Chimeric RNA: Methods and Protocols

263 Pages·2020·7.225 MB·English

by  Hui Li, Justin Elfman

Methods in
Molecular Biology 2079
Hui Li · Justin Elfman Editors
Chimeric
RNA
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
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JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
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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
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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