Regulatory elements in the 3 ' untranslated region of the GP82 glycoprotein are responsible for its stage-specific expression in Trypanosoma cruzi metacyclic trypomastigotes

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ActaTropica123 (2012) 230–233

ContentslistsavailableatSciVerseScienceDirect

Acta

Tropica

j ou rn a l h o m e pa g e: w w w . e l s e v i e r . c o m / l o c a t e/ a c t a t r o p i c a

Regulatory

elements

in

the

3

′

_untranslated

_region

_of

_the

_GP82

_glycoprotein

_are

responsible

for

its

stage-specific

expression

in

Trypanosoma

cruzi

metacyclic

trypomastigotes

Ethel

Bayer-Santos

1

_,

_Luciana

_Girotto

_Gentil

1,2

_,

_Esteban

_Maurício

_Cordero

2

_,

Paulo

Roberto

Ceridório

Corrêa, José

Franco

da

Silveira

∗

DepartamentodeMicrobiologia,ImunologiaeParasitologia,UniversidadeFederaldeSãoPaulo,UNIFESP,RuaBotucatu862,CEP04023-062SãoPaulo,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25January2012

Receivedinrevisedform13March2012 Accepted14March2012

Available online 8 May 2012

Keywords:

Untranslatedregion Post-transcriptionalcontrol

Trans-sialidase

Surfaceprotein

Trypanosomacruzi

a

b

s

t

r

a

c

t

GeneexpressioninTrypanosomacruziisregulatedatthepost-transcriptionallevelandcis-acting

ele-mentspresentinthe3′_untranslated_region₍₃′_UTR)_play_an_important_role_by_interacting_with_regulatory

proteins.PreviousstudiesdemonstratedthattheGP82surfaceglycoprotein,whichisinvolvedinhostcell

invasion,isup-regulatedintheinfectivemetacyclictrypomastigoteform,andthatGP82mRNAhalf-life

islongerinthisformcomparedtothenon-infectiveepimastigoteform.Here,wedemonstratethatthe

3′_UTR_of_the_GP82_transcript_is_involved_in_this_{developmental}_regulation,_promoting_higher_expression

ofthegreenfluorescentprotein(GFP)reporterinmetacyclictrypomastigotesthaninepimastigotes.A

seriesofstepwisedeletionsinthe3′_UTR_was_created_and_results_suggest_that_the_mechanism_regulating

GP82expressioninvolvesmultipleelementsinthe3′_UTR.

1. Introduction

Transcriptionintrypanosomatidsoccursinaconstitutive fash-ionandinitiatesbi-directionallyinregionsbetweentwodivergent geneclusters(Martinez-Calvilloetal.,2003).Differentsetsofgenes are transcribed as large polycistronic pre-mRNA units that are processedintomaturemRNAsbya coupledmechanism

involv-ing trans-splicing and polyadenylation (Matthews et al., 1994).

As a consequence of polycistronic transcription, regulation of gene expression in trypanosomatids takes place at the post-transcriptionallevel,wheremRNAsinteractwithdifferentsetsof regulatoryproteinsthatadjustmRNAlevelsaccordingtocellular demands.Transcriptomicmicroarrayanalysisrevealedthat50%of

Trypanosomacruzigenesare regulatedduring theparasite’slife

cycle(Minningetal.,2009).RegulatoryelementsinmRNA5′_and

3′ _untranslated_regions _(UTRs)_have_been_shown_to_be_involved

incontrollingtranscriptabundancethroughinteractionwith RNA-bindingproteins(RBPs)(reviewedinAraujoandTeixeira,2011).

∗Correspondingauthorat:UniversidadeFederaldeSãoPaulo,RuaBotucatu,862 –6thfloor,CEP04023-062SãoPaulo,Brazil.Tel.:+551155711095.

E-mailaddress:[email protected](J.F.daSilveira).

1 _Both_authors_contributed_equally_to_this_work.

2 _Present_address:_Department _of_Biological_Sciences,_The_Border_Biomedical

ResearchCenter,UniversityofTexasatElPaso,ElPaso,TX79968,USA.

Metacyclictrypomastigotesexpressasurfaceglycoproteinwith anapparentmolecularweightof82kDacalledGP82(Teixeiraand Yoshida,1986), whichisa memberofthetrans-sialidasefamily andisinvolvedintheparasiteattachmenttoandinternalization intomammaliancells(Ramirezetal.,1993,1999).Itwas demon-stratedthatGP82transcriptsaccumulateinmetacyclicformsand thattreatmentswithtranslationinhibitorsincreasedGP82mRNA half-lifein epimastigotes,suggestingthat proteinfactorsactby destabilizingtranscriptsin theepimastigotestage(Gentiletal., 2009). To better understand the mechanisms regulating GP82 stage-specificgeneexpression,wetransfectedepimastigoteswith constructscontainingthenativeandtruncatedversionsofGP82

3′_UTR_fused_down-stream_of_the_green_fluorescent_protein_(GFP)

reporter,andsubmittedthemtoinvitrometacyclogenesis.Their GFPmRNAandproteinlevelswerethenanalyzed.

2. Materialsandmethods

AllGFP reporter constructs were generated by PCR amplifi-cationusingasetofprimersdescribed inSupplementalTableI. AmpliconswereclonedintopGEM®_-T_easy_vector_(Promega)_and

sequenced.Insertsweredirectionallysub-clonedintothepTEX vec-tor(Kellyetal.,1992)usinguniquerestrictionsites.Togenerate thepGFPconstruct,theGFPgenewasamplifiedfromthe pEGFP-C2plasmid(BDBiosciencesClontech) withtheoligonucleotides GFP-FwdandGFP-Rev.ThisconstructcontainstheGAPDH3′_UTR

http://dx.doi.org/10.1016/j.actatropica.2012.03.014

Open access under the Elsevier OA license.

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E.Bayer-Santosetal./ActaTropica123 (2012) 230–233 231

Fig.1.SchematicrepresentationofplasmidconstructsandGP823′_UTR_sequence._(A)_Structure_of_the_pTEX_vector_containing_the_green_fluorescent_protein₍_GFP₎_coding

sequenceandtheselectionmarkerneomycinphosphotransferasegene(Neo)flankedbyglyceraldehyde3-phosphatedehydrogenaseintergenicsequences(GAPDH),which containmRNAprocessingsites(trans-splicingandpolyadenylation).TheGP823′_UTR_sequences_were_inserted_after_the_GFP_coding_sequence_as_indicated_(arrow)._(B)_Schematic

representationofconstructsclonedintothepGFPplasmid.TheGP823′_UTR_was_divided_into_four_parts_and_alternate_regions_were_deleted_in_the_indicated_positions._(C)_The GP823′_UTR_sequence_used_in_the_study_was_derived_from_the_GP82_gene_under_GenBank_ID:_L14824._Major_deleted_sequences_are_indicated_in_black_and_gray._The_AU₃_A_motif

andTcUBP1putativebindingmotifareunderlined.

sequencedownstreamoftheGFPcodingregionandwasusedas a control.The p3′_UTR_construct _was_created_by _amplifying_the

GP823′_UTR_from_the_recombinant_plasmid_pGEX-J18b_(GenBank

ID:L14824)witholigonucleotides3UTR-Fand3UTR-R,and subse-quentlycloneddownstreamoftheGFPgeneinpGFP.Constructs p1,p2,p3andp4werecreatedusingthep3′_UTR_construct

astemplate.Thep1constructwasamplifiedusingtheprimers

1-Fwdand3UTR-R.Tocreatethep4construct,the3UTR-Fand

4-Revprimerswereused.Thep2andp3deletionswere pre-paredbyspliceoverlapextensionPCR (SOE-PCR)(Hortonetal., 1989).Briefly,theGP823′_UTR_was_split_in_two_separate_PCRs_into

twofragmentssharingacomplementaryregion.Thesewere puri-fied,combinedinequimolarquantitiesandamplifiedinasecond PCRusingtheouterprimers,3UTR-Fand3UTR-R.Theprimersused inthefirstPCRswere3UTR-F,2-Rev,2-Fwdand3UTR-Rforthe p2constructand3UTR-F,3-Rev,3-Fwdand3UTR-Rforthe p3construct.Allampliconswerecloneddownstreamfromthe

GFPgeneintotheBglII/HindIIIrestrictionsitesofthepGFPvector. EpimastigotesfromT.cruziGstrainweretransfectedby elec-troporation with 30␮g of circular plasmid DNA as previously

described(Ramirezetal.,1999).Metacyclictrypomastigoteswere obtainedfromtransfectedepimastigoteinstationarygrowthphase, cultivatedinliverinfusiontryptosemedium(LIT)supplemented with5% fetal calf serum (FCS) at 28◦_C, _and _purified _by

DEAE-cellulosechromatography (Yoshida, 1983), resultingin samples containingmorethan95%metacyclics.Northernblotanalyseswere performedwithtotalRNAextractedfrom1×108parasitesas

pre-viously described (Gentil et al., 2009). Autoradiographies were digitalizedandanalyzedbydensitometryusingtheImageJ soft-ware1.38(http://rsbweb.nih.gov/).GFPfluorescencewasanalyzed onaFACSCaliburIIcytometer(BectonDickinson),anddata analy-sisperformedusingCellQuestsoftware.Parasitestransfectedwith thepTEXvectorwithouttheGFPsequencewereusedtoadjustthe backgroundfluorescence.Westernblotwasperformedwithtotal proteinextract(7×106 parasites)resolvedon10%SDS-PAGEas

described(Corderoetal.,2009).Nitrocellulosemembraneswere incubated with a mouse anti-GFPpolyclonal antibody. To rule

outvariations inplasmid copynumber and proteinloading,an immunoblottingusinganti-NPTII(neomycinphosphotransferase II)antibody(Sigma)wasperformedtonormalizethesignals.

3. Results

PreviousstudiesrevealedthattheGP82mRNAandproteinare up-regulatedinthemetacyclicstageandthatitsmRNAhalf-life (6h)islongerthanthatinepimastigotes(30min) (Gentiletal., 2009).ToinvestigatewhethertheGP823′_UTR_may_be_involved_in

thestage-specificexpressionwecreatedaconstructcontainingthe

GFPreportergeneinfusionwiththeGP823′_UTR._We_also_generated

fouradditionalGFPconstructsinwhicha∼90bpregionwasdeleted

fromtheGP823′_UTR_to_search_for_specific_cis_-acting_elements._A

schematicrepresentationoftheconstructsusedinthisstudyand thenucleotidesequencefromtheGP823′_UTR_are_shown_in_Fig.₁_.

Epimastigotestransfectedwiththeconstructsdescribedabove weredifferentiatedintometacyclictrypomastigotesandhadtheir

GFPmRNAand proteinlevelsquantifiedand compared. Fig.2A showsa northernblotandtheeffectoftheGP823′_UTR_on_GFP

mRNAaccumulation.Inepimastigotes,theplasmidcontainingthe

GP823′_UTR_(p3′_UTR)_reduced_reporter_mRNA_levels_2.8-fold

com-paredwiththecontrolplasmid(pGFP).Theopposite effectwas observedinmetacyclictrypomastigotes,whereGFPmRNAlevels weretwiceashighinthep3′_UTR_as_in_the_pGFP_construct._GFP

pro-teinlevelswereanalyzedusingtwomethods,measurementofGFP fluorescencebyflowcytometry(Fig.2B)andwesternblot(Fig.2C), andapositivecorrelationwasobservedbetweenmRNAand pro-tein levels.In epimastigotes,thenegative effectexertedbythe

GP823′_UTR_on_mRNA_accumulation_was_also_observed_for_protein

expression,asGFPfluorescencewas13.5-foldlowerinp3′_UTR_than

in pGFP, whilein metacyclic trypomastigotes, GFPfluorescence increased16-foldinthep3′_UTR_compared_to_the_pGFP_construct.

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232 E.Bayer-Santosetal./ActaTropica123 (2012) 230–233

Fig.2.TheGP823′_UTR_contains_cis_-elements_that_direct_green_fluorescent_protein_(GFP)_{stage-specific}_gene_expression._Parasites_were_transfected_with_the_indicated

constructsandtheeffectofGP823′_UTR_on_reporter_mRNA_and_protein_levels_were_determined_in_{epimastigotes}_and_metacyclic_{trypomastigotes.}_(A)_Northern_blot_performed

withtotalRNAextractedfrom108_cells_using_the_GFP_coding_region_as_probe._Ethidium_bromide_staining_(EtBr)_of_the_same_gel_and_{densitometric}_{quantifications}_(ratio

topGFP)areshownimmediatelybelow.Arepresentativeexperimentisshown.(B)GFPfluorescencewasquantifiedbyflowcytometryandvalueswerenormalizedto theautofluorescenceofparasitestransfectedwithpTEX.DataareshownasmeanandSDvaluesderivedfromthreeindependentexperiments.Significantdifferencesin fluorescencewereobservedbetweenpGFPandp3′_UTR._Of_the_plasmids_with_deletions,_only_p1andp3frommetacyclicformsdifferedsignificantlyfromp3′_UTR_(Student t-test,p<0.05).(C)Westernblotwasperformedwith7×106cells;membraneswereincubatedwithapolyclonalantibodyagainstGFPtoassessproteinlevelsandNPTIIwas

usedasaloadingcontrol.Densitometricquantifications(ratiotopGFP)ofGFPexpressionnormalizedbyNPTIIareshownimmediatelybelow.

construct.Cycloheximidetreatmentofepimastigotescarryingthe p3′_UTR_plasmid_increased_the_amount_of_GFP_mRNA_3-fold

com-paredwithuntreatedepimastigotes(datanotshown).

Epimastigotes transfected with plasmids containing GFP in fusion with shorter 3′_UTRs _(p_1–₄₎ _were _unable _to _reach

mRNAandproteinlevelsobservedforthecontrolplasmidpGFP (Fig.2A–C). Althougha slightincreasecouldbeobservedinthe constructlackingadistalregion(p4),nostatisticallysignificant differencewas observedbetween p3′_UTR_and _p_1–_4. _Unlike

epimastigotes,inmetacyclictrypomastigotes,deletioninthefirst (p1)andthird(p3)segmentsresultedinasubstantialand sta-tisticallysignificantdecreaseinGFPmRNAandproteinlevels.To confirmthatchangesintheabundanceoftheGFPreportergene were indeeddue toa regulationprocess and not theresult of changesinplasmidcopynumber,neomycinphosphotransferase (NPT)geneexpressionwasanalyzedbywesternblot.NPTisthe selectionmarkerinthepTEXvectorandisunderthecontrolofthe

GAPDH3′_UTR._Fig.₂_C_shows_the_NPT_{immunoblotting,}_where_no

markeddifferenceswerefound,thusexcludinganyeffectbasedon differencesinplasmidcopynumber.

4. Discussion

Inthiswork,wedemonstratetheinvolvementoftheGP823′_UTR

instage-specificexpressionoftheGFPreportergene.Similar mech-anisms for controllingmRNAstability andtranslation by 3′_UTR

sequenceshavealsobeendescribedforothermembersofthetrans -sialidasesuperfamily,aswellasforotherT.cruzigenes,suchas amastin,betatubulin,mucinandheatshockprotein(Araujoetal., 2011;Coughlinetal.,2000;daSilvaetal.,2006;DiNoiaetal.,2000; Jageretal.,2008;Rodriguesetal.,2010;Westonetal.,1999).Inour study,noevidenceoftranslationalcontrolwasobservedsinceboth

GFPmRNAandproteinlevelswereup-regulatedbytheGP823′_UTR

inthemetacyclicforms(Fig.2A–C),thussuggestingthatregulation ofgeneexpressionmediatedbytheGP823′_UTR_occurs_primarily

viamRNAstability.

Althoughtheroleofthetrans-sialidase3′_UTR_in_{stage-specific}

expressionhasalreadybeenshown,theregulatorysequencesand controlmechanisminvolvedinthisregulationremainundefined. Regulatorymotifsofvariablesizeswereidentifiedin the3′_UTR

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E.Bayer-Santosetal./ActaTropica123 (2012) 230–233 233

procyclin,touptomorethanahundrednucleotidesforamastin (reviewedinHaileand Papadopoulou,2007).We tried to iden-tifyputativecis-elementsintheGP823′_UTR_by_performing_serial

deletionsofabout90nt(constructsp1–p4)andanalyzingtheir effectontheexpressionoftheGFPreportergene(Fig.2A–C).Since inepimastigotes,noneofthedeletionswassufficienttorestore theGFPreporterlevelstothoseobservedinparasitescarryingthe controlplasmidpGFP, eventhedeletionof4 regionthat con-tainsthedestabilizingAUUUAmotif(D’OrsoandFrasch,2001a),it isreasonabletosuggestthatmorethanoneregulatorycis-element ispresentintheGP823′_UTR_and_that_these_act_in_coordination_to

destabilizeGP82mRNA.Recently,insilicoanalyseshaveshownthat theGP823′_UTR_carries_a_putative_binding_motif_for_the_RNA

bind-ingproteinTcUBP1(Noeetal.,2008),afactorinvolvedinSMUG

mucinmRNAdestabilization(D’OrsoandFrasch,2001b),whichis

locatedattheendof1region(Fig.1C).Thus,TcUBP1couldbe oneoftheproteinfactorsinvolvedinthedestabilizationofGP82

mRNAinepimastigotes.Inmetacyclictrypomastigotes,deletions performedinconstructsp1andp3resultedinstatistically sig-nificantdecreasesinGFPreporterlevelssimilartothoseinparasites transfectedwithpGFP,indicatingthattheseregionscontaincis -elementsresponsibleformRNAstabilizationinmetacyclicforms. Thesedataagreewiththehypothesisraisedforregulationinthe epimastigotestagethatmorethanonecis-elementispresentinthe

GP823′_UTR._The_lack_of_the₄_region_slightly_increased_reporter

levelsinmetacyclictrypomastigotesandepimastigotes,although thisincreasewasnotstatisticallysignificant,bothresultsmaybe relatedtothelackofadestabilizingmotif.Theseresultsmakeus speculatethatthemRNAsteady-statelevelinagiven developmen-talstagecouldbetheresultofa balancebetweenpositive and negativeproteinfactorsactingsimultaneouslythroughdifferent

cis-elementsinthe3′_UTR.

Acknowledgments

ThisworkwassupportedbytheFundac¸ãodeAmparoàPesquisa doEstadodeSão Paulo(FAPESP)andtheConselho Nacionalde DesenvolvimentoCientíficoeTecnológico(CNPq),Brazil.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, in the online version, at http://dx.doi.org/10.1016/j.actatropica. 2012.03.014.

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