MuchofthebasicbiologyofmRNAeditingremainsunknowndespitedecadesofstudy,largelydueto thelimitationsofexistingexperimentalmethods.AdenosinetoInosine(AtoI)editingaresultofadenosine deaminationcatalyzedbydeaminasessuchasADARenzymesisoneofthemostcommonformsof posttranscriptionalchemicalmodificationofRNA,generallyknownasRNAediting.AtoIeditingisessential fornotonlynoncodingRNAfunction,suchasrRNAandtRNAhigherorderstructurestabilization,butalsofor posttranscriptionalmRNAregulation.Forexample,GRIA2,whichencodestheGluR2subunitoftheAMPA glutamatereceptor,hasahighlyconservedADAR2targetededitingsiteinitscodingsequence.Perturbationof theorthologousratGria2editingeventleadstoneurologicaldysfunction.InhumanswithAmyotrophicLateral Sclerosis(ALS),manydiseaseaffectedmotorneuronsappeartodieasaresultofglutamatergiccalcium toxicityandhavedeficientGRIA2editing,andAdar2knockoutmicedemonstrateanALSlikephenotype,which isrescueduponexogenousexpressionofeditedGria2.GRIA2andseveralothermRNAAtoIeditingtargets havebeenstudiedformorethantwentyyears,andoverthelastfouryearstranscriptomewidesurveysofAto IeditinghaveprovidedevidenceformanymoremRNAtargets.Despitethismotivation,muchbasicinformation aboutthebiologyofthisimportantmRNAregulatoryprocess,includingitssubcellularlocalization,timing relativetotranscription,andassociationwithotherregulatoryfactors,remainsunknownlargelyduetothelimits ofexperimentaltechniques. Recently,ourlaboratorydescribedafluorescence?insitu?hybridizationmethodforvisualizingand quantifyingsinglenucleotidevariants(SNVs)insinglecellsusinganovelprobehybridizationstrategyknown asSNVFISH.Weareleveraginginosine'sstructureasaguanosineanalogtoadaptSNVFISHtotheanalysis ofAtoIeditinginaprotocolwerefertoasinosineFISH(iFISH).Ourpreliminarydatashowthatwecanuse iFISHtospecificallyidentifyeditedanduneditedGRIA2transcriptsi?nsitu?inculturedcells.Inthisproject,we arequantifyingsubsinglecellulartrendsinAtoIeditingofknowntargets,suchasGRIA2andmRNAtargets newlyidentifiedintranscriptomewidescreens.Specifically,weareusingiFISHtomeasuresinglecellediting ratedistributions,tovisualizesubcellularlocalizationpatternsofeditedmRNAs,andtomeasureeditingtiming relativetotranscription.WearealsostudyingtheeffectofADAR2onsubcellularGRIA2trafficking.Lastly,we areapplyingournovelmethodtowardbettercharacterizingperturbationsofGRIA2editinginindividualmotor neuronsinALSlesionsbyextendingtheuseofiFISHtofixedsectionsofpostmortembrainandspinalcord tissuesamplesfromhealthyanddiseaseddonors.
RNAeditingisafamilyofenzymemediatedchemicalchangestoRNA,perturbationsofwhichare associatedwithseveraldiseases,includingAmyotrophicLateralSclerosis(ALS)andepilepsy.Current molecularbiologicaltechniquesusedtostudyRNAeditingarelimitedinresolution?theycanonlydetectgross changesinlargepopulationsofcellsandinoverfifteenyearsofusehavenotbeenabletorevealprecise mechanismsunderlyingtheseintriguingdiseaseassociations.Wehavedevelopedatooltovisualizeindividual RNAeditingeventsinsinglecells,weareengineeringtissueprocessingtechniquestoenableitsuseinfixed brainandspinalcordsamples,andwewillapplyourmethodtowardbettercharacterizingtheregulationof RNAeditingingeneralaswellasspecificeditingchangesthatmayleadtomotorneurondeathinpatientswith ALS.
Rouhanifard, Sara H; Mellis, Ian A; Dunagin, Margaret et al. (2018) ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification. Nat Biotechnol : |
Mellis, Ian A; Gupte, Rohit; Raj, Arjun et al. (2017) Visualizing adenosine-to-inosine RNA editing in single mammalian cells. Nat Methods 14:801-804 |