, we predict that understanding the molecular mechanismsregulatingreversibleproteinaggregationthroughLCRswillprovideimportantfunctional insightsintotheregulationofproteinaggregationinnormalphysiologyandindiseases.
112 P501
FunctionalandstructuralanalysesofnativelypurifiedrDNA chromatindomainsfromS.cerevisiae
SchächnerC.,PilslM.,MaierA.,LiS.,HermansN.,vanNoortJ.,TschochnerH.,MilkereitP., GriesenbeckJ.
LehrstuhlBiochemieIII.UniversitätRegensburg
RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the main contributor to transcription in proliferating eukaryotic cells. Pol I produces rRNA precursor molecules which are finally processed into the 3 largest ribosomal RNAs (rRNAs) forming the structural scaffold of the ribosome. To reach the high transcriptional output Pol I is a specialized enzyme with an efficient transcriptionmachinery.Additionally,rRNAgenesarepresentinseveralhundredcopiesineachcell. Interestingly,notallofthesecopiesaretranscribed.Individingyeastcellsabout50%oftherRNA genes are kept in a nucleosomal, transcriptionally inactive state (“closed”). The other half of the copiesareusuallyheavilytranscribedandlargelydevoidofnucleosomes,whichisreferredtoasthe “open”chromatinstate.Thedifferentchromatinstatescanundergodynamicchanges.Replication, forexample,convertsrDNAcopiesinthe“closed”chromatinstate,whereastranscriptionofPolIis requiredtoconvert“closed”into“open”copies.Withourresearch,weaimtoelucidatetheinterplay between Pol I transcription and rDNA chromatin structure. We chose S. cerevisiae as model organism,sincethereisarichknowledgeabouttheinterrelationshipbetweenPolItranscriptionand chromatin structure in vivo. To analyse these processes in molecular detail, we established a purification approach to isolate native rDNA chromatin and analyse it in its composition and structureinvitro(Hamperletal.2014).Previousanalyseswerelimitedbythefactthattheisolated rDNAchromatindomainswereamixtureofbothchromatinstates,andthatthenativechromatin had not been rigorously used in functional assays. Here we present strategies to separate both chromatin states biochemically. We show preliminary results of single molecule analyses yielding insights into heterogeneity and individual properties of the rDNA chromatin states. Besides, we presentfirst resultsofin vitrotranscriptionexperimentsfrom native chromatin templatesusinga minimalpurifiedPolItranscriptionsystem.Webelievethattheuseofadefinedinvitrosystemwill addtoourknowledgeaboutanimportantbiologicalprocess
113 P511
AssemblyanddisassemblyoftheSSUprocesome
ChristinaSchmidt,MichaelBrummerandJorgePerezͲFernandez
LehrstuhlBiochemieIII.UniversitätRegensburg
Thesynthesisofribosomesisanessentialprocessinallgrowingcells.Duringribosomebiogenesis, morethan200assemblyfactors(AFs)facilitatetheprocessingandfoldingoftheribosomal(r)RNAas wellastheincorporationoftheribosomalproteins.Inyeastcells,therRNAconstitutesmorethan 60% of the transcribed RNA, and around 80% of total cellular transcription is dedicated to the synthesisofribosomes.Therefore,ribosomebiogenesisconsumesmostofthecellularresourcesand requiresatightregulation.
The correct production of the small subunit involves the formation of the SSU processome. The assembly of the SSU processome requires the transient association of more than 50 AFs with the nascent RNA. The AFs establish hierarchical interdependencies for their association in the SSU processome.However,themolecularmechanismsbehindthehierarchicalrecruitmentofAFsarenot clear. Some of these AFs have been found in large protein complexes not engaged in ribosome assembly.Amongthem,theUTPͲA/tͲUTPcomplexhasbeencharacterizedastheprimarybinderof thenascentrRNA.
Interestingly, the protein composition of the UTPͲA/tͲUTP complex is not clear. Among the UTPͲA components,Pol5hasnotbeenidentifiedin the proteomesofSSU processomepurified particles. AlthoughPol5hasbeenrelatedwiththesynthesisofribosomesitsroleduringribosomebiogenesis hasnotbeencharacterized.WepresentadetailedfunctionalanalysisofPol5onpreͲrRNAprocessing andsubunitassembly.OurresultssuggestaroleofPol5inthesynthesisofbothribosomalsubunits. We discuss possible roles of Pol5 in recycling the UTPͲA complex and adjusting the production of smallandlargesubunits.
114 P521
Characterizationofthecooperativefunctionofthe METTL3/METTL14methyltransferasecomplexandinvestigationof theputativeRNAmethyltransferaseMETTL8
E.Schöller1,F.Weichmann1,T.Treiber1,N.Treiber1,A.Bruckmann1,R.Hett1,R.Merkl1,R.Feederle2, E.Kremmer2andG.Meister1
1UniversityofRegensburg,Universitätsstraße31,93053Regensburg2HelmholtzZentrumMünchen,Ingolstädter Landstraße1,85764Neuherberg
Morethan hundred differentpostͲtranscriptionalRNAmodificationscanbefoundinallclassesof RNA.ProcessingandmodificationstepsinmRNAsinclude5`capping,additionofthepoly(A)tail, splicingaswellastheincorporationofbasemodificationssuchasN6Ͳadenosinemethylation(m6A) whoseinvestigationwasreignitedinthelastfewyears.Dynamicm6Amodificationsareinvolvedin fundamental cellular processes including gene expression, meiosis and stemness by regulating translation as well as inducing mRNA instability. The m6A landscape in mRNA is catalyzed by a heterodimericcorecomplexcomposedofMETTL3ͲMETTL14togetherwiththesplicingfactorWTAP. HereweconfirmthatMETTL3isthecatalyticallyactivesubunit,whiletheRGGdomainofMETTL14is essential for substrate recognition. Furthermore, we show that the phosphorylation site S399 of METTL14 does not influence the interaction between METTL3 and METTL14 neither methylation activityofthiscomplex.Interestingly,phylogeneticanalysisindicatesaclusteroftheMETTLproteins 2,6and8,whichmightrepresentafunctionallyrelatedclassofmethyltransferasesactingonRNA.So far,thefunctionsandthebiologicalrelevanceoftheseproteinsareunknownandthusweplanto characterizethemethyltransferaselikeprotein8inmoleculardetailbyusingdifferentbiochemical assays.TobetterunderstandthebiologicalroleoftheMETTL8,weestablishmonoclonalantibodies againstthisMETTLprotein,FlpͲIn™TͲRex™celllinesandgeneknockoutbytheCRISPR/Cas9system. Thistoolboxwillbeidealstartingpointstoobtainindicationsofbindingpartners,promisingtarget RNAsandpotentialconsensusbindingsequences.
115 P531
Asynergisticnetworkofinteractionspromotestheformationofin vitroprocessingbodiesandprotectsmRNAagainstdecapping
StefanSchütz,ErikNöldeke,RemcoSprangers
UniversityofRegensburg
Cellularliquid–liquidphaseseparation(LLPS)resultsintheformationofdynamicgranulesthatplay animportantroleinmanybiologicalprocesses.Onamolecularlevel,theclusteringofproteinsintoa confinedspaceresultsfromanindefinitenetworkofintermolecularinteractions.Here,weintroduce and exploit a novel highͲthroughput bottomͲup approach to study how the interactions between RNA,theDcp1:Dcp2mRNAdecappingcomplexandthescaffoldingproteinsEdc3andPdc1resultin the formation of processing bodies. We find that the LLPS boundaries are close to physiological concentrationsuponinclusionofmultipleproteinsandRNA.Withininvitroprocessingbodiesthe RNA is protected against endonucleolytic cleavage and the mRNA decapping activity is reduced, which argues for a role of processing bodies in temporary mRNA storage. Interestingly, the intrinsicallydisorderedregion(IDR)intheEdc3proteinemergesasacentralhubforinteractionswith bothRNAandmRNAdecappingfactors.Inaddition,theEdc3IDRplaysaroleintheformationof irreversibleproteinaggregatesthatarepotentiallydetrimentalforcellularhomeostasis.Insummary, our data reveal insights into the mechanisms that lead to cellular LLPS and into the way this influencesenzymaticactivity.
116 P541
PlasmodiumfalciparumNucleosomesExhibitReducedStabilityand LostSequenceDependentNucleosomePositioning
ElisabethSilberhorn1,UweSchwartz1,PatrickLoeffler2,SamuelSchmitz2,AnneSymelka1, TaniadeKoningͲWard3,RainerMerkl2,GernotLaengst1
BiochemistryIII;BiochemistryCentreRegensburg(BCR),UniversityofRegensburg,Regensburg
The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased diͲnucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by ATͲrepeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its ATͲrich genome. Human and parasite histones bind with higher affinity to GCͲrich DNA and with lower affinity to ATͲrich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GCͲrich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest thatthebiochemicalpropertiesoftheparasitehistonesaredistinctfromthetypicalcharacteristics of other eukaryotic histones and these properties reflect the increased accessibility oftheP.falciparumgenome
117 P551
Newplayersinvolvedinplantfertilization
NicoleSpitzlbergerandAndreaBleckmann
CellBiologyandPlantBiochemistry,BiochemieͲZentrumRegensburg,UniversityofRegensburg,DͲ93053Regensburg, GermanyEͲmail:[email protected]
Thelaunchofseeddevelopmentinfloweringplantsisinitiatedbytheprocessofdoublefertilization. Two immobile sperm cells are delivered by the vegetative pollen tube towards the female gametophytebyguidanceandattractionmechanisms.Afterpollentubeburstthetwospermcells arereleaseandfusewiththeeggandthecentralcelltoformtheprecursorcellsofthetwomajor seedcomponents,theembryoandendosperm,respectively.Theunderlyingmechanismsinvolvedin pollentubeguidanceandspermcellreleasearenotfullyunderstoodandwearelookingfornew regulatoryelements.Sofar,wecouldidentifytwopeptides,expressedbythesynergids,themajor producerofpollentubeattractants,whichmightbeinvolvedinpollentubeguidanceorspermcell release.Bothpeptideslocalizetothefiliformapparatusandarelikelytobesecreted.Tounraveltheir functionsduringtheprocessofpollentubeguidanceanddoublefertilization,wewillgenerateand characterizemutantsandusesynthesizedpeptidesinfertilizationexperiments.
118 P561
FunctionalcharacterizationofanRNaseIIIͲprocessedsmallRNA withanantisensepartnerinthefoodͲbornepathogen Campylobacterjejuni
SarahL.Svensson,MonaAlzheimer,GauravDugar,andCynthiaM.Sharma
InstituteofMolecularInfectionBiologyMolecularInfectionBiologyIIUniversityofWürzburg,Würzburg,Germany
TranscriptomemappingusingdeepsequencinghasrevealedmanyconservedcandidatesmallRNAs (sRNAs) in Campylobacter jejuni (1), a major cause of foodborne illness, as well as in the related gastric pathogen Helicobacter pylori (2). These sRNAs represent an uncharacterized layer of postͲ transcriptional regulation that might control pathogenesis. Here, we are characterizing a pair of overlappingantisensesRNAsofC.jejuni,CJͲRepGandantisenseͲCJͲRepG(asͲCJͲRepG).Weshowthat CJͲRepG represents a functional homologue of H. pylori RepG (HPͲRepG), an sRNA which directly targetsalengthͲvariableGͲrepeatinatargetmRNA5’UTRandtherebylinksphasevariationtopostͲ transcriptional regulation (3). Deletion of the locus encoding CJͲRepG and asͲCJͲRepG upregulates PtmG,anenzymeinthelegionaminicacidflagellinͲglycosylationpathway.Regulationinthedeletion straincanbecomplementedbyeitherCJͲRepGalone(withoutitspartnerasͲCJͲRepG)orbyHPͲRepG. CJͲRepG and HPͲRepG directly interact with a GͲrich sequence near the ribosomal binding site of ptmG mRNA, suggesting an analogous regulatory mechanism. Although CJͲRepG and HPͲRepG appear to be functionally related sRNAs that target GͲrich sequences, they have very different biogenesis pathways. While HPͲRepG is transcribed as a mature primary transcript from a standͲ alonegene,CJͲRepGisgeneratedbyprocessingandalsohasaprocessedantisensepartner.BothCJͲ RepGandasͲCJͲRepGareprocessedbyRNaseIIItosmallersRNAsderivedfromthe3’Ͳand5’Ͳendsof their primary transcripts, respectively.Processing of asͲCJͲRepG, but surprisingly not of CJͲRepG, requires its antisense partner. CJͲRepG is also expressed from two promoters to produce primary transcripts with different 5’Ͳend lengths that are processed to the same 3’Ͳderived ~70Ͳnt sRNA product.UnderstandingthebiogenesisofCJͲRepGanditsantisensepartnerwillprovideinsightinto RNaseEͲindependentsRNAprocessing,aswellashowsRNAsthemselvesmightbetranscriptionally andpostͲtranscriptionallyregulatedbymultiplesignals.VaryingconservationoftheasRNApromoter in Campylobacter raises questions about the function of the asRNA and whether it can regulate additionaltargetgenesindependentofCJͲRepG.Finally,CJͲRepGanditstargetptmGtargetaffectC. jejuniͲcoloniccellinteractionsinanewtissueͲengineeredmodelforstudyingintestinalpathogens, indicatingaroleforthesRNAinvirulence.
119 P571
StructuralandFunctionalStudiesontheRoleofNoc3pforLarge RibosomalSubunitMaturationinSaccharomycescerevisiae
FabianTeubl,ThomasHierlmeier,JanLinnemann,GiselaPöll,JoachimGriesenbeck,Herbert TschochnerandPhilippMilkereit
UniversityofRegensburgFacultyforBiologyandPreclinicalMedicineBiochemistryIIIProf.Dr.HerbertTschochnerWorking GroupMilkereit
Ribosomesarecomplexandhighlyconservedribonucleoproteinparticlesthattranslateincellsofall domains of life messenger RNA into proteins. During ribosome production ribosomal RNA (rRNA) precursorsundergoextensiveprocessingandfoldingeventsandassemblewithalargenumberof ribosomal proteins (rͲprotein) in a highly dynamic and defined manner. A multitude of small nucleolar RNAs (snoRNA) and biogenesis factors transiently interact with the maturing eukaryotic preͲribosomal particles. Most of these factors are conserved from yeast to human and play importantrolesinribosomematuration.Examplesarethe“NocͲproteins”Noc1pandNoc3pwhich containacommonhomologydomainandwhichareessentialinyeastforearlytointermediatesteps oflargeribosomalsubunitrRNAmaturation.Here,weanalyzedtheeffectofNoc3pinvivodepletion ontheassemblyofyeastlargeribosomalsubunitprecursors.Furthermore,weinvestigatedhowthe rͲprotein assembly state influences preͲribosomal recruitment and release of Noc3p. Our findings indicatethatNoc3pcoordinatesrRNAprocessingwithsubunitassemblyeventsinintermediatelarge ribosomal subunit precursors. We are currently studying by tertiary structure probing approaches possible structural transitions accompanying these Noc3p mediated maturation events and will reportontheresults.
120 P581
Animproved,versatileRNAbindͲandͲseqmethodfordirect assessmentofRBPspecificity
ThomasTreiber,NoraTreiber,GerhardLehmann,NorbertEichnerandGunterMeister
BiochemieZentrumRegensburg,BiochemieI,UniversitätRegensburg,
RNAbindingproteins(RBPs)playcrucialrolesintheregulationofgeneexpression,RNAmetabolism and the response to viruses. Most RBPs contain RNA binding domains (RBDs) that recognize a coherentshortsequencemotifontheboundRNAandthusdefinethespecificityoftheinteraction. Frequently,RBPsconsistofamodulararrayofmorethanoneRBD,whichleadstogreaterflexibility ofbindingmodes.AmongthemostcommonRBDsaretheRNArecognitionmotif(RRM),zincfingers, KHͲdomains and cold shock domains. Although the binding specificity of an increasing number of RBPs has been elucidated in recent years, the binding mode and specificity of the majority of canonicalandevenmoresoofnonͲcanonicalRBPsremainsunknown.
Several techniques for the unbiased experimental assessment of the binding preferences of RBPs have been described including SELEX, RNA compete and RNA bind and seq (RBNS), in which a complex pool of RNA sequences is selected by an immobilized RBP and subsequently analyzed by microarrayanalysisorsequencing.WehaveestablishedanimprovedRBNSprotocolusingveryshort (8Ͳ14mer)randomsequenceswithonly4invariantnucleotidesforselection.Afterselectionbythe RBP of interest, the pool is read out by adapter ligation and deep sequencing. Specially designed adaptorsallowfortranscriptionoftheinsertandadditionalroundsofselectionandsequencing.
Wehavetestedourprotocolwithanumberofpurified,recombinantRBPsandfindthattheshort insertlengthallowsforaneasyandrobustdetectionofenrichedmotifswithouttherequirementfor extensivesequencingorbioinformaticanalysis.Inadditionwecouldshowthatitworkswithawhole range of protein formats and selection protocols using differently tagged or even untagged RBP preparations.
Importantly, we have been able to extend our analysis to full length RBPs overexpressed in mammaliancellculturesandevenendogenousRBPsfromculturedcellsortissuelysates.Tothisend, weimmunopurifytheRBPofinterestusingaspecificantibodyagainstthetagorproteinandperform theRNApoolselectiononthebeads.Thisoffersthepossibilitytodirectlyanalyzethespecificityof RBPs purified from their native environment. A clear difference in the selected motif by the wellͲ studiedRBPhnRNPA1betweenrecombinantandendogenousproteinsemphasizestheimportance ofthisapproach.
121 P591
Preliminaryinsightsintobicoid–Exuperantiainteraction
DianaVieira1,JoséTrincão2,IvoA.Telley1
1IGC,FundaçãoCalousteGulbenkian,Oeiras,Portugal2DiamondLightSource,HarwellScienceandInnovationCampus, Didcot,UnitedKingdom.
Targeted mRNA localization in cells enables a spatially and temporally tight regulation of protein expression. This regulatory mechanism is crucial in Drosophila melanogaster oogenesis, first in determining the future oocyte within the stem cell niche, and subsequently in defining the anatomical axes of the egg leading to the body axes of the future adult fly. One of the canonical mRNAsinbodyaxesformationisbicoid,whichisproducedinthenursecellsandtransportedtothe oocyteanterior.bicoidisresponsibleforheadͲtoͲtaildevelopmentoftheembryoanditstransportis accomplishedinaRNAͲproteincomplexdependentonthemicrotubulecytoskeletonandassociated molecularmotors.Inthisproject,weaimatdeterminingtheroleofoneproteinͲExuperantia(Exu)Ͳ whichispartofthebicoidͲproteincomplexandisinvolvedinthelocalizationmechanismofbicoid mRNAintheDrosophilamelanogasteroocyte.Ourstrategyistoobtainbothstructuralinsightsfrom purifiedproteinaswellaskineticinformationofmRNAͲproteininteractionwithinthephysiological settingoftheoocyte.Tofulfilthefirstaim,weclonedfourdifferentconstructsofthetargetgenein plasmidsofpETexpressionsystemsandexpressedtheproteininE.coli.Wewereabletoexpressand purifyoneofExu’sconstructs,aftertheoptimizationoftheexpressionprotocolbygenesynthesis, usingnickelaffinitychromatography(IMAC)andionicͲexchangechromatography(IEC).Theobtained protein was then concentrated to take the biomolecule into a supersaturation state and several crystallizationconditions,fromhomemadeandcommerciallyavailablekitswerescreenedusingthe vapor diffusion method. Obtaining good morphological diffracting crystals is considered the bottleneckofXͲraycrystallography.Onlyafteratwomonthsincubationperiodandover3500drops, fewpromisinghitswereobtained.TheputativeproteincrystalsobtainedwerecryoͲprotectedand sentfordiffractionexperimentsatDiamondLightSource,UnitedKingdom.Forthemajorityofthe crystalsitssizewasalimitingstep,forsomeotherstheinternalorderwaspoorandthediffraction datadidnotyieldadetailedstructure.Presently,thebiochemicalinformationobtainedallowsusto upliftthestudyintoaphysiologicalsituationanddeterminetheinteractionkineticsofExuandbicoid in the oocyte, determine the dependence on the microtubule cytoskeleton more thoroughly and testingloadingandtransportusingaexvivoreconstitutionapproach.
122 P601
EstablishmentofmonoclonalantibodiesagainstmodifiedRNAͲ bases
FranziskaWeichmann1,SamRingle1,RobertHett1,KaoutharSlama2,AndrewFlately3,Aloys Schepers3,MarkHelm2,ReginaFeederle3andGunterMeister1
1UniversityofRegensburg,Universitätsstraße31,93053Regensburg2JohannesGutenbergUniversitätMainz, Staudingerweg5,55128Mainz3HelmholtzZentrumMünchen,DeutschesForschungszentrumfürGesundheitundUmwelt (GmbH),IngolstädterLandstr.1,85764Neuherberg
MorethanonehundreddifferentposttranscriptionalmodificationsarefoundinallclassesofRNA. Someareratherabundantandrelativelywellinvestigated,butothersareinfrequentandonlypoorly characterised.Tobetterunderstandthebiologicalrolesofsuchmodifications,westartedtoestablish specificmonoclonalantibodiesagainstavarietyofRNAbasemodifications.Asantigens,wecoupled theoxidizedmodifiednucleosidesofinteresttothelysinesofovalbuminandinjectedthemintorats. Afterimmunizationandcellfusion,thequalityoftheresultinghybridomasproducedbytheobtained fusioncloneswasdeterminedinELISAͲscreens,dotblotandRNAͲIPexperimentsoftotalRNA.For furthervalidation,wedeterminedtheenrichmentfactorsofthedifferentcandidateantibodies.We performedRNAͲIPsusinginvitrotranscribedRNAoligoscontainingeither32PͲUTPandtherespective modified nucleotides or 32PͲATP and unmodified bases. After complete digestion of the enriched RNA, the nucleotides were analysed via thin layer chromatography. The signals for the differently labellednucleotidescouldthenbecomparedandanenrichmentfactorcouldbedefined.Withthis data, we could further characterize the candidate antibodies. Stable hybridoma cell lines were generated from the most specific and strongest binders. Using this very useful toolbox we aim at detecting specifically modified RNAs. Using RNAseq we plan to identify potential consensus sequenceswithinmodifiedRNAcandidates,potentiallynecessaryforthebindingofwriterorreader proteins. Putative consensus sequences might be ideal starting points for the identifications of catalytic enzymes that establish these modifications (writers) or proteins that recognize and bind them(readers).
123 P611
TheribosomebiogenesisfactoryUtp23/hUTP23coordinateskey interactionsintheyeastandhumanpreͲ40SparticleandhUTP23 containsanessentialPINdomain
GraemeR.Wells,FranziskaWeichmann,KatherineE.Sloan,DavidColvin,NicholasJ.Watkins, ClaudiaSchneider
InstituteforCellandMolecularBiosciences,NewcastleUniversity,UK
OneofthebiggestchallengesandenergyͲdemandingprocessesinthecellistheproductionofthe four mature ribosomal (r)RNA components of the small (40S) and large (60S) ribosomal subunits. During preͲrRNA processing, a number of timely and spatially regulated endoͲ and exonucleolytic cleavagesreleasethemature18S,5.8Sand25S/28SrRNAsfromasingleprecursormolecule.Three earlypreͲrRNAcleavagesinyeast(y)andhuman(h)18SrRNAmaturation(atsitesA0,A1andA2in yeastandsitesA0,1and2ainhumans)requirethecorrectassemblyofalargeribonucleoprotein complexknownastheSSUprocessome,whichcontainstwoproteinswithevolutionarilyconserved PINendonucleasedomains,yUtp24(Fcf1)/hUTP24andyUtp23/hUTP23.
TheyUtp24/hUTP24PINendonucleaseisproposedtocleaveatsitesA1/1andA2/2a,buttheenzyme cleavingatsiteA0isnotknown.Inbuddingyeast,yUtp23containsadegeneratePINdomain(only2 outof3requiredcatalyticresiduesarepresent)andlikelyplaysanonͲenzymaticrole.Incontrast, hUTP23 harbours three PIN domain active site residues, which could be sufficient for enzymatic activity. The yUtp23 protein functions together with a H/ACA snoRNP, snR30, while hUTP23 is associatedwithU17,thehumansnR30counterpart.
Here,usinginvivoRNAͲproteincrosslinkingandgelshiftexperiments,weshowthatyUtp23/hUTP23 makesdirectcontactswithexpansionsequence6(ES6)inthe18SrRNAsequenceandthatyUtp23 interacts with the 3’ half of the snR30 snoRNA. ProteinͲprotein interaction studies further demonstratethatyUtp23/hUTP23directlyinteractswiththeH/ACAsnoRNPproteinyNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52) and the ribosome biogenesis factors yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and releaseofES6bindingfactorsandlikelyplaysapivotalroleinremodellingthispreͲrRNAregionin bothyeastandhumans.StudiesusingRNAiͲrescuesystemsinhumancellsfurtherrevealthatintact PINdomainandZincfingermotifsinhUTP23areessentialfor18SrRNAmaturation.Thisraisesthe excitingpossibilitythathUTP23maybeanactivenuclease.
124 P621
Smallbuttough:structuralandfunctionalimportanceofthe Argonautelinkerhelix
SarahWillkomm1,ChristineA.Oellig2,AdrianZander1,SabineSchneider2,DinaGrohmann1
1nstituteofMicrobiology,UniversityofRegensburg,93053Regensburg,Germany2CenterforIntegratedProteinScience MunichCIPSM,DepartmentofChemistry,TechnicalUniversityMunich,85748Garching,Germany
Argonaute (Ago) proteins are key players in nucleic acid silencing processes in all domains of life. WhereaseukaryoticAgosaremostlyrestrictedtoRNAͲguidedsilencingofcomplementarymRNAs, the substrates used by prokaryotic Agos are more diverse. The euryarchaeal Ago variant from Methanocaldococcus jannaschii (MjAgo) for example is able to slice target DNAs in a DNA guideͲ dependentandguideͲindependentmanner.AlthoughthesubstratesandbiologicalfunctionsofAgo proteinsfromdifferentdomainsoflifearedisparate,theyarestructurallyhighlysimilar.Agoproteins areorganizedinabilobalfashionwiththeNͲterminalandthePAZdomainpositionedintheNͲlobe and the CͲlobe consisting of the Mid and the catalytically active PIWI domain. Comparison of the human Ago2Ͳguide RNA structure with the crystal structure of guideͲbound MjAgo revealed the presenceofaconservedhelixpositionedinthelinkerregionbetweentheCͲandtheNͲlobeofAgo, calledhelixͲ7inhumanAgo2(hAgo2)andhelixͲ8inMjAgo1,2.Thiselementwasshowntobedecisive for efficient target search of hAgo2 in the enormous amount of possible RNA targets in living organisms2,3.Furthermore,wewereabletosolvethecrystalstructureoftheMjAgoapoenzyme1. Here,helixͲ8ispresentinabipartitestate.Wethereforeconcludethatguidebindinginducesthe formation of a continuous helix. Mutation of a leucine to a proline in the bipartite motif disturbs formationofthefullͲlengthhelixͲ8anddisruptionofthehelixnegativelyaffectstargetcleavage1,4. Furthermore,ifhelixͲ8isdisrupted,substratesequenceandtheidentityofthe5’Ͳnucleotideofthe guidebecomeimportantparametersthatdeterminetargetcleavageefficiency1,4.Thissuggeststhat helixͲ8 is not only important for an efficient target search, but also for a correct positioning of a variety of guideͲtarget duplexes within Ago. Since our biochemical data revealed that hAgo2 and MjAgosharemanymechanisticfeatures5,wehypothesizethathelixͲ7mightplayanequallycrucial roleinhAgo2.
1Willkommetal.,Nat.Microbiol.(2017) 2Schirleetal.,Science(2014) 3Chandradossetal.,Cell(2015) 4Zanderetal.,Nat.Microbiol.(2017) 5Willkommetal.,PloSOne(2016)
125 P631
Theconformationallandscapeandactiveconformationofthe mRNAdecappingcomplex
JanPhilipWurm,IrisHoldermann,JanH.Overbeck,PhilippH.O.Mayer&RemcoSprangers
DepartmentofBiophysicsI,UniversityofRegensburg
Crystalstructuresofenzymesareindispensabletounderstandingtheirmechanismsonamolecular level. It, however, remains challenging to determine which structures are adopted in solution, especiallyfordynamiccomplexes.Here,westudythebilobeddecappingenzymeDcp2thatremoves the5഻capstructurefromeukaryoticmRNAandtherebyefficientlyterminatesgeneexpression.The numerousDcp2structurescanbegroupedintosixstateswherethedomainorientationbetweenthe catalyticandregulatorydomainssignificantlydiffers.Despitethiswealthofstructuralinformationit isnotpossibletocorrelatethesestateswiththecatalyticcycleortheactivityoftheenzyme.Using methyltransverserelaxationͲoptimizedNMRspectroscopy,wedemonstratethatonlythreeofthe six domain orientations are present in solution, where Dcp2 adopts an open, a closed, or a catalyticallyactivestate.WeshowhowmRNAsubstrateandtheactivatorproteinsDcp1andEdc1 influence the dynamic equilibria between these states and how this modulates catalytic activity. Importantly,theactivestateofthecomplexisonlystablyformedinthepresenceofbothactivators andthemRNAsubstrateorthem7GDPdecappingproduct,whichwerationalizebasedonacrystal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interestingly, we find that the activating mechanisms in Dcp2 also result in a shift of the substrate specificity from bacterial to eukaryotic mRNA.
126 P641
PhosphorylationofArgonauteProteinsAffectsmRNABindingandis EssentialformicroRNAͲguidedGeneSilencing
DanielaM.Zeitler1,JohannesDanner1,JudithHauptmann1,AstridBruckmann1,NicholasStrieder2, JuliaC.Englmann2,RainerDeutzmann1,MiguelQ.Huberdeau3,LucileFressigné3,SandraPiquet3, GuillaumeJannot3,MartinJ.Simard3andGunterMeister1
1BiochemistryCenterRegensburg(BZR),LaboratoryforRNABiology,UniversityofRegensburg,Universitätsstraße31,93053 Regensburg,Germany2DepartmentofStatisticalBioinformatics,UniversityofRegensburg,Regensburg,Germany3StͲ PatrickResearchGroupinBasicOncology,CentreHospitalierUniversitairedeQuébecͲUniversitéLaval(L’HôtelͲDieude Québec),LavalUniversityCancerResearchCentre,QuebecCity,Québec,G1R2J6,Canada
ArgonauteproteinsassociatewithmicroRNAsandarekeycomponentsofgenesilencingpathways. Withsuchapivotalroleingenesilencing,theseproteinsrepresentidealtargetsforregulatorypostͲ translational modifications. Using quantitative mass spectrometry, we find that a CͲterminal Serine/Threonineclusterisphosphorylatedatfivedifferentresiduesinhuman.ThisconservedhyperͲ phosphorylationdoesnotaffectmicroRNAbinding,localizationorcleavageactivityofhumanAgo2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAsormRNAs,theclusterremainsunphosphorylatedindicatingaroleatlatestagesofgene silencing.Interestingly,thismutantretainsitscapacitytoproduceandbindmicroRNAsandrepresses expression when artificially tethered to an mRNA. Altogether, our data suggest that the phosphorylationstateoftheSerine/ThreonineclusterisimportantforArgonauteͲmRNAinteractions
127 P651
ThenovellongnonͲcodingRNAEDClnc1isindispensableforproper terminalkeratinocytedifferentiation
ChristianZiegler1,AstridBruckmann1,NicholasStrieder2,BiancaFörstl1,RainerMerkl3,Julia Engelmann2,SonjaKretzͲHombach1,MarkusKretz1
1InstituteofBiochemistry,GeneticsandMicrobiology,BiochemistryI,UniversityofRegensburg,Regensburg,Germany 2InstituteofFunctionalGenomics,StatisticalBioinformatics,UniversityofRegensburg,Regensburg,Germany3Instituteof BiophysicsandPhysicalBiochemistry,BiochemistryII,UniversityofRegensburg,Regensburg,Germany
Several thousand long nonͲprotein coding transcripts >200 nt are encoded within the human genome. Although this large, newly discovered portion of the human transcriptome is still poorly characterizedtodate,anumberoffunctionalstudiesstronglyindicatethatlongnonͲcodingRNAs (lncRNAs) are highly functional relevant and not mere transcriptional waste. Full transcriptome sequencing of differentiated versus human progenitor keratinocytes revealed the strong upregulation of the previously undescribed lncRNA EDClnc1 (Epidermal Differentiation Complex lncRNA1) in differentiated keratinocytes. During further studies, we were able to pinpoint the transcriptstructureofEDClnc1andshowedthatrecentannotationswithinthisgenelocusarenot valid for keratinocytes. The strong induction of EDClnc1 during epidermal differentiation already suggestsafunctionalimportanceofEDClnc1forkeratinocytedifferentiationandindeed,knockdown of EDClnc1 in human keratinocytes results in a severe downregulation of several key epidermal differentiation markers. Furthermore, an RNAͲsequencing approach with EDClnc1 depleted organotypictissuesconfirmedthenecessityofEDClnc1forproperdifferentiationandpointedtoa possible epigenetic regulation mechanism of EDClnc1. In summary, we report the novel long nonͲcoding RNA EDClnc1 which is indispensable for keratinocyte differentiation, including a comprehensive locus analysis and preliminary results regarding the mechanism of EDClnc1. These studies will help us to understand the exact molecular role of EDClnc1 during keratinocyte differentiationaswellasthegenerationofskindiseases.
128 P661
ViewingpreͲ60Smaturationataminute’stimescale
GertrudeZisser,UliOhmayer,ChristinaMauerhofer,IsabellaKlein,ValentinMitterer,GeraldN. Rechberger,HeimoWolinski,MichaelPrattes,BrigittePertschy,PhilippMilkereitandHelmutBergler
InstituteofMolecularBiosciences,Humboldtstrasse50,UniversityGraz,AͲ8010Graz,Austria
Theformationofribosomalsubunitsisahighlydynamicprocessthatisinitiatedinthenucleusand involvesmorethan200transͲactingfactors,someofwhichaccompanythepreͲribosomesintothe cytoplasmandhavetoberecycledintothenucleus.Theinhibitordiazaborinepreventscytoplasmic releaseandrecyclingofshuttlingpreͲ60SmaturationfactorsbyinhibitingtheAAAͲATPaseDrg1.The failuretorecycletheseproteinsresultsintheirdepletioninthenucleolusandhaltsthepathwayat anearlymaturationstep.Herewemadeuseofthefastonsetofinhibitionbydiazaborinetochase thematurationpathinrealͲtimefrom27SA2containingpreͲribosomeslocalizedinthenucleolusup tonearlymature60Ssubunitsshortlyaftertheirexportintothecytoplasm.Thisallowsforthefirst time to put protein assembly and disassembly reactions as well as preͲrRNA processing into a chronologicalcontextunravelingtemporalandfunctionallinkagesduringribosomematuration.
129 P671
StructureandfunctionoftheKOW4andKOW6domainsofthe humantranscriptionfactorDSIF
PhilippZuber,LukasHahn,AnneReinl,PaulRösch,StefanKnauer,KristianSchweimer,BirgittaM. Wöhrl
UniversitätBayreuth,LehrstuhlBiopolymere,95448Bayreuth,Germany
EukaryotictranscriptionbytheenzymeRNApolymeraseII(RNAPII)istightlyregulatedbydifferent mechanisms. One important regulatory step is promoter proximal transcriptional pausing, which introduces an early block to RNAP II elongation after ca. 20 to 70 transcribed bases. The general transcriptionfactorDRB(5,6,ͲdichloroͲ1ͲɴͲDͲribofuranosylbenzimidazole)sensitivityinducingfactor (DSIF) is involved in this rate limiting step of RNAP II transcription of various cellular promoters. HumanDSIFisaheterodimer,composedofa14kDa(hSpt4)anda120kDa(hSpt5)subunitwhichare thehumanhomologuesofSpt4andSpt5fromSaccharomycescerevisiae.ThehSpt5subunitcontains a region that is homologous to the NͲterminal domain of the bacterial transcription factor NusG (NGN).NusG/Spt5proteinsareconservedinallthreekingdomsoflife.hSpt4,whichisnotpresentin bacteria,interactswithhSpt5viatheNGNͲdomain.Furthermore,theNGNdomaininteractswiththe Rpb1and2subunitsofRNAPII.ThepositionofDSIFboundtoRNAPIIcouldbelocalizedoverthe activecentercleftintheclampdomainofRNAPolII.BacterialNusGandthearchaealSpt5proteins consist of the NGNͲdomain and a CͲterminal KypridesͲOuzounisͲWoese (KOW) domain. Eukaryotic Spt5harborsseveralcopiesofKOWdomainswhosefunctionshavenotbeenstudiedinfulldetail. Upon phosphorylation DSIF appears to have an activating effect on RNAP II elongation. hSpt5 is knowntointeractwithRNAPIIthroughtheregionharbouringtheKOWmotifs.TheKOW1domainis flexibleandislocatedinaregionbetweentheclampandtheRNAexittunnelofRNAPII.Although thestructureofmammalianRNAPIIhasbeensolved,thefunctionofthedifferentKOWdomainsof human DSIF remain elusive.Structures of KOW 1, 2, 3 and 5 are available from yeast or homo sapiens, however the structures of KOW 4 and KOW 6 are still missing. To learn more about the function of human KOW 4 and 6, we determined their structures by solution nuclear magnetic resonance(NMR)analysesandperformedinteractionstudieswithnucleicacidsandRpb4/7.Both KOWdomainsexhibitacentralɴͲbarrelmotif,however,additionalstructuralfeaturesarepresent. FluorescenceandNMRͲbasedtitrationsshowedthatonlyKOW4bindstonucleicacids,whereasno bindingtonucleicacidscouldbedetectedforKOW6.
130 P681
Regulationofbacterialtranscriptionbythetransformerprotein RfaH
PhilippK.Zuber,KristianSchweimer,PaulRöschandStefanH.Knauer
LehrstuhlBiopolymereundForschungszentrumfürBioͲMakromoleküle,UniversitätBayreuth,Universitätsstraße30,95447 Bayreuth,Germany
TranscriptionisthefirststepingeneexpressionandRNApolymerase(RNAP)ishighlyregulatedbya varietyoftranscriptionfactorsofwhichNusGproteinsrepresenttheonlyuniversallyconservedclass. RfaHisanoperonͲspecificparalogofNusGinEscherichiacoli.ItsNͲterminaldomain(NTD)exhibits thetypicalNusGtopology,butincontrasttothebetaͲbarrelconformationoftheCͲterminaldomain (CTD)ofNusGproteinstheRfaHͲCTDisanalphaͲhelicalhairpinthattightlybindstotheRfaHͲNTD, maskingtheRNAPbindingsiteonRfaHͲNTDandrenderingRfaHautoinhibited.Uponrecruitmentof RfaH to elongating RNAP (transcription elongation complex, TEC) the domains dissociate and the RfaHͲCTDtransformsfromitsalphaͲhelicalstateintothebetaͲbarreltypicalforNusGproteins(Fig. 1)1.RfaHactivationdependsonanoperonpolaritysuppressor(ops)elementintheleaderregionof the RfaHͲcontrolled operon. The ops sequence pauses the TEC so that the ops site in the nonͲ template strand is exposed and accessible2. So far, the structural basis of RfaH regulation and its recruitmentinparticularareonlypoorlyunderstood.Hereweprovidemoleculardetailsofthelife cycle of RfaH obtained by applying solution state NMR spectroscopy on a supramolecular, multicomponentsystem.Duetothesizeofthesystem(RNAP:390kDa)[1H,13C]ͲlabeledIle,Leu,and Val methyl groups in perdeuterated protein were used as NMR active probes. First, we aimed to identify the signal for RfaH recruitment. We showed that both opsDNA and RNAP bind to RfaH individually,butneitheroftheisolatedinteractionpartnerswasabletotriggerdomaindissociation. WethenassembledaTECpausedattheopssite(opsTEC)anddemonstratedtherecruitmentofRfaH to this opsTEC by directly following RfaH binding and the transformation of its CTD. Quantitative analysis of the signal intensity allowed us to determine the opsTEC interaction site on RfaH. In combinationwiththecrystalstructureoftheRfaH:opscomplexthatwesolvedwecreatedamodel of RfaH bound the opsTEC. Moreover, we showed that TECͲbound RfaH can bind to ribosomal protein S10, an interaction that is essential for the activation of translation as RfaHͲcontrolled operonslackaShineͲDalgarnosequence.Finally,displacementexperimentsconfirmedthatRfaHcan berecycledintoitsautoinhibitedstateupondissociationfromtheTEC.
[1]Burmann,B.M.;Knauer,S.H.;Sevostyanova,A.,Schweimer,K.,Mooney,R.A.;Landick,R.;Artsimovitch,I.; Rösch,P.Cell2012,150,291Ͳ303 [2]Artsimovitch,I.;Landick,R.Cell2002,109,193Ͳ203
131 P691
RNPͲIDgeneratesapictureofthemolecularinteractionsduring shuttlingandmRNPexport
FlorianRichter,AnfisaSolovyeva,IIlkaWittig,MichaelaͲMüllerMcNIcoll
GoetheUniversityFrankfurt
SRproteinsbelongtoafamilyofRNAͲbindingproteins(RBPs)withessentialfunctionsasregulators ofsplicing.Additionally,somefamilymembersshuttlecontinuouslybetweennucleusandcytoplasm andactasexportadaptersforthemainmRNAexportreceptorNXF1(MüllerͲMcNicoll,2016;Botti, 2017).WehaveshownthatshuttlingofSRproteinsisinfluencedbytheextentofNXF1interaction, methylationoftheNXF1interactionregion,bindingtodifferentRNAclassesandthephosphorylation stateoftheirRSdomain(Botti,2017).RecruitedNXF1subsequentlydimerizeswithitscoͲfactorNXT1 toformabindingplatformfornuclearexportofcellularmRNAsviathenuclearporecomplex.
To understand the regulation of mRNA export by SR proteins and NXF1 we developed an MS approach,basedontheendogenousexpressionofGFPͲtaggedbaitproteinsfromgenomicloci,UVͲ crosslinking,differentialRNasedigestandstringentpulldown.Thismethod,termedRNPͲID,allows thequantitativecomparisonofRNPsbetweencelltypesorindifferentcellularconditionsandthe extractionofdiverseparametersdescribingthecomposition,modificationanddynamicsofRNPs.We applied this approach to SRSF5Ͳ and NXF1ͲGFP endogenously expressed in murine P19 cells. Comparing +UV toͲUV samples in RNaseͲsensitive and RNaseͲresistant fractions we were able to devise protein:protein interactors and protein:RNA:protein interaction partners that bind in close proximitytothebaitonthesameRNApiece.MoreoverweidentifiedandquantifiedPTMsandRNA crosslinks as well as UVͲinduced disulfide bridges that provide information on transient protein interactionsandtheresponsibleproteindomains.UsingNXF1asbait,wepurifiedanactiveshuttling complexcontainingmanynuclearporeproteins(NUPs)andexportfactors.Ofinterest,oursample preparation strategy allowed us for the first to monitor the complex path of NXF1 during its way throughthenuclearporeincludingNUP,RBPandRNAinteractionsusingUVinducedRNAcrosslinks and transient disulfide bridges. Modeling and integrating these transient interactions and RNA contacts including data for SRSF5 will help to understand the different shuttling capacities of SR proteinsandtheirimpactotheregulationofmRNPexportbyNXF1.
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133 LIST OF PARTICIPANTS
A AnastasiiaBazhenova UniversityofRegensburg IngridAraya CellBiologyandPlantBiochemistry UniversityofRegensburg Universitätsstrasse31 BiochemistryIII 93053Regensburg Universitätsstrasse31 Germany 93053Regensburg [email protected] Germany [email protected] HelmutBergler UniversityGraz GeorgAuburger MolecularBiology GoetheUniversity Humboldtstrasse50 MedicalSchoolExp.Neurology 8010Graz TheodorSternKai7 Austria 60590FrankfurtamMain helmut.bergler@uniͲgraz.at Germany [email protected]Ͳfrankfurt.de PriyaBhutada UniversityofGraz InstituteofMolecularBiosciences B Humboldtstrasse50 8010Graz NenadBan Austria ETHZurich priya.bhutada@uniͲgraz.at MolecularBiologyandBiophysics OttoSternWeg5 AndreaBleckmann 8093Zurich UniversityofRegensburg Switzerland CellBiology/PlantBiochemistry [email protected] Universitätsstrasse31 93053Regensburg MiriamBanas Germany UniversityHospitalRegensburg [email protected] Nephrology Annaweg7 MarkusT.Bohnsack 93161Sinzing GöttingenUniversity Germany InstituteofMolecularBiology [email protected] Humboldtallee37073 Göttingen SusanBaserga Germany YaleUniversity [email protected]Ͳgoettingen.de MolecularBiophysics&Biochemistry 333CedarStreet SimonBraun 6510NewHaven InstituteofMolecularBiology(IMB) UnitedStates AGKönig [email protected] Ackermannweg4 55128Mainz Germany s.braun@imbͲmainz.de 134 AstridBruckmann ThomasDresselhaus UniversityofRegensburg UniversityofRegensburg BiochemistryI CellBiology&PlantBiochemistry Universitätsstrasse31 Universitätsstrasse31 93051Regensburg 93053Regensburg Germany Germany [email protected] [email protected] FlorianBusch AleksejDrino TheOhioStateUniversity MedicalUniversityofVienna ChemistryandBiochemistry CenterforAnatomyandCellBiology 196EFrambesAve Schwarzspanierstrasse17 43201Columbus 1090Vienna UnitedStates Austria [email protected] [email protected] BenjaminDudenhöffer C UniversityofBayreuth LehrstuhlBiopolymere PabloCastroͲHartmann Universitätsstrasse30 UniversityofCambridge 95447Bayreuth Haematology Germany HillsRoad [email protected] CB20XYCambridge UnitedKingdom DavidDulin [email protected] FAUErlangenͲNürnberg IZKF GeaCereghetti Hartmannstrasse14 ETHZürich 91052Erlangen DepartmentofBiology,InstituteofBiochemistry Germany OttoͲSternͲWeg3 david.dulin@ukͲerlangen.de 8093Zürich Switzerland E [email protected] AndreaEberle UniversityofBern D DepartmentofChemistryandBiochemistry Freiestrasse3 SenthilKumarDevan 3012Bern InstituteforMicrobiologyBiology Switzerland Universitätsstrasse [email protected] 40225Duesseldorf Germany HansFriedrichEhrnsberger [email protected] UniversityofRegensburg Cellbiologyandplantbiochemistry SvenDiederichs Universitätsstrasse31 GermanCancerResearchCenter(DKFZ) 93053Regensburg RNABiology&Cancer Germany ImNeuenheimerFeld280(B150) HansͲ[email protected]Ͳregensburg.de 69120Heidelberg Germany [email protected]
135 SaraEisenbart AndreasFuchs UniversityofWürzburg UniversityofRegensburg ResearchCentreforInfectiousDiseases BiochemistryIII JosefͲSchneiderͲStrasse2/BauD15 Universitätsstrasse31 97080Würzburg 93053Regensburg Germany Germany sara.eisenbart@studͲmail.uniͲwuerzburg.de [email protected]Ͳregensburg.de MariaEnglhart AnnaͲLisaFuchs UniversityofRegensburg UniversityofRegensburg CellBiologyandPlantBiochemistry BiophysikI Universitätsstrasse31 Universitätsstrasse31 93053Regensburg 93053Regensburg Germany Germany [email protected] annaͲ[email protected] SilviaEsposito G UniversityofRegensburg PlantBiochemistryandCellBiology MinakshiGandhi Universitätsstrasse31 DKFZ 93053Regensburg RNABiologyandCancer(B150) Germany ImNeuenheimerFeld280 [email protected] 69120Heidelberg Germany DariaEsyunina [email protected] InstituteofMolecularGenetics LaboratoryofMolecularGeneticsofMicroorganisms JohannesGraf Kurchatovsquare,2 UniversityofRegensburg 123182Moscow Biologyandpreclinicalmedicine,biochemistryI RussianFederation Universitätsstrasse31 [email protected] 93053Regensburg Germany F [email protected]Ͳregensburg.de SébastienFerreiraͲCerca MarionGrasser UniversityofRegensburg UniversityofRegensburg Lst.BiochemistryIII Biologie Universitätsstrasse31 Universitätsstrasse31 93053Regensburg 93053Regensburg Germany Germany sebastien.ferreiraͲ[email protected] [email protected] UtzFischer KlausGrasser UniversityofWürzburg UniversityofRegensburg Biochemistry CellBiologyandPlantBiochemistry Biocenter/AmHubland Universitätsstrasse31 97074Wuerzburg 93053Regensburg Germany Germany [email protected]Ͳwuerzburg.de [email protected]
136 AliceGriselin H UniversityofRegensburg BiochemistryI StevenHahn Universitätsstrasse31 FredHutchinsonCancerCenter 93053Regensburg DivisionofBasicSciences Germany 1100FairviewAveN [email protected]Ͳregensburg.de 98109Seattle UnitedStates JoachimGriesenbeck [email protected] UniversityofRegensburg BiochemistryIII DanieleHasler Universitätsstrasse31 UniversityofRegensburg 93053Regensburg BiochemistryI Germany Universitätsstrasse31 [email protected] 93053Regensburg Germany DinaGrohmann [email protected] UniversityofRegensburg LehrstuhlfürMikrobiologie WinfriedHausner Universitätsstrasse31 UniversityofRegensburg 93059Regensburg LehrstuhlfürMikrobiologie Germany Universitätsstrasse31 [email protected] 93053Regensburg Germany FelixGrünberger [email protected] UniversityofRegensburg Microbiology LeonhardHeizinger Universitätsstrasse31 UniversityofRegensburg 93053Regensburg Universitätsstrasse31 Germany 93053Regensburg [email protected] Germany [email protected] NishthaGulati TechnicalUniversityMunich AlanHinnebusch BayerischeNMRCenter NationalInstitutesofHealth Lichtenbergstrasse4 LaboratoryofGeneRegulationandDevelopment 85747Garching Building6,Room230,9000RockvillePike Germany 20892Bethesda [email protected] UnitedStates [email protected] AlexanderGust UniversityofRegensburg HungHoXuan Mikrobiologie UniversityofRegensburg Universitätsstrasse31 FakultätfürBiologieundVorklinischeMedizin 93053Regensburg Universitätsstrasse31 Germany 93053Regensburg [email protected] Germany XuanͲ[email protected] TonyGutschner MartinͲLutherͲUniversityHalleͲWittenberg MedicalFaculty HeinrichͲDamerowͲStrasse1 06120Halle Germany tony.gutschner@ukͲhalle.de 137 HelenHoffmeister J UniversityofRegensburg BiochemistryIII RolandJacob Universitätsstrasse31 MartinͲLutherͲUniversityHalleͲWittenberg 93053Regensburg FacultyofMedicineͲZAMED Germany HeinrichͲDamerowͲStrasse1 [email protected]Ͳregensburg.de 06120Halle(Saale) Germany DavidHonys roland.jacob@ukͲhalle.de InstituteofExperimentalBotanyASCR PollenBiology NicolasJaé Rozvojova263 GoetheͲUniversityFrankfurt 16500Praha6 InstituteforCardiovascularRegeneration CzechRepublic TheodorͲSternͲKai7 [email protected] 60590Frankfurt Germany JensHör [email protected]Ͳfrankfurt.de UniversityofWürzburg InstituteforMolecularInfectionBiology LeonhardJakob JosefͲSchneiderͲStrasse2Ͳ4/D15 UniversityofRegensburg 97080Würzburg Mikrobiologie Germany Universitätsstrasse31 jens.hoer@uniͲwuerzburg.de 93053Regensburg Germany AndreasHorn [email protected] UniversityofRegensburg BiochemistryI LeemorJoshuaͲTor Universitätsstrasse31 HHMI/ColdSpringHarborLaboratory 93053Regensburg KeckStructuralBiologyLab Germany 1BungtownRd. [email protected] 11724ColdSpringHarbor UnitedStates EdHurt [email protected] UniversitätHeidelberg BiochemistryͲZentrum(BZH) K ImNeuenheimerFeld328 69120Heidelberg VasileiosKargas Germany UniversityofCambridge [email protected]Ͳheidelberg.de DepartmentofHaematology CambridgeBiomedicalCampus I CB20XYCambridge UnitedKingdom AxelInnis [email protected] IECBͲUniversityofBordeaux INSERMU1212 StefanKnauer 2,rueRobertEscarpit UniversityofBayreuth 33607Pessac LehrstuhlBiopolymere France Universitätsstrasse30 [email protected] 96447Bayreuth Germany stefan.knauer@uniͲbayreuth.de
138 RobertKnüppel AndreyKulbachinskiy UniversityofRegensburg Institute of Molecular Genetics, Russian Academy of BiochemistryIII Sciences Universitätsstrasse31 LaboratoryofMolecularGeneticsofMicroorganisms 93053Regensburg Kurchatovsquare2 Germany 123182Moscow [email protected] RussianFederation [email protected] LisaKofler UniversityofGraz L DepartmentofMolecularBiosciences Humboldtstrasse50/EG GernotLängst 8010Graz UniversityofRegensburg Austria BichemistryIII lisa.kofler@uniͲgraz.at Universitätsstrasse31 93053Regensburg JulianKönig Germany InstituteofMolecularBiology(IMB) [email protected] Ackermannweg4 55128Mainz ClaudiaLatini Germany UniversityofRegensburg j.koenig@imbͲmainz.de Biochemistry1 Universitätsstrasse31 KevinKramm 93053Regensburg UniversityofRegensburg Germany Microbiology [email protected]Ͳregensburg.de Universitätsstrasse31 93053Regensburg DanielaLazzaretti Germany MaxPlanckInstituteforDevelopmentalBiology [email protected] ResearchGroupFulviaBono Spemannstrasse35 MarkusKretz 72076Tuebingen UniversityofRegensburg Germany BiochemistryI [email protected] Universitätsstrasse31 93053Regensburg ReinhardLührmann Germany MaxPlanckInstituteforBiophysicalChemistry [email protected] CellularBiochemistry AmFassberg11 SonjaKretzͲHombach 37077Göttingen UniversityofRegensburg Germany BiochemistryI [email protected] Universitätsstrasse31 93053Regensburg M Germany [email protected] CameronMackereth InsermU1212/Univ.Bordeaux ClausKuhn InstitutEuropeendeChimieetBiologie UniversityofBayreuth 2rueRobertEscarpit BIOmacResearchCenter 33706Pessac Universitätsstrasse30 France 95447Bayreuth [email protected]Ͳbordeaux.fr Germany claus.kuhn@uniͲbayreuth.de 139 LisaͲKatharinaMaier RebeccaMoschall UniversityofUlm UniversityofRegensburg BiologieII BiochemistryI AlbertͲEinsteinͲAllee11 Universitätsstrasse31 89069Ulm 93053Regensburg Germany Germany lisaͲkatharina.maier@uniͲulm.de [email protected] RodrigoMaldonado OliverMühlemann UniversityofRegensburg UniversityofBern VKL ChemistryandBiochemistry Universitätsstrasse31 FreieStrasse3 93053Regensburg 3012Bern Germany Switzerland [email protected]Ͳregensburg.de [email protected] JanMedenbach AdrianMüller UniversityofRegensburg UniversityofRegensburg BiochemistryI Biochemistry3 Universitätsstrasse31 Universitätsstrasse31 93053Regensburg 93053Regensburg Germany Germany [email protected] [email protected]Ͳregensburg.de GunterMeister ChristophMüller UniversityofRegensburg EuropeanMolecularBiologyLaboratory(EMBL) BiochemistryI StructuralandComputationalBiologyUnit Universitätsstrasse31 Meyerhofstrasse1 93053Regensburg 69117Heidelberg Germany Germany [email protected]Ͳregensburg.de [email protected] RainerMerkl MichaelaMüllerͲMcNicoll UniversityofRegensburg GoetheUniversityFrankfurt BiochemistryII InsituteforCellBiologyandNeuroscience Universitätsstrasse31 MaxͲvonͲLaueͲStrasse13 93083Regensburg 60438FrankfurtamMain Germany Germany [email protected] [email protected] PhilippMichlͲHolzinger N UniversityofRegensburg CellBiologyandPlantBiochemistry AlenaNáprstková Universitätsstrasse31 InstituteofExperimentalBotanyASCR,v.v.i. 93053Regensburg PollenBiology Germany Rozvojová263 [email protected] 16500Prague6 CzechRepublic PhilippMilkereit [email protected] UniversityofRegensburg LehrstuhlfürBiochemistryIII Universitätsstrasse31 93053Regensburg Germany [email protected]Ͳregensburg.de 140 NagammalNeelagandan MerveÖztürk ZMNH IMBͲMainz NeuronalTranslationalcontrol Biology Falkenried94 Ackermannweg4 20251Hamburg 55128Mainz Germany Germany [email protected] M.Oeztuerk@imbͲmainz.de LeaNeumannͲArnold P UniversityofRegensburg Genetics BalagopalPai Universitätsstrasse31 UniversityofRegensburg 93053Regensburg BiochemistryI Germany Universitätsstrasse31 lea.neumannͲ[email protected] 93053Regensburg Germany EvgenyNudler [email protected] NewYorkUniversity Biochemistry RoyParker 450E29thStreet,Room303 HHMI/UniversityofColoradoBoulder 10009NewYork ChemistryandBiochemistry UnitedStates 3415ColoradoAve [email protected] 80303Boulder UnitedStates O [email protected] DirkOstareck VicentPelechano UniversityHospitalRWTHAachen KarolinskaInstitutetͲSciLifeLab Intensive Care and Intermediate Care, Experimental Microbiology,TumorandCellBiology ResearchUnit Tomtebodavägen23 Pauwelsstrasse30 17165Solna 52074Aachen Sweden Germany [email protected] [email protected] JorgePerezͲFernandez AntjeOstareckͲLederer UniversityofRegensburg UniversityHospitalRWTHAachen BiochemistryIII Department of Intensive Care and Intermediate Care, Universitätsstrasse31 ExperimentalResearchUnit 93053Regensburg Pauwelsstrasse30 Germany 52074Aachen [email protected] Germany [email protected] MelaniePertl UniversityofGraz JanOverbeck MolekulareBiowissenschaften UniversityofRegensburg Humboldtstrasse50/EG FakultätfürBiologieundVorklinischeMedizin 8010Graz Universitätsstrasse31 Austria 93053Regensburg melanie.pertl@uniͲgraz.at Germany [email protected]Ͳregensburg.de
141 BrigittePertschy RobertReichelt UniversityofGraz UniversityofRegensburg InstituteforMolecularBiosciences MicrobiologyandArchaeenzentrum Humboldtstrasse50/EG8010 Universitätsstrasse31 Graz 93053Regensburg Austria Germany brigitte.pertschy@uniͲgraz.at [email protected] BenjaminPillet SimoneReicheltͲWurm UniversityofFribourg UniversityHospitalRegensburg Biology AbteilungfürNephrologie CheminduMusée10 FranzͲJosefͲStraußͲAllee11 1700Fribourg 93053Regensburg Switzerland Germany [email protected] Simone.ReicheltͲ[email protected] MichaelPilsl FlorianRichter UniversityofRegensburg Heusingerstraße64 BiochemistryIII 65934FrankfurtamMain Universitätsstrasse31 [email protected] 93053Regensburg Germany IngridRössler [email protected] UniversityofGraz InstituteofMolecularBiosciences KristynaPoncova Humboldtstrasse50 Laboratoryofregulationofgeneexpression 8010Graz Videnska1083 Austria 14200Praha4 ingrid.roessler@uniͲgraz.at CzechRepublic [email protected] S MichaelPrattes PeterSarnow UniversityofGraz StanfordUniversity InstituteofMolecularBiosciences Microbiology&Immunology Humboldtstrasse50 299CampusDrive 8010Graz 94305Stanford Austria UnitedStates michael.prattes@uniͲgraz.at [email protected] R MichaelSattler HelmholtzCenterMunich StefanReich InstituteofStructuralBiology UniversityofRegensburg IngolstädterLandstrasse1 Biochemistry1 85764Neuherberg Universitätsstrasse31 Germany 93053Regensburg sattler@helmholtzͲmuenchen.de Germany [email protected]
142 ChristopherSchächner ElisabethSilberhorn UniversityofRegensburg UniversityofRegensburg BiochemistryIII BiochemistryIII Universitätsstrasse31 Universitätsstrasse31 93053Regensburg 93053Regensburg Germany Germany [email protected] [email protected] ChristinaSchmidt NahumSonenberg UniversityofRegensburg McGillUniversity Universitätsstrasse31 Biochemistry 93053Regensburg 1160PineAvenueWest Germany H3A1A3Montreal [email protected] Canada [email protected] ClaudiaSchneider NewcastleUniversity JörgSoppa InstituteforCellandMolecularBiosciences GoetheUniversity FramlingtonPlace Biocentre,InstituteforMolecularBiosciences NE24HHNewcastleuponTyne MaxͲvonͲLaueͲStrasse9 UnitedKingdom 61476Frankfurt [email protected] Germany [email protected]Ͳfrankfurt.de EvaSchöller UniversityofRegensburg NicoleSpitzlberger BiochemistryI UniversityofRegensburg Universitätsstrasse31 CellBiologyandPlantBiochemistry 93053Regensburg Universitätsstrasse31 Germany 93053Regensburg [email protected]Ͳregensburg.de Germany [email protected]Ͳregensburg.de StefanSchütz UniversityofRegensburg RemcoSprangers Dept.forBiologyandPreclinicalMedicine UniversityofRegensburg Universitätsstrasse31 Biophysik1 93053Regensburg Universitaetsstrasse31 Germany 93053Regensburg [email protected] Germany [email protected]Ͳregensburg.de WolfgangSeufert UniversityofRegensburg StefanieSprunck InstituteofBiochemistry,GeneticsandMicrobiology UniversityofRegensburg Universitätsstrasse31 ZellbiologieundPflanzenBiochemistry 93053Regensburg Universitätsstrasse31 Germany 93053Regensburg [email protected] Germany [email protected] CynthiaSharma Institute of Molecular Infection Biology, University of DorotheeStaiger Würzburg BielefeldUniversity MolecularInfectionBiologyII RNABiologyandMolecularPhysiology JosefͲSchneiderͲStrasse2/D15 Universitaetsstrasse25 97080Würzburg 33615Bielefeld Germany Germany cynthia.sharma@uniͲwuerzburg.de dorothee.staiger@uniͲbielefeld.de 143 SarahSvensson U UniversityofWürzburg InstituteforMolecularInfectionBiology JernejUle JosefͲSchneiderͲStrasse2/BauD15 TheFrancisCrickInstitute 97080Würzburg 1MidlandRoad Germany NW11ATLondon sarah.svensson@uniͲwuerzburg.de UnitedKingdom [email protected] T V DylanTaatjes UniversityofColorado,Boulder StepankaVanacova Biochemistry MasarykUniversityCEITEC CampusBox596 Kamenice5/A35 80303Boulder 62500Brno UnitedStates CzechRepublic [email protected] [email protected] NadjaTedesco SvenVanselow UniversityofRegensburg UniversityofRegensburg BiochemistryIII/AGLängst LehrstuhlfürGenetik Universitätsstrasse31 Universitätsstrasse31 93053Regensburg 93053Regensburg Germany Germany [email protected] [email protected] FabianTeubl DianaVieira UniversityofRegensburg InstitutoGulbenkiandeCiência BiochemistryIII FundaçãoCalousteGulbenkian Universitätsstrasse31 RuadaQuintaGrande,6 93053Regensburg 2780Ͳ156Oeiras Germany Portugal [email protected] [email protected] ThomasTreiber ZoltanVillanyi UniversityofRegensburg UniversityofGeneva BiochemistryI DepartmentofMicrobiologyandMolecularMedicine Universitätsstrasse31 1rueMichaelServet 93053Regensburg 1211Geneva Germany Switzerland [email protected]Ͳregensburg.de [email protected] HerbertTschochner JörgVogel UniversityofRegensburg UniversityofWürzburg BiochemistryͲZentrumRegensburg IntituteforMolecularInfectionBiology Universitätsstrasse31 JosefͲSchneiderͲStrasse2,D15 93053Regensburg 97080Würzburg Germany Germany [email protected] joerg.vogel@uniͲwuerzburg.de
144 W SarahWillkomm UniversityofRegensburg AlanWarren DepartmentofMicrobiology UniversityofCambridge Universitätsstrasse31 CIMR 93053Regensburg HillsRoad Germany CB20XYCambridge [email protected] UnitedKingdom [email protected] DanielWilson UniversityofHamburg NickWatkins InstituteforBiochemistryandMolecularBiology NewcastleUniversity MartinͲLutherͲKingͲPlatz6 InstituteforCellandMolecularBiosciences 20146Hamburg FramlingtonPlace Germany NE303DXNewcastleuponTyne [email protected]Ͳhamburg.de UnitedKingdom [email protected] BirgittaWöhrl UniversityofBayreuth MariaTheresiaWatzlowik LehrstuhlBiopolymere UniversityofRegensburg Universitätsstrasse30 VKL 95447Bayreuth Universitätsstrasse31 Germany 93053Regensburg birgitta.woehrl@uniͲbayreuth.de Germany mariaͲ[email protected] FranziskaWojciech UniversityofRegensburg FranziskaWeichmann Genetics UniversityofRegensburg Universitätsstrasse31 BiochemistryI 93053Regensburg Universitätsstrasse31 Germany 93059Regensburg [email protected] Germany [email protected] PhilipWurm UniversityofRegensburg AlbertWeixlbaumer Biophysik IGBMC Universitätsstrasse31 IntegratedStructuralBiology 93053Regensburg 1,rueLaurentFries Germany 67404Strasbourg JanͲ[email protected]Ͳregensburg.de France [email protected] Z FinnWerner SindyZander UniversityCollegeLondon MartinͲLutherͲUniversityHalleͲWittenberg StructuralandMolecularBiology FacultyofMedicineͲZAMED GowerStreet HeinrichͲDamerowͲStrasse1 WC1E6BTLondon 06120Halle(Saale) UnitedKingdom Germany [email protected] sindy.zander2@ukͲhalle.de
145 KathiZarnack ChristianZiegler BuchmannInstituteofMolecularLifeSciences(BMLS) UniversityofRegensburg MaxͲvonͲLaueͲStrasse15 BiochemistryI 60438Frankfurt Universitätsstrasse31 Germany 93053Regensburg [email protected] Germany [email protected] DanielaZeitler UniversityofRegensburg PhilippZuber BiochemistryI UniversityofBayreuth Universitätsstrasse31 LehrstuhlBiopolymere 93053Regensburg Universitätsstrasse30 Germany 95447Bayreuth [email protected] Germany philipp.zuber@uniͲbayreuth.de
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153 VENUE INFORMATION &MAPS
NetworkaccessintheThonͲDittmerͲPalais
Informationtoaccessthelocalnetwork‘StadtRegensburg’willbeprovidedonsite.
TheaudioͲvisualteam
Touploadyourtalksontheprovidedcomputers,ortoconnectyourown computer to the system, please contact Felix Grünberger (pictured) or anyothermemberoftheAVteam.
Amapoftheoldtownwiththeconferencesitesmarkedcanalsobefoundhere:
154 DowntownRegensburg
Cathedral
Heuport Haus
Dollingersaal
Thon-Dittmer-Palais
155