Chimeric RNA: Methods and Protocols PDF
Preview Chimeric RNA: Methods and Protocols
Methods in Molecular Biology 2079 Hui Li · Justin Elfman Editors Chimeric RNA 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 methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Chimeric RNA Methods and Protocols Edited by Hui Li Department of Pathology, School of Medicine, University of Virginia, Charlottesville, VA, USA; Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA, USA Justin Elfman Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA Editors HuiLi JustinElfman DepartmentofPathology DepartmentofBiochemistry SchoolofMedicine andMolecularGenetics UniversityofVirginia UniversityofVirginia Charlottesville,VA,USA Charlottesville,VA,USA DepartmentofBiochemistry andMolecularGenetics SchoolofMedicine UniversityofVirginia Charlottesville,VA,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-9903-3 ISBN978-1-4939-9904-0 (eBook) https://doi.org/10.1007/978-1-4939-9904-0 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2020 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. 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Preface Gene fusions are considered hallmarks of many cancer types, associated with particular cytogenetic signatures, which can serve as markers for disease detection or targets for treatment. These are perhaps best characterized by the first published recurring chromo- somal abnormality in cancer: the Philadelphia chromosome, comprised by the fusion of BCR and ABL1. The abnormality was first discovered in 1960 as an undersized chromo- some in chronic myeloid leukemia patient samples. While the reciprocal translocation that gives rise to the Philadelphia chromosome provides a drastic change to chromosome structure,themostsignificanteffectstemsfromtheBCR-ABL1genefusion,whichencodes foranaltered,constitutivelyactiveABL1kinase. The chimeric RNA and protein products which result from gene fusion, such as the BCR-ABL1transcriptanditscorrespondingfusionprotein,oftenhaveimportantimpactsin the progression of neoplasia. Other examples include TMPRSS2-ETS in prostate cancer, PAX3-FOXO1 in alveolar rhabdomyosarcoma, MYC-IGH in Burkitt’s lymphoma, and JAZF1-JJAZ1inendometrialstromalsarcoma.Oncogenicgenefusionsmostofteninclude transcription factors or kinases, whose dysregulation can lead to substantial downstream effects. Importantly, chimeric RNAs have increasingly been found without corresponding changes to the genome. These non-canonical, intergenically spliced chimeric RNAs can ariseviatrans-splicingofprecursor mRNAsor viacis-splicingofadjacentgenes(cis-SAGe) (Fig. 1). Perhaps most interestingly, intergenically spliced chimeric RNAs have been detected in a variety of healthy tissues and cell lines, and some have demonstrated impor- tancetocellhealth,proliferation,andmotility.Additionally,chimericRNAsdonotstrictly followtheexpressionpatternsoftheirparentalgenes,andsomeshowconsiderablecell-type specificity. In parallel to alternative splicing, this particular class of chimeric RNAs presents yetanother meansforexpansionanddiversificationofthefunctionalgenome. A common thread that runs through these findings is simply the pervasiveness of chimericRNAs.Theydonotseemtoberestrictedtoanyparticularfunctionalityorpurpose; rather, theyseem to play rolesin anabundance ofnormaland abnormalcellularprocesses. Our current knowledge stems from a combination of candidate-based studies and larger scale studies utilizing RNA-seq datasets from projects such as TCGA and GTEx. In fact, interest in the field has increased substantially with the advent of and accessibility to next- generation sequencing technologies (Fig. 2). Notably, over 40 software tools have been developedtofacilitatetheeffort.DespitethevarietyofapproachesappliedtochimericRNA researchthusfar,thereremainsignificantgapsinourknowledge. Much of this research can be approached through careful application of standard methods in molecular biology and biochemistry. However, the inherent homology of chimeric RNAs to their parental genes presents notable hurdles which must be overcome. First, bioinformatic predictions of chimeric RNAs are susceptible to false-positive predic- tions. We have found that these are often due to inconsistency in transcript annotations or regionsofcommonhomologyamongpredictedparentalgenes.Additionally,non-canonical chimericRNAstendtoexhibitlowlevelsofexpressionwhencomparedtomostothergenes, whichcanplacemorepredictiveweightonfewer mappablereadsandcontributetounder- estimation of chimeric RNA detection in individual samples. This homology presents vi Preface Fig. 1 Mechanisms of chimeric RNA generation. (a) Canonical chimeric RNA. Chromosomal rearrangement resulting in a gene fusion. Transcription from the gene fusion creates a chimeric RNA. (b) Trans-spliced chimeric RNA. Two precursor mRNAs are transcribed from parental genes A and B and are trans-spliced together to form a chimeric transcript. (c) Cis-spliced chimeric RNA. A readthrough transcript is generated fromgeneXintogeneYandisalternativelysplicedintoachimericRNA further challenges with regard to specificity in sequence-targeted assays, such as RNAi or RNA-FISH, as the only section of truly unique sequence occurs at the chimeric junction. Moreover, chimeric RNAs have the potential to arise due to template-switching during elongation processes in reverse transcription and PCR. Each of these limitations warrants carefulconsiderationinexperimentaldesign. Perhapsduetothefield’sinfancy,terminologyregardingchimericRNAscanbeincon- sistent.Numerousother termshavebeenappliedtochimericRNAs,suchastranscription- mediated fusions, gene fusions, conjoined genes, complex genes, cotranscribed genes, spanning genes, hybrid genes, tandem chimerism, and fusion transcripts. Within this book, we define chimeric RNAs or fusion transcripts as any transcript which contains the nucleotide sequence of two distinct parental genes. Gene fusions, on the other hand, refer specificallytochangesintheDNAsequence,whichresultsinageneconsistingofnucleotide sequence from two parental genes. Our definition for chimeric RNA includes both trans- splicedandcis-splicedtranscripts,aswellasRNAstranscribedfromgenefusions. In this book, we provide numerous methods for identification, validation, and func- tionalcharacterizationofchimericRNAs.Tocovertheseareas,wehaveorganizedthebook into four parts. In Part I, we present five chapters dedicated to the identification and characterizationofchimericRNAsusingbioinformatictools.PartIIincludesfourchapters relatedtoexperimentalvalidationusingRT-PCR,hybridizationmethodsforRNA,andthe Preface vii Fig.2PublicationrateofarticlesrelatedtochimericRNA.AnumberofpublicationswereobtainedviaGoogle Scholar using search terms including any of the following: chimeric RNA, chimeric transcript, fusion RNA, fusiontranscript use of mass spectrometry data for fusion protein validation. Five chapters in Part III are relatedtofunctionalandmechanisticstudiesofchimericRNAs,andthefinalfourchapters provide a sampling of translational applications ranging from exosome-based liquid biopsy andSMaRTfortherapytotwocasestudiesofchimericRNAsinbladdercancerandnormal physiology. Each chapter contains advice for overcoming common hurdles associated with studying chimeric RNAs, as well as specific criteria we use to enrich for likely true targets, drawing from the summed wealth of experience from experts in the field. These chapters describenovelapproaches tochimericRNAstudyand demonstratehowmanyof thebasic procedurescontainedhereincanbeexpandedupontoachieveremarkableresults. Wehopethatthisbookservesasavaluableresourceforthoseinterestedinenteringthe fieldofchimericRNAsaswellasthoselookingtoexpandupontheirexpertise. Weextendourgratitudetoalloftheauthorsfor theircontributionandtime,aswellas to Prof. John Walker from Humana Press for the opportunity to compile and share our knowledgewiththescientificcommunity.Lastly,ourfieldofstudyisexpansive,andthereis anexcessofoutstandingresearchthatwecouldnothopetocoverinthisvolume.Weurge thereadertoexploreandlearnfromthewealthoftalentedresearcherswhoarenotfeatured herein. Charlottesville,VA,USA HuiLi JustinElfman Contents Preface ..................................................................... v Contributors................................................................. xi PART I IDENTIFICATION AND CHARACTERIZATION 1 Prediction,Characterization,andInSilicoValidationof ChimericRNAs......................................................... 3 SandeepSinghandHuiLi 2 IdentificationofFusionTranscriptsfromUnalignedRNA-Seq ReadsUsingChimeRScope .............................................. 13 NeethaNanothVellichirammal,AbrarAlbahrani,YouLi, andChittibabuGuda 3 IdentificationofChimericRNAsUsingRNA-SeqReadsand Protein–ProteinInteractionsofTranslatedChimeras........................ 27 MilanaFrenkel-Morgenstern 4 GeneFusionDiscoverywithINTEGRATE ................................ 41 JinZhangandChristopherA.Maher 5 CaseStudy:SystematicDetectionandPrioritizationofGene FusionsinCancerbyRNA-Seq:ADIYToolkit............................. 69 PankajVats,ArulM.Chinnaiyan,andChandanKumar-Sinha PART II EXPERIMENTAL VALIDATION 6 DetectionandMeasurementofChimericRNAsbyRT-PCR................. 83 JustinElfmanandHuiLi 7 DetectionofGroupIIIntron-GeneratedChimericmRNAs inBacterialCells........................................................ 95 Fe´lixLaRoche-Johnston,CarolineMonat,andBenoitCousineau 8 RNaseProtectionAssay.................................................. 109 JianzhuZhao,JunTang,JustinElfman,andHuiLi 9 ValidationofChimericFusionPeptidesUsingProteomicsData .............. 117 SandeepSinghandHuiLi 10 NanoStringnCounterTechnology:High-ThroughputRNAValidation ....... 125 AngelaGoytainandTonyNg PART III FUNCTIONAL AND MECHANISTIC STUDIES 11 KnockdownofChimericRNAbyRNAi................................... 143 FujunQin,XinruiShi,andHuiLi 12 OverexpressionofChimericRNAbyRetroviralTransduction................ 155 HaoWuandHuiLi ix x Contents 13 SeparationofNuclearandCytoplasmicFractionsforChimeric RNACharacterization................................................... 167 FujunQin,XinruiShi,andHuiLi 14 ConfirmationofTranscriptionalRead-ThroughEventsbyRT-PCR........... 177 XinruiShi,FujunQin,andHuiLi 15 ValidatingGeneFusionastheSourceofChimericRNAs .................... 187 SachinKumarGupta,JocelynDuen-YaJea,andLaisingYen PART IV TRANSLATIONAL APPLICATIONS 16 ChimericRNAandExosomes-BasedLiquidBiopsy......................... 211 XiurongKe,XiaoXiong,YushengLin,andHaoZhang 17 SMaRTforTherapeuticPurposes......................................... 219 LisaM.Riedmayr 18 CaseStudy:LandscapeofChimericRNAsinBladderCancer ................ 233 DingjunZhuandHuiLi 19 CaseStudy:TheRecurrentFusionRNADUS4L-BCAP29 inNoncancerHumanTissuesandCells ................................... 243 YueTang,FangxiaGuan,andHuiLi Index ...................................................................... 259
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