Table Of ContentNucleic Acids and Molecular Biology
Volume 28
Series Editor
JanuszM. Bujnicki
International InstituteofMolecular
and Cell Biology
Laboratory ofBioinformatics and
Protein Engineering
Trojdena 4
02-109 Warsaw
Poland
Forfurthervolumes:
http://www.springer.com/series/881
.
Albrecht Bindereif
Editor
RNA Metabolism in
Trypanosomes
Editor
AlbrechtBindereif
DepartmentofBiologyandChemistry
JustusLiebigUniversityofGiessen
Giessen
Germany
ISSN0933-1891 ISSN1869-2486(electronic)
ISBN978-3-642-28686-5 ISBN978-3-642-28687-2(eBook)
DOI10.1007/978-3-642-28687-2
SpringerHeidelbergNewYorkDordrechtLondon
LibraryofCongressControlNumber:2012940659
#Springer-VerlagBerlinHeidelberg2012
Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartof
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation,broadcasting,reproductiononmicrofilmsorinanyotherphysicalway,andtransmissionor
informationstorageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilar
methodologynowknownorhereafterdeveloped.Exemptedfromthislegalreservationarebriefexcerpts
inconnectionwithreviewsorscholarlyanalysisormaterialsuppliedspecificallyforthepurposeofbeing
enteredandexecutedonacomputersystem,forexclusiveusebythepurchaserofthework.Duplication
ofthispublicationorpartsthereofispermittedonlyundertheprovisionsoftheCopyrightLawofthe
Publisher’s location, in its current version, and permission for use must always be obtained from
Springer.PermissionsforusemaybeobtainedthroughRightsLinkattheCopyrightClearanceCenter.
ViolationsareliabletoprosecutionundertherespectiveCopyrightLaw.
Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublication
doesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevant
protectivelawsandregulationsandthereforefreeforgeneraluse.
Whiletheadviceandinformationinthisbookarebelievedtobetrueandaccurateatthedateofpublication,
neithertheauthorsnortheeditorsnorthepublishercanacceptanylegalresponsibilityforanyerrorsor
omissionsthatmaybemade.Thepublishermakesnowarranty,expressorimplied,withrespecttothe
materialcontainedherein.
Printedonacid-freepaper
SpringerispartofSpringerScience+BusinessMedia(www.springer.com)
Contents
1 RNAPolymerasesandTranscriptionFactorsofTrypanosomes. . . 1
ArthurG€unzl
2 SLRNABiogenesisinKinetoplastids:ALongandWinding
Road. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
NancyR.Sturm,JesseR.Zamudio,andDavidA.Campbell
3 Pre-mRNASplicinginTrypanosomabrucei:Factors,
Mechanisms,andRegulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
ChristianPreußer,NicolasJae´,ArthurG€unzl,andAlbrechtBindereif
4 mRNATurnoverinTrypanosomes. . . . . . . . . . . . . . . . . . . . . . . . . 79
ChristineClayton
5 tRNABiogenesisandProcessing. . . . . . . . . . . . . . . . . . . . . . . . . . . 99
JessicaL.Spears,MaryAnneT.Rubio,PaulJ.Sample,
andJuanD.Alfonzo
6 rRNABiogenesisinTrypanosomes. . . . . . . . . . . . . . . . . . . . . . . . . 123
ShulamitMichaeli
7 RNAEditinginAfricanTrypanosomes:AU-ser’sG-U-ide. . . . . . 149
H.UlrichG€oringer
8 TheRNAInterferencePathwayinTrypanosomabrucei. . . . . . . . . 167
ElisabettaUllu,NikolayG.Kolev,RebeccaL.Barnes,
andChristianTschudi
9 TranslationinTrypanosomatids. . . . . . . . . . . . . . . . . . . . . . . . . . . 187
MichalShapiraandAlexandraZinoviev
v
vi Contents
10 MitochondrialTranslationinTrypanosomatids. . . . . . . . . . . . . . . 215
DmitriA.MaslovandRajendraK.Agrawal
11 RNA-SeqAnalysisoftheTranscriptomeofTrypanosomabrucei. . . 237
JanMani,KapilaGunasekera,andIsabelRoditi
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Chapter 1
RNA Polymerases and Transcription Factors
of Trypanosomes
ArthurGu€nzl
Abstract RNAsynthesisintrypanosomatidorganismsdeviatessubstantiallyfrom
what we see in model organisms. In these parasites, protein-coding genes are
arranged in long tandem arrays that are polycistronically transcribed by an unre-
solvedmodeoftranscriptioninitiation.Moreover,theAfricanTrypanosomabrucei
hasevolvedamultifunctionalRNApolymeraseIsystemwhichitemploysforpre-
rRNAsynthesisaswellasfortranscriptionofspecificgeneunitsencodingitsmajor
cell surface antigens. Additionally, the trypanosomatid RNA polymerase III sys-
tem, by relying mostly on bidirectional tRNA gene promoters, exhibits clear
differences to other eukaryotic systems. Interestingly, annotation of completed
trypanosomatidgenomesrevealedonlyasmallsubsetofbasaltranscriptionfactors
(BTFs),whichsuggestedthattrypanosomeshaveasimplifiedtranscriptionmachin-
ery. Recent research, however, has demonstrated that trypanosomes possess
extremely divergent orthologs of most BTFs and that these factors deviate in
manyaspectsfromtheirhumancounterparts;theyarethefocusofthischapter.
1.1 Introduction
Trypanosomatids represent a phylogenetically early diverged, protistan family of
parasiteswhichincludethehuman-pathogenicTrypanosomabrucei,Trypanosoma
cruzi, and Leishmania major (“Tritryp”). The transcriptional processes in these
organismsdiffersubstantiallyfromthosedescribedinthemodelsystemsofhuman
cellsandofthebuddingyeastSaccharomycescerevisiae.Inthemodelsystems,the
A.G€unzl(*)
DepartmentofGeneticsandDevelopmentalBiology,UniversityofConnecticutHealthCenter,
400FarmingtonAvenue,Farmington,CT06030,USA
DepartmentofMolecular,MicrobialandStructuralBiology,UniversityofConnecticutHealth
Center,263FarmingtonAvenue,Farmington,CT06030,USA
e-mail:[email protected]
A.Bindereif(ed.),RNAMetabolisminTrypanosomes,NucleicAcids 1
andMolecularBiology28,DOI10.1007/978-3-642-28687-2_1,
#Springer-VerlagBerlinHeidelberg2012
2 A.G€unzl
threecanonicalnuclearRNApolymerases(pols)I,II,andIIIperformdistincttasks:
RNA pol I transcribes exclusively the large ribosomal gene units (RRNA) in the
nucleolus, RNA pol II all protein-coding genes and some small RNA genes, and
RNA pol III genes encoding tRNAs, 5S rRNA, and remaining small RNAs.
Transcriptionistypicallymonocistronicandeachgeneincludesspecifictranscrip-
tion initiation and termination sites. In each case, conserved basal transcription
factors (BTFs) bind to core promoters, recruit RNA pols to specific transcription
initiation sites (TISs), and facilitate correctly initiated transcription. Conversely,
trypanosomatid protein-coding genes are organized in long tandem arrays, which
aretranscribedpolycistronicallywithtranscriptioninitiationatthesearraysappar-
ently not depending on classical promoters and BTF assembly. Furthermore, the
African trypanosome T. brucei has evolved a multifunctional RNA pol I system,
which, in addition to rRNA synthesis, is utilized for the transcription of specific
protein-coding gene units. Finally, small nuclear and cytoplasmic RNA genes are
transcribedbyRNApolIIIfromunique,bidirectionaltRNAgenepromoters.
Polycistronic as well as RNA pol I-mediated transcription of protein-coding
genesintrypanosomesisbasedonanatypicalmodeofmRNAprocessinginwhich
polycistronic precursor RNAs are resolved into individual mRNAs by spliced
leader (SL) trans splicing and polyadenylation. In trans splicing, the capped 50
terminalregionofthesmallnuclearSLRNA,referredtoastheSLormini-exon,is
transferred onto the 50 end of each mRNA. SL trans splicing is therefore a
posttranscriptionalcappingmechanismwhichisincontrasttotheco-transcriptional
cappingmodeinyeastandhighereukaryotes.Inthosesystems,themRNAcapping
enzymephysicallyinteractswiththecarboxy-terminaldomain(CTD)ofthelargest
RNA pol II subunit RPB1 (from here on referred to as “CTD”) and cotranscrip-
tionally caps pre-mRNA (Bentley 2005). This enzyme association has two
consequences: pre-mRNA is exclusively synthesized by RNA pol II, because the
cappingenzymedoesnotassociatewithotherRNApols,andprotein-codinggenes
are transcribed monocistronically because the capping enzyme requires a free 50
end.However,intrypanosomatids,SLtranssplicinguncouplescappingfromRNA
polIItranscriptiontherebyeliminatingtherequirementforafreeRNAendaswell
as enabling RNA polIto productively express functional mRNA. This difference
betweentrypanosomatidsandothereukaryotesbecomesapparentwhentheRRNA
promoterisused toexpressareporter gene.Inthemousesystem,thisexperiment
yielded virtually no reporter protein despite high transcription rates (Grummt and
Skinner 1985), whereas in trypanosomes the RRNA promoter directed the expres-
sion of functional mRNA and very high reporter levels (Zomerdijk et al. 1991;
Rudenkoetal.1991).
Transcriptionalstudiesintrypanosomatidsfirstfocused onpromotercharacter-
izations. These studies were conducted more than a decade ago and have been
reviewed several times since, most recently by Martinez-Calvillo et al. (2010),
G€unzletal.(2007),andPalencharandBellofatto(2006).Promoterstructureswill,
therefore, only be briefly discussed in this chapter. In contrast, our knowledge of
RNA pol subunits and transcription factors stems predominantly from recent
research.WhilethelargestRNApolsubunitswereidentifiedearlyon(K€ocketal.
1 RNAPolymerasesandTranscriptionFactorsofTrypanosomes 3
1988;Jessetal.1989;Eversetal.1989;Smithetal.1989a,b),thefirsttranscription
factorwasonlycharacterizedin2003(DasandBellofatto2003).Sincethen,RNA
pol complexes and several transcription factors have been characterized in detail;
together they currently account for an impressive repertoire of 80 proteins
(Tables 1.1 and 1.2). Finding such a large number of transcriptional proteins was
unexpectedbecauseannotationofthecompletedTriTrypgenomes(Berrimanetal.
2005;ElSayedetal.2005;Ivensetal.2005)revealedonlyveryfeworthologsof
knowntranscriptionfactors.Nowweknowthatthesefactorsequencesareextremely
divergentfromtheireukaryoticcounterparts,whichhasmadebioinformaticidenti-
ficationnearlyimpossible.Thus,mostofthetranscriptionalproteinswereidentified
biochemicallyaspartofproteincomplexes.Theadventoftandemaffinitypurifica-
tiontechnologyenabledefficientproteincomplexisolationatnearlyphysiological
conditionsandinamountsthatcouldbeanalyzedbyliquidchromatography–tandem
massspectrometry(LC/MS/MS)(Rigautetal.1999;G€unzlandSchimanski2009).
Whiletheoriginalmethodprovedtobeeffectiveforsometranscriptioncomplexes,
mosttranscriptionfactorswerepurifiedbyamodifiedapproach,whichdependedon
a new epitope combination, termed PTP, that was specifically developed for the
isolation of complexes from transcriptionally active extracts (Schimanski et al.
2005b). Most of these studies were carried out on T. brucei and therefore the
trypanosometranscriptionmachineryisthefocusofthischapter.
1.2 RNA pol II Transcription
Except for gene units encoding the major cell surface antigens variant surface
glycoprotein (VSG) and procyclin in T. brucei, trypanosomatid RNA pol II
transcribes protein-coding gene arrays, which also contain genes encoding small
nucleolarRNAsand,asrecentlyshown,asingletRNAgene(Aebyetal.2010).The
onlyotherknownRNApolIItranscriptionunitistheSLRNAgene(SLRNA).
1.2.1 RNA pol II
Across eukaryotes, RNA pol II consists of 12 highly conserved subunits termed
RPB1-12. The two largest proteins RPB1 and RPB2 form the catalytic core, the
subunitsRPB3,10,11,and12representanassemblyplatformoftheenzyme,while
theremainingsubunitshavespecializedfunctionsinthetranscriptionprocess.The
subunitsRPB5,6,9,10,and12aresharedbetweenallthreeeukaryoticRNApols,
whereasthereareparalogoussubunitsoftheothersevensubunitsinRNApolsIand
III. RPB1-12 are highly conserved with orthologs present in archaea (Werner
2007),andthereforeallofthemcouldbeidentifiedinsilicointheTritrypgenomes
(Table1.1)(Kellyetal.2005).Interestingly,trypanosomatidsharbortwodivergent
sets of the shared subunits RPB5, 6, and 10, and due to trypanosomatid-specific
4 A.G€unzl
0 B.
Others BDF3Tb11.01.1830 FACTSSRP1Tb927.10.1439FACTSpt16Tb927.3.5620TFIIS1Tb11.02.2600TFIIS2-1Tb927.2.3580TFIIS2-2Tb927.2.3480 p://www.GeneD
htt
at
dgs
eveolo
Med-T Med-T1Tb11.01.7350 Med-T2Tb927.4.3030Med-T3Tb927.3.5170Med-T4Tb927.8.2360Med-T5Tb11.03.0430Med-T6Tb11.02.4940 Med-T7Tb09.211.2270Med-T8Tb927.4.2960cMed-T9XP_951672 cescanberetrinosomatidorth
na
ep
uy
eqtr
so
dt
aTFIIH XPBTb927.3.5100 XPDTb927.8.5980b‘p62’Tb11.01.1200b‘p52’Tb10.70.1900b‘p44’Tb927.8.6540b‘p34’Tb11.01.7730 TFB5Tb10.61.2600TSP1Tb927.1.1080TSP2Tb11.01.5700 ed.Nucleotideandaminoacithesewebsitesprovidelinks sizesofthehumanproteinsTritrypDBdatabases
T.bruceiRNApolIIsubunitsandtranscriptionfactorsTable1.1 /TFIIATFIIBRNApolIITRF4/SNAPc RPB1TRF4TFIIBTb927.4.5020Tb927.10.15950Tb09.160.4220Tb927.8.7400RPB2SNAP50Tb927.4.3810Tb09.211.1510RPB3SNAP2Tb927.3.5500Tb927.5.3910RPB4SNAP3Tb927.3.5270Tb927.10.7070RPB5TFIIA-2Tb927.10.13320Tb927.10.4840RPB6TFIIA-1/TAF?Tb927.4.3490Tb927.10.15570Tb927.4.3510RPB7Tb11.01.6090RPB8Tb11.02.5790RPB9Tb11.02.5180dRPB10“Tb11.02.1185”RPB11Tb11.57.0004RPB12Tb927.1.1170grayProteinshighlightedinhavebeenexperimentallyidentified/characterizorgandhttp://TritrypDB.orgwithaccessionnumberscited.Foreachgene,aHumannomenclaturebTheapparentsizesofthetrypanosomeorthologsdonotcorrespondtothecNCBIaccessionnumber;thegenehasnotbeenidentifiedintheGeneDB/RPB10T.bruceidThegenehasnotbeenannotatedinthegenome
