RNA Tagging: Methods and Protocols

485 Pages·2020·13.6 MB·English

by  Manfred Heinlein

Methods in
Molecular Biology 2166
Manfred Heinlein Editor
RNA Tagging
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
Forfurther volumes:
http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
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indexedinPubMed.
RNA Tagging
Methods and Protocols
Edited by
Manfred Heinlein
Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France
Editor
ManfredHeinlein
InstitutdeBiologieMole´culairedes
Plantes(IBMP-CNRS)
Universite´deStrasbourg
Strasbourg,France
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-0711-4 ISBN978-1-0716-0712-1 (eBook)
https://doi.org/10.1007/978-1-0716-0712-1
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Preface
RNA molecules play diverse roles in the cell owing to their secondary structure and
interactionwithvariousproteinsandnucleicacids.Tostudythecomposition,localization,
dynamics,andfunctionofthesefunctionalRNAcomplexesinvitroorinlivecellsandtissues
ofanimalsandplantsrequiresthespecifictaggingoftheRNAorofRNA-bindingproteins
with suitable reporter molecules. This book provides a compendium of state-of-the-art
methodsfor thelabeling,detection,andpurificationofRNAandRNA–proteincomplexes
and thereby constitutes an important toolbox for researchers interested in understanding
thecomplexrolesofRNAmoleculesindevelopment,signaling,anddisease.Inadditionto
studyingthenaturalfunctionofRNAmoleculeswithincells,numerouslabshavedeveloped
methods to actually apply RNA molecules as guides for the sequence-specific cleavage,
modification, or imaging of RNA and DNA. Therefore, this book also includes protocols
thatapplyRNAmoleculesassequence-specificguidemolecules.
Theprotocolchaptersofthisbookareorganizedinsixparts.PartIprovidesprotocols
for the in situ detection of RNA molecules using fluorescent in situ hybridization (FISH)
techniques,whereasPartIIprovidesprotocolsforthetaggingofRNAmoleculesforspecific
detection in live cells by fluorescent RNA-binding proteins. A dedicated review article
describesthe imagingofRNAmoleculeswithfluorogensthat bind toRNAtargets tagged
with specific light-up RNA aptamers. Part III contains protocols for monitoring RNA
uptake by cells or for addressing RNA transport between cells. Important protocols useful
for the characterization of RNA-binding proteins and protein complexes are presented in
PartIV.PartV isdedicatedtoprotocolsfor theapplicationofRNAmoleculesasguidesin
RNA-mediatededitingand imagingofchromosomeloci.Starting withanoverviewarticle
about CRISPR guide RNA design for genome editing, this part continues by presenting
protocols in which RNA molecules are applied to guide nuclease-deactivated CRISPR-
associated protein 9 (dCas9) for the imaging of specific genome loci. Another interesting
protocolinthispartofthebookallowstheinvivomonitoringoftranscribedlocibyimaging
tagged nascent messenger RNA molecules emerging during gene transcription. The final
Part VI provides advanced protocols for the functional analysis of RNA molecules. These
includethedetectionofsmallRNAsinvolvedinRNAsilencingbyNorthernblothybridiza-
tion, the monitoring of miRNA-mediated RNA silencing events with an in vivo reporter
system,theisolationandcharacterizationofRNAmoleculesassociatedwithpolyribosomes,
aprotocolfortransfectionofRNAmoleculesintoplantprotoplasts,andalsothebiochemi-
calinvitromodificationandtaggingofRNAmoleculesforimagingandstructuralanalysis.
Theexperimentalprotocolsareprovidedbyleadingexpertswithhands-onexperiencein
the respective method. I hope that the provision of these protocols will further stimulate
researchinRNAbiologyandthattheresearchcommunityinterestedinthisfieldwillaccept
thisbookasanimportantreference.
Iamverygratefultoalltheauthorswhocontributedtothisbook.Ialsothanktheseries
editor,JohnM.Walker,forhiscontinuoussupportindevelopingthisvolume.
Strasbourg,France Manfred Heinlein
v
Contents
Preface ..................................................................... v
Contributors................................................................. xi
PART I IN SITU DETECTION OF RNA MOLECULES USING FISH TECHNIQUES
1 TaggingandApplicationofRNAProbesforSequence-Specific
VisualizationofRNAsbyFluorescentInSituHybridization ................. 3
ThomasDresselhausandAndreaBleckmann
2 QuantitativeFluorescenceInSituHybridizationDetection
ofPlantmRNAswithSingle-MoleculeResolution .......................... 23
KunHuang,MonaBatish,ChongTeng,AlexHarkess,
BlakeC.Meyers,andJeffreyL.Caplan
3 VisualizationofEndoplasmicReticulum-AssociatedmRNA
inMammalianCells..................................................... 35
JingzeJ.WuandAlexanderF.Palazzo
4 SimultaneousDetectionofmRNAandProteininS.cerevisiae
bySingle-MoleculeFISHandImmunofluorescence......................... 51
EvelinaTutucciandRobertH.Singer
PART II IN VIVO IMAGING OF RNATRANSPORT AND LOCALIZATION
5 DevelopmentandApplicationsofFluorogen/Light-UpRNAAptamer
PairsforRNADetectionandMore ....................................... 73
MichaelRyckelynck
6 VisualizationofTransientlyExpressedmRNAinPlantsUsingMS2........... 103
EduardoJose´Pen˜aandManfredHeinlein
7 NewGenerationsofMS2VariantsandMCPFusionstoDetect
SinglemRNAsinLivingEukaryoticCells.................................. 121
XavierPichon,Marie-Ce´cileRobert,EdouardBertrand,
RobertH.Singer,andEvelinaTutucci
8 InVivoImagingofMobilemRNAsinPlantsUsingMS2.................... 145
Kai-RenLuo,Nien-ChenHuang,andTien-ShinYu
9 RNAImagingwithRNase-InactivatedCsy4inPlantsandFilamentous
Fungi.................................................................. 157
DavidBurnett,AlexanderLichius,andJensTilsner
PART III IMAGING AND ANALYSIS OF RNA UPTAKE AND TRANSPORT
BETWEEN CELLS
10 UtilizingPotatoVirusXtoMonitorRNAMovement....................... 181
ZhimingYu,SungKiCho,PengchengZhang,
YiguoHong,andDavidJ.Hannapel
vii
viii Contents
11 AProtocolforNon-biasedIdentificationofRNAsTransferred
BetweenHeterologousMammalianCellTypesUsingRNATagging,
CellSorting,andSequencing............................................. 195
SandipanDasguptaandJeffreyE.Gerst
12 SynthesizingFluorescentlyLabeleddsRNAsandsRNAs
toVisualizeFungalRNAUptake ......................................... 215
RachaelHamby,MingWang,LuluQiao,andHailingJin
13 LabelingofdsRNAforFungalUptakeDetectionAnalysis ................... 227
MatteoGalli,JafargholiImani,andKarl-HeinzKogel
PART IV IDENTIFICATION AND ANALYSIS OF RNA-BINDING PROTEINS
14 UsingRNAAffinityPurificationFollowedbyMassSpectrometry
toIdentifyRNA-BindingProteins(RBPs) ................................. 241
MenggeShanandBrianD.Gregory
15 PlantIndividualNucleotideResolutionCross-Linking
andImmunoprecipitationtoCharacterizeRNA-ProteinComplexes .......... 255
TinoK¨osterandDorotheeStaiger
16 ASingle-MoleculeRNAMobilityAssaytoIdentifyProteins
thatLinkRNAstoMolecularMotors ..................................... 269
VarunBhaskar,MinJia,andJeffreyA.Chao
17 Proximity-CLIPProvidesaSnapshotofProtein-OccupiedRNA
ElementsatSubcellularResolutionandTranscriptome-Wide Scale............ 283
DanielBenhalevyandMarkusHafner
18 Double-StrandedRNAPull-DowntoCharacterizeViralReplication
ComplexesinPlants..................................................... 307
MarcoIncarboneandChristopheRitzenthaler
PART V RNAS AS GUIDES FOR GENOME EDITING AND IMAGING
OF CHROMOSOME LOCI
19 CRISPRGuideRNADesignGuidelinesforEfficientGenomeEditing........ 331
PatrickSchindele,FelixWolter,andHolgerPuchta
20 Live-CellCRISPRImaginginPlantCellswithaTelomere-Specific
GuideRNA............................................................ 343
SolmazKhosravi,StevenDreissig,PatrickSchindele,FelixWolter,
TwanRutten,HolgerPuchta,andAndreasHouben
21 Live-CellImagingofGenomicLociUsingCRISPR/Molecular
BeaconHybridSystems.................................................. 357
XiaotianWu,YachenYing,ShiqiMao,ChristopherJ.Krueger,
andAntonyK.Chen
22 UsingRNATagsforMulticolorLiveImagingofChromatinLoci
andTranscriptioninDrosophilaEmbryos ................................. 373
HongtaoChenandThomasGregor
Contents ix
PART VI SPECIFIC TECHNIQUES FOR THE ANALYSIS OF RNA FUNCTIONS
23 IntegratedGenome-ScaleAnalysisandNorthernBlotDetection
ofRetrotransposonsiRNAsAcrossPlantSpecies............................ 387
MarcelB¨ohrer,BartRymen,ChristopheHimber,AudeGerbaud,
DavidPflieger,DebbieLaudencia-Chingcuanco,AmyCartwright,
JohnVogel,RichardSibout,andToddBlevins
24 TransfectionofSmallNoncodingRNAsintoArabidopsisthaliana
Protoplasts............................................................. 413
Ste´phanieLalande,MarjorieChery,ElodieUbrig,GuillaumeHummel,
JulieKubina,Ange`leGeldreich,andLaurenceDrouard
25 InVivoReporterSystemfor miRNA-MediatedRNASilencing............... 431
Ka´rolyFa´tyol
26 TRAP-SEQofEukaryoticTranslatomesAppliedtotheDetection
ofPolysome-AssociatedLongNoncodingRNAs ........................... 451
SoledadTraubenik,FlavioBlanco,Marı´aEugeniaZanetti,
andMauricioA.Reynoso
27 PosttranscriptionalSuzuki-MiyauraCross-CouplingYieldsLabeled
RNAforConformationalAnalysisandImaging ............................ 473
ManishaB.WalunjandSeergazhiG.Srivatsan
Index ...................................................................... 487
Contributors
MONABATISH • DepartmentsofBiologicalSciencesandMedical&MolecularSciences,
UniversityofDelaware,Newark,DE,USA
DANIELBENHALEVY • LaboratoryofMuscleStemCellsandGeneRegulation,National
InstituteofArthritisandMusculoskeletalandSkinDiseases,NationalInstitutesofHealth,
Bethesda,MD,USA
EDOUARDBERTRAND • InstitutdeGe´ne´tiqueMole´culairedeMontpellier,Universite´de
Montpellier,CNRS,Montpellier,France;DepartmentofAnatomyandStructuralBiology,
AlbertEinsteinCollegeofMedicine,Bronx,NY,USA
VARUNBHASKAR • FriedrichMiescherInstituteforBiomedicalResearch,Basel,Switzerland
FLAVIO BLANCO • InstitutodeBiotecnologı´ayBiologı´aMolecular,FacultaddeCiencias
Exactas,UniversidadNacionaldeLaPlata,CentroCientı´ficoyTecnol(cid:1)ogico-LaPlata,
ConsejoNacionaldeInvestigacionesCientı´ficasyTe´cnicas,LaPlata,Argentina
ANDREA BLECKMANN • CellBiologyandPlantBiochemistry,RegensburgCenter for
Biochemistry,UniversityofRegensburg,Regensburg,Germany
TODDBLEVINS • InstitutdeBiologieMole´culairedesPlantes,CNRSUPR2357,Universite´de
Strasbourg,Strasbourg,France
MARCELBO¨HRER • InstitutdeBiologieMole´culairedesPlantes,CNRSUPR2357,Universite´
deStrasbourg,Strasbourg,France
DAVIDBURNETT • CellandMolecularSciences,TheJamesHuttonInstitute,Dundee,UK;
BiomedicalSciencesResearchComplex,TheUniversityofStAndrews,St.Andrews,UK
JEFFREYL.CAPLAN • DepartmentofPlantandSoilSciences,UniversityofDelaware,
Newark,DE,USA;DelawareBiotechnologyInstitute,UniversityofDelaware,Newark,
DE,USA
AMYCARTWRIGHT • DOEJointGenomeInstitute,LawrenceBerkeleyNationalLaboratory,
Berkeley,CA,USA
JEFFREYA.CHAO • FriedrichMiescherInstituteforBiomedicalResearch,Basel,Switzerland
ANTONYK.CHEN • DepartmentofBiomedicalEngineering,CollegeofEngineering,Peking
University,Beijing,China
HONGTAOCHEN • Lewis-SiglerInstituteforIntegrativeGenomics,PrincetonUniversity,
Princeton,NJ,USA
MARJORIECHERY • InstitutdeBiologieMole´culairedesPlantes—CNRS,Universite´de
Strasbourg,Strasbourg,France
SUNG KICHO • Dura-LineCorporation,Clinton,TN,USA
SANDIPANDASGUPTA • DepartmentofMolecularGenetics,WeizmannInstituteofScience,
Rehovot,Israel
STEVENDREISSIG • LeibnizInstituteofPlantGeneticsandCropPlantResearch(IPK),
Gatersleben,Germany
THOMASDRESSELHAUS • CellBiologyandPlantBiochemistry,RegensburgCenter for
Biochemistry,UniversityofRegensburg,Regensburg,Germany
LAURENCEDROUARD • InstitutdeBiologieMole´culairedesPlantes—CNRS,Universite´de
Strasbourg,Strasbourg,France
KA´ROLYFA´TYOL • AgriculturalBiotechnologyInstitute,NationalAgriculturalResearchand
InnovationCentre,Go¨do¨ll˝o,Hungary
xi