Mitochondrial Gene Expression: Methods and Protocols

335 Pages·2021·7.962 MB·English

by  Michal Minczuk, Joanna Rorbach

Methods in
Molecular Biology 2192
Michal Minczuk
Joanna Rorbach Editors
Mitochondrial
Gene Expression
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
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Mitochondrial Gene Expression
Methods and Protocols
Edited by
Michal Minczuk
Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK
Joanna Rorbach
Division of Molecular Metabolism, Department of Medical Biochemistry and Biophysics, Karolinska
Institutet, Solna, Sweden; Max Planck Institute Biology of Ageing - Karolinska
Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden
Editors
MichalMinczuk JoannaRorbach
MedicalResearchCouncil DivisionofMolecularMetabolism
MitochondrialBiologyUnit DepartmentofMedicalBiochemistryandBiophysics
UniversityofCambridge KarolinskaInstitutet
Cambridge,UK Solna,Sweden
MaxPlanckInstituteBiologyofAgeing-Karolinska
InstitutetLaboratory
KarolinskaInstitutet
Stockholm,Sweden
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-0833-3 ISBN978-1-0716-0834-0 (eBook)
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Preface
From the discovery of the mitochondrial genome (mtDNA) in 1963 to the most recent
cryo-EMmodel of the human mitochondrial ribosome, the scientific community has been
gaining better understanding of how the expression of mtDNA is regulated. In mammals,
this pathway is necessary for the synthesis of 13 proteins that are encoded by mtDNA and
translated by a specialized,membrane-attached mitoribosome to form the catalytic core of
the oxidative phosphorylation complexes. The significantly compacted mitochondrial
genome, the limited set of uniquely structured transcripts, and the need for the proteins
tobeco-translationallyinsertedintothemembraneandco-assembledwithnuclear-encoded
subunitshavesubstantialconsequencesforthemitochondrialgeneexpressionmachinery.In
fact, all stages of mitochondrial gene expression differ significantly from the analogous
stagesforbacterialpathwaysandeukaryoticpathwaysfor nuclear-encodedgenes.
Mitochondrial dysfunction has been implicated in many metabolic and degenerative
diseases,cancer,andaging;therefore,adeeperunderstandingoftheprocessesofmitochon-
drialgeneexpressionhasagreatimportancenotonlyforuncoveringfundamentalaspectsof
organelleandcellfunction,butalsoforhumandisease.
This volume presents the recent advancements in the field of mitochondrial gene
expression: from mtDNA replication and transcription, through translation to membrane
insertion ofthe finalproteinproducts. Each chapter provides anoverviewofthe particular
process and a detailed protocol that can be applied to study this process by a variety of
research laboratories. Investigators who are new to the field are offered an opportunity to
accessafocusedcollectionofprotocolsforanentirerangeofmethods,whereasinvestigators
with previous experience in the field will find the volume useful for the perspective on
complementaryapproachesandfortheplethoraofpracticalguidelines,ofteninvaluablefor
asuccessfulexamination.
Wearemostgratefultotheauthorsfor theireffortsincontributingtothisvolumeand
generoussharingoftheirprotocolsandtechnicalexpertise.Wewouldliketoalsoacknowl-
edgethehelpofDelia-DenisaDunkaduringthefinalstagesofpreparationofthisvolume.
Cambridge,UK MichalMinczuk
Solna,Sweden JoannaRorbach
Stockholm,Sweden
v
Contents
Preface ..................................................................... v
Contributors................................................................. ix
1 InVitroAnalysisofmtDNAReplication................................... 1
JayP.UhlerandMariaFalkenberg
2 InVivoAnalysisofmtDNAReplicationattheSingleMoleculeLevel
andwithHighResolution................................................ 21
MarcoTigano,AaronFraserPhillips,andAgnelSfeir
3 InVitroReconstitutionofHumanMitochondrialTranscription.............. 35
AzadehSarfallahandDmitryTemiakov
4 InvestigatingMitochondrialTranscriptomesandRNAProcessingUsing
CircularRNASequencing................................................ 43
IrinaKuznetsova,OliverRackham,andAleksandraFilipovska
5 Detectionof5-formylcytosineinMitochondrialTranscriptome............... 59
LindseyVanHauteandMichalMinczuk
6 VisualizationofMitochondrialRNAGranulesinCulturedCells
Using5-BromouridineLabeling.......................................... 69
VanessaJoanneXavierandJean-ClaudeMartinou
7 QuantitativeProteomicsinDrosophilawithHolidicStable-Isotope
LabelingofAminoAcidsinFruitFlies(SILAF)............................. 75
FlorianA.Schober,IlianAtanassov,ChristophFreyer,andAnna
Wredenberg
8 MassSpectrometricAnalysisofMitochondrialRNAModifications............ 89
YumaIshigami,TsutomuSuzuki,andTakeoSuzuki
9 mito-Ψ-Seq:AHigh-ThroughputMethodforSystematicMapping
ofPseudouridineWithinMitochondrialRNA .............................. 103
AldemaSas-Chen,RonitNir,andSchragaSchwartz
10 High-ThroughputDetectionofmtDNAMutationsLeading
totRNAProcessingErrors............................................... 117
MaritaAnnikaIsokallioandJamesBruceStewart
11 High-ThroughputMeasurementofMitochondrialRNA
TurnoverinHumanCulturedCells....................................... 133
AnnaV.Kotrys,LukaszS.Borowski,andRomanJ.Szczesny
12 RNACrosslinkingtoAnalyzetheMitochondrialRNA-BindingProteome..... 147
SelmaL.vanEsveldandJohannesN.Spelbrink
13 VisualizingMitochondrialRibosomalRNAandMitochondrial
ProteinSynthesisinHumanCellLines.................................... 159
MatthewZorkau,YasminProctor-Kent,RolandoBerlinguer-Palmini,
AndrewHamilton,ZofiaM.Chrzanowska-Lightowlers,andRobertN.
Lightowlers
vii
viii Contents
14 MitoribosomeProfilingfromHumanCellCulture:AHighResolution
ViewofMitochondrialTranslation........................................ 183
SarahF.Pearce,MiriamCipullo,BettyChung,IanBrierley,
andJoannaRorbach
15 ApplicationofCryo-EMforVisualizationofMitoribosomes................. 197
VivekSinghandAlexeyAmunts
16 SucroseGradientSedimentationAnalysisofMitochondrialRibosomes........ 211
AustinChoiandAntoniBarrientos
17 TheAnalysisofYeastMitochondrialTranslation............................ 227
AndreasCarlstr¨om,MagdalenaRzepka,andMartinOtt
18 InSituStudiesofMitochondrialTranslationbyCryo-Electron
Tomography ........................................................... 243
RobertEnglmeierandFriedrichF¨orster
19 MitochondrialComplexomeProfiling..................................... 269
HeikoGiese,JanaMeisterknecht,JulianaHeidler,andIlkaWittig
20 Blue-NativeElectrophoresistoStudytheOXPHOSComplexes.............. 287
ErikaFernandez-VizarraandMassimoZeviani
21 IdentificationofPutativeMitochondrialProteaseSubstrates ................. 313
EduardHofsetz,PitterF.Huesgen,andAleksandraTrifunovic
Index ...................................................................... 331
Contributors
ALEXEYAMUNTS • ScienceforLifeLaboratory,DepartmentofBiochemistryandBiophysics,
StockholmUniversity,Solna,Sweden;DepartmentofMedicalBiochemistryandBiophysics,
KarolinskaInstitutet,Stockholm,Sweden
ILIANATANASSOV • ProteomicsCoreFacility,MaxPlanckInstituteforBiologyofAgeing,
Cologne,Germany
ANTONI BARRIENTOS • DepartmentofNeurology,UniversityofMiamiMillerSchoolof
Medicine,Miami,FL,USA;DepartmentofBiochemistryandMolecularBiology,
UniversityofMiamiMillerSchoolofMedicine,Miami,FL,USA
ROLANDO BERLINGUER-PALMINI • NewcastleUniversityBioimagingUnit,Newcastle
University,MedicalSchool,NewcastleUponTyne,UK
LUKASZS.BOROWSKI • InstituteofBiochemistryandBiophysics,PolishAcademyofSciences,
Warsaw,Poland;InstituteofGeneticsandBiotechnology,FacultyofBiology,Universityof
Warsaw,Warsaw,Poland
IANBRIERLEY • DepartmentofPathology,UniversityofCambridge,Cambridge,UK
ANDREASCARLSTRO¨M • DepartmentofBiochemistryandBiophysics,StockholmUniversity,
Stockholm,Sweden
AUSTIN CHOI • DepartmentofNeurology,UniversityofMiamiMillerSchoolofMedicine,
Miami,FL,USA
ZOFIAM.CHRZANOWSKA-LIGHTOWLERS • WellcomeCentreforMitochondrialResearch,
NewcastleUniversityBiosciencesInstitute,NewcastleUniversity,MedicalSchool,Newcastle
UponTyne,UK
BETTYCHUNG • DepartmentofPathology,UniversityofCambridge,Cambridge,UK
MIRIAMCIPULLO • DivisionofMolecularMetabolism,DepartmentofMedicalBiochemistry
andBiophysics,KarolinskaInstitutet,Solna,Sweden;MaxPlanckInstituteBiologyof
Ageing—KarolinskaInstitutetLaboratory,KarolinskaInstitutet,Stockholm,Sweden
ROBERTENGLMEIER • Cryo-ElectronMicroscopy,BijvoetCenter forBiomolecularResearch,
UtrechtUniversity,Utrecht,TheNetherlands
MARIAFALKENBERG • DepartmentofMedicalBiochemistryandCellBiology,Universityof
Gothenburg,Gothenburg,Sweden
ERIKAFERNANDEZ-VIZARRA • MRCMitochondrialBiologyUnit,UniversityofCambridge,
Cambridge,UK
ALEKSANDRAFILIPOVSKA • HarryPerkinsInstituteofMedicalResearch,Nedlands,WA,
Australia;ARCCentreofExcellenceinSyntheticBiology,QEIIMedicalCentre,Nedlands,
WA,Australia;CentreforMedicalResearch,TheUniversityofWesternAustralia,QEII
MedicalCentre,Nedlands,WA,Australia;TelethonKidsInstitute,NorthernEntrance,
PerthChildren’sHospital,Nedlands,WA,Australia;SchoolofMolecularSciences,The
UniversityofWesternAustralia,Crawley,WA,Australia
FRIEDRICH FO¨RSTER • Cryo-ElectronMicroscopy,BijvoetCenter forBiomolecularResearch,
UtrechtUniversity,Utrecht,TheNetherlands
CHRISTOPHFREYER • MaxPlanckInstituteBiologyofAgeing—KarolinskaInstitutet
Laboratory,DivisionofMolecularMetabolism,DepartmentofLaboratoryMedicine,
KarolinskaInstitutet,Stockholm,Sweden;DepartmentofMedicalBiochemistryand
ix
x Contributors
Biophysics,KarolinskaInstitutet,Stockholm,Sweden;CentreforInheritedMetabolic
Diseases,KarolinskaUniversityHospital,Stockholm,Sweden
HEIKOGIESE • MolecularBioinformatics,InstituteofComputerScience,Goethe-University,
FrankfurtamMain,Germany
ANDREWHAMILTON • SchoolofMedicine,DentistryandNursing,GlasgowUniversity,
Glasgow,UK
JULIANAHEIDLER • FunctionalProteomics,ZBC,Goethe-University,FrankfurtamMain,
Germany
EDUARDHOFSETZ • CologneExcellenceClusteronCellularStressResponsesinAging-
AssociatedDiseases(CECAD),UniversityofCologne,Cologne,Germany;Institutefor
MitochondrialDiseasesandAging,MedicalFaculty,UniversityofCologne,Cologne,
Germany
PITTERF.HUESGEN • CentralInstituteforEngineering,ElectronicsandAnalytics,ZEA-3,
ForschungszentrumJu¨lich,Ju¨lich,Germany
YUMAISHIGAMI • DepartmentofChemistryandBiotechnology,GraduateSchoolof
Engineering,UniversityofTokyo,Tokyo,Japan
MARITAANNIKAISOKALLIO • MaxPlanckInstituteforBiologyofAgeing,Cologne,Germany
ANNAV.KOTRYS • InstituteofBiochemistryandBiophysics,PolishAcademyofSciences,
Warsaw,Poland
IRINAKUZNETSOVA • HarryPerkinsInstituteofMedicalResearch,Nedlands,WA,Australia;
ARCCentreofExcellenceinSyntheticBiology,QEIIMedicalCentre,Nedlands,WA,
Australia;CentreforMedicalResearch,TheUniversityofWesternAustralia,QEII
MedicalCentre,Nedlands,WA,Australia
ROBERTN.LIGHTOWLERS • WellcomeCentreforMitochondrialResearch,Newcastle
UniversityBiosciencesInstitute,NewcastleUniversity,MedicalSchool,NewcastleUpon
Tyne,UK
JEAN-CLAUDEMARTINOU • DepartmentofCellBiology,UniversityofGeneva,Gene`ve,
Switzerland
JANA MEISTERKNECHT • FunctionalProteomics,ZBC,Goethe-University,Frankfurtam
Main,Germany
MICHAL MINCZUK • MedicalResearchCouncilMitochondrialBiologyUnit,Universityof
Cambridge,Cambridge,UK
RONITNIR • DepartmentofMolecularGenetics,WeizmannInstituteofScience,Rehovot,
Israel
MARTINOTT • DepartmentofBiochemistryandBiophysics,StockholmUniversity,Stockholm,
Sweden
SARAH F.PEARCE • DivisionofMolecularMetabolism,DepartmentofMedicalBiochemistry
andBiophysics,KarolinskaInstitutet,Solna,Sweden;MaxPlanckInstituteBiologyof
Ageing—KarolinskaInstitutetLaboratory,KarolinskaInstitutet,Stockholm,Sweden
AARONFRASERPHILLIPS • DepartmentofDevelopmentalGenetics,SkirballInstituteof
BiomolecularMedicine,NewYorkUniversitySchoolofMedicine,NewYork,NY,USA
YASMINPROCTOR-KENT • WellcomeCentreforMitochondrialResearch,NewcastleUniversity
BiosciencesInstitute,NewcastleUniversity,MedicalSchool,NewcastleUponTyne,UK