Journal of Cell Science h rgnlwr spoel ie n l ute itiuin ftewr raatto r ujc to subject are adaptation or work terms. the License of distributions Commons further Creative Attribution all same and Commons that cited the provided Creative properly medium is any the work in of original reproduction the and which terms distribution use, the (http://creativecommons.org/licenses/by-nc-sa/3.0/), non-commercial under unrestricted License permits distributed Alike article Share Access trans Non-Commercial Open by an is capped This are mRNAs I trypanosome polymerase all RNA because by possible driven being in (Gu the of unusual case to is the transcription linked both In being of inactivation 2007). also the and promoter this gene the procyclin molecules, a the by of procyclin replaced activation is of coat for monoallelic VSG responsible the coat ensures vector, trypanosomes, fly which of tsetse transmission 2007), the the physical In al., 2002). their et (Borst, expression and Navarro to (Navarro 2001; body linked site Gull, expression sub-nuclear apparently a with being association The this time. one any transcription, at active of is which activity of one only a be sites, of expression can representatives of coat different repertoire this of large immunity, expression coat evade the (VSG) to through through glycoprotein changed order hosts surface In variable mammalian 2011). a their (Rudenko, of that of expression Africa bloodstream their sub-Saharan the of in parasites survive are trypanosomes African Introduction proteins. regulatory words: with Key interactions parasite. for the important of is forms developmental structure distinct RNA in that factors bifunctional suggesting regulatory this conserved, positive that predict and We negative 3 forms. the competing stumpy of by in Analysis expression targeted gene is activating surface element positively, a sequence acts for variant also RNA signals but control telomeric forms post-transcriptional function. slender the cryptic some in forms, of silencing proteins stumpy of expression secreted in genome. of activation component group the and a a forms in conserved encoding slender elsewhere well forms, as in transcribed stumpy silencing transmissible also identified developmental in are their genes originally understand family regulated To diverse highly were most this the genes of each among chronicity. members during are whose infection many genes sustain accumulate although proteins, to forms or sites, stumpy transmission ESAG9 expression for quiescent gene adaptation are while glycoprotein an as genes group either many upregulated this are of genes repression Among of the subset their a by to However, transmission characterised parasitaemia. for of is adaptation wave an transition as This forms ‘slender’ vector. proliferative fly from forms tsetse ‘stumpy’ generate trypanosomes host mammalian their In Summary 10.1242/jcs.126011 doi: 2294–2304 126, ß Science Cell of Journal 2013 February 25 Accepted Edinb of University UK Sciences, 3JT, Biological ( EH9 of correspondence School Edinburgh, for Research, Road, *Author Infection and Mains Matthews* Immunology West for R. Buildings, Institute Evolution, King’s Keith and Infection and Immunity, for Simmonds Centre Peter Monk, L. Stephanie of forms developmental or positively to regulate operates negatively element bifunctional short A 2294 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. nle l,20;Rdnoe l,18) hsbigmade being this 1989), al., et Rudenko 2003; al., et ¨nzl vsg eeepeso iepooe Lner n Navarro, and (Landeira promoter site expression gene ifrnito,Gn expression, Gene Differentiation, vsg ESAG9 9 xrsinstsi otolda h ee of level the at controlled is sites expression T euaoyrgosfakn h ihydiverse highly the flanking regions regulatory UTR vsg [email protected] eehv enmpe.Ti dniidapeieRAsqec lmn f3 uloie htcnrbtst gene to contributes that nucleotides 34 of element sequence RNA precise a identified This mapped. been have gene ee,ta r xrse rmtelomeric from expressed are that genes, vsg n rcci eeexpression, gene procyclin and ) rpnsm brucei Trypanosoma ESAG9 rpnsm brucei Trypanosoma ESAG9 xrsini different in expression ua eu Py ta. 01 oge l,19) Although 1998). al., the et co-association Xong their with by of 2001; defined are component genes al., associated trypanolytic site et expression the (Pays to serum resistance enables human that 1992) gene al., a et a (Paindavoine in activity exclusively encode and, cyclase 1994), promoter) adenylate al., an site et encodes (Salmon expression receptor although the transferrin unknown, heterodimeric to largely closest The is 1989). (positioned al., these et Pays 1987; of al., et function Kooter 1985; al., also et (Cully gene are ( upstream genes the associated genes the between site expression positioned notably, of other number Most a contains units, locus. site each transcription in respective co-transcribed their 1991). al., of et sequence Zomerdijk leader 1987; transcribed al., et II Kooter polymerase 2002; RNA (Clayton, an of splicing o xml Slo ta. 02;Slo ta. 2012b). al., et Salmon 2012a; al., host, et mammalian (Salmon the example in for infections trypanosome of establishment (gene the of GRESAGs outside positioned termed Although 2001). often vsg al., et are (Pays ESAGs) genes to related These protein site genes. non-expression polycistronic all coding for are RNA-polymerase-II-driven mRNAs the generate These of that arrays part sites. as expression transcribed outside positioned representatives Although xrsinsts RSG a lob ucinl members functional; be also can GRESAGs sites, expression aiyrvasta h ierrgltr eunei o highly not is sequence regulatory linear the that reveals family ESAG4 vsg vsg aiyhv ucini ohctknssadearly and cytokinesis both in function a have family ee several gene, n rcci RA r h ao products major the are mRNAs procyclin and rpnsm rcirhodesiense brucei Trypanosoma ESAG vsg rmtradtelomeric and promoter eefmle lohave also families gene eerhArticle Research vsg ESAG6 , SRA expression encodes ESAG4 ESAG9 ESAG and urgh, vsg 7 ) Journal of Cell Science xml fasrnetyadcodntl euae eefamily gene regulated coordinately the and stringently Hence, a of 2010). gene the example al., of copies et associated expression to (Barnwell the the restricted not involves different is during so This many and mRNAs cells. of expressed stumpy expression to elevated highly intermediate genes most from these transition the with formation, among stumpy during being events earliest be the Vassella of 2012; can Matthews, 1997). which and al., MacGregor et of 2006; al., developmental aspects et defined (Laxman some a bloodstream, via MacGregor from mimicked the to occur 2011; events in al., forms progression These pathway et 2012). intermediate (MacGregor morphological al., 1A) et through (Fig. their forms forms, stumpy (stumpy and slender signal arrest proliferative cycle parasites, cell parasite-derived for the stimulating a of parasite this from detected, which the is parasite of factor) in induction prepare form the a sensing to prevent involve quorum also processes to developmental and both These transmission. host serves the This Vassella the 1997; overwhelming 1997). that al., the al., et mechanism ensure (Reuner et thereby stages sensing and transmission parasitaemia density of the production a monitor 2009), to to use al., parasites response of event et wave differentiation in This each Jensen 2012). occurs of al., peak 2010; et toward the (MacGregor al., at parasitaemia parasites accumulate et that bloodstream expressed stages (Barnwell transmission the being forms of bloodstream, stumpy development mammalian upon the only in assist to expression Most however, 2010). potentially al., et parasites, interestingly, (Barnwell transmission form or chronicity bloodstream infection of products by protein The secreted 2008). (Barnwell al., regions et ESAG9 Hertz-Fowler sub-telomeric 2010; and al., et internal positioned units chromosomal transcription at as polycistronic found within rarely located and being diverse conventional highly of are genes components These 1991). al., et om rsnssm neetn hlegst h aaie Firstly, parasite. the to challenges interesting some stumpy presents in forms expression gene developmental forms, midgut tsetse and parasites’ the to response in signal. expressed quorum-sensing developmentally is that ncnrs odfeeta eerglto ewe bloodstream between regulation gene differential to contrast In unusual One ee r fukonfnto,atog hyare they although function, unknown of are genes nvitro in nvivo In ESAG sn elprebecM rAPanalogues AMP or cAMP permeable cell using h peuainof upregulation the , aiyi the is family ESAG9 vsg xrsinsts otcopies most sites, expression ee hwdevelopmental show genes ESAG9 ESAG9 ESAG9 ESAG9 eefml (Florent family gene ee rvd an provide genes rncit sone is transcripts aiymembers family xrsinst opnn in component site to expression related closely Tb nodrt analyse to order In forms slender in silencing 3 ESAG9-EQ The motneo t 3 the this confirmed its For signals 2009). regulatory al., of the et of (Dean importance analysis fly tsetse an the molecule, as signal enter differentiation forms the of stumpy perception the in transporter important enriched carboxylate stumpy protein a PAD1, one – date, characterised To been has forms. molecule stumpy in be enriched set genes in small must of the expression on repression gene focusing particularly developmental bloodstream, this mammalian understand the to and sought have forms, reasons, we these For slender bloodstream. the in in development upon also alleviated silent must forms held stumpy in wide-ranging activated be are that the al., genes et Finally, (Schwede 2011). etc.) the with pH levels, to glucose temperature, entry contrasting (e.g. fly upon tsetse development intrinsic, govern that signals parasite environmental pathway signalling is developmental P. a that of Ivens, product the R. A. exclusively K. is M., and expression Smith gene stumpy-enriched S. T. Secondly, Bringaud, data). unpublished Capewell, F. M., Walrad, P. P. Fenn, genes 1998; K. MacGregor, most Parsons, on and enforced repression (Brecht translational the molecules expressed escape stumpy must that such quiescent are forms stumpy ope n ipre McrgradMthw,2012). Matthews, and (MacGregor being dispersed define, and precisely complex to difficult were regulation developmental Results bloodstream element relevant parasite. characterised transmission the well and of disease first forms precisely in the the in most and identified operating date the elements to among regulatory trypanosomes as post-transcriptional element such identifies defined RNA itself, This forms. SIF bifunctional stumpy to intersect in this expression also that gene and promotes analogues pathway it cAMP that induction stumpy to the responsive with is that motif element RNA same regulatory short defined This ensures highly bloodstream. a mammalian identified the has in regulation developmental co-regulated the ee ehv xlie h tiigdvlpetlcnrlof control developmental striking the exploited have we Here, 2..60(ecfrhtermed (henceforth 927.5.4620 ESAG9 sntt scale. 5 to the not from is UTR create the to along designed deletions primers sequential 3 forward nt using 250 PCRs nt, with 300 nt, (350 series 1997). al., Bam 3 et the (Biebinger regulated of developmentally Replacement not is it since 3 rnae 3 truncated sa,soigtergo fteCT4 etrwt the resistance with phleomycin (BLE vector (CAT), CAT449 transferase the acetyl of chloramphenicol region the showing assay, 3 for strategy subcloning ( in bloodstream. development mammalian trypanosome during the forms stumpy study. to this slender from of basis experimental and ( biological The 1. Fig. A 9 T ofrigcntttv xrsin(ah ta. 2006) al., et (Mayho expression constitutive conferring UTR ersnaino h vnsacmayn h differentiation the accompanying events the of Representation ) Iand HI R .brucei T. ,and ), eeslnigi lne om.Mroe,the Moreover, forms. slender in silencing gene 9 ESAG9 9 T ( UTR T otiue ogn expression gene to contributes UTR Bbs ESAG9 ESAG9 b tblngns h loae(L)5 (ALD) aldolase The genes. -tubulin etito nyests h 3 The sites. enzyme restriction I 9 T,btsqecsipratin important sequences but UTR, D L)fakteCTgn,wt the with gene, CAT the flank ALD) eeomna oto 2295 control developmental 9 eerglto,w sda model a as used we regulation, gene eefml obte understand better to family gene T iha xeietlURue the used UTR experimental an with UTR ee rgnlyietfe san as identified originally genes 9 T nlsswt A eotrgene reporter CAT a with analysis UTR rpnsm equiperdum Trypanosoma B ESAG9-EQ 9 T eune)wr generated were sequences) UTR ceai iga fthe of diagram Schematic ) ,wihi most is which ), 9 n.Tediagram The end. 9 T deletion UTR 9 T and UTR , 0nt 50 D ALD [67% Journal of Cell Science itr47i aallwt h aecntutbaigthe bearing construct same the with parallel in form bloodstream 427 monomorphic Lister to in transfected was of downstream annotated and to successfully used been 3 previously map has the approach of This contributions 1B). the (Fig. assess reporter to CAT used a ESAG9 was forms Initially, approach shown). stumpy not assay (data in gene forms expression slender in mRNA than higher fivefold an gene strains indicating This different to in similarity). stumpy-enriched related (88% being of as 2008) highly confirmed al., also et in was also (Young copy and cloning linked TAR respectively], site 1991), expression al., to similarity et acid amino 84% and 2296 oo;K .adS . nulse bevtos.This observations). unpublished M., S. 3 ‘ESAG9-EQ and construct the M. generated the K. for (nt) site codon; polyadenylation nucleotides mapped 400 the ESAG9-EQ a beyond only large extending too this manipulation, provided the of this further downstream Since for bp. is 3057 region of sequence region intergenic genome 927/4 .brucei T. PAD1 9 T oto lmnsof elements control UTR 3 ora fCl cec 2 (10) 126 Science Cell of Journal 9 T eunet h oto fgn expression gene of control the to sequence UTR McrgradMthw,21) h ers gene nearest The 2012). Matthews, and (MacGregor rncit(oiindat (positioned transcript Brwl ta. 00,wt iia AEanalysis SAGE Digital with 2010), al., et (Barnwell ESAG9-EQ ESAG9-EQ eewsicue o analysis, for included was gene COX Tb .brucei T. ESAG9u 2..60 eeaigan generating 927.5.4630, , ee Myoe l,2006) al., et (Mayho genes nthe in 8 trltv otestop the to relative nt 380 9 T ullnt’ which length’, full UTR and 47ietfe by identified s427 .b brucei b. T. ESAG9c .brucei T. 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Fig. vector; rnae loae3 aldolase truncated L,wihwsnraie o10 nalexperiments; all in 100% to normalised was which ALD, ncmaio othe to comparison In oydnlto ie neetnl,ivsiaino the of investigation Interestingly, site. polyadenylation eetduigti osrc eg ah ta. 2006). al., et Mayho always (e.g. transcript construct bicistronic this a using upper represent detected The lanes vector. all unmodified in the bands for a obtained of as that values rRNA of are to percentage blot normalisation the lower following indicate the abundance to Below mRNA analysed lane. sample, each each of nt in loading 250 staining present and bromide rRNA ethidium nt the the 300 of shows nt, panel 350 lower full-length, The the constructs. of each of the replicates from RNA with mRNA, oydnlto ielcto n h e ieidctsthe indicates ( line location. red the element the indicates regulatory and ‘A’ location where construct, site of representations each polyadenylation schematic in are analysed graphs sequence the derived of the being left the these At expression, B). mRNA from for shown protein is the replicates for replicates means biological and with of expression, each (s.e.m.) 3; mean nt: means the 250 are 3, of nt: Values 300 replicates. 2, experimental nt: 1–2 350 5, clones) full-length: derived was: (independently used replicates biological of number ae)epeso rmtecntut hw,a ecnaeof the percentage of a clone as one shown, from constructs expression the from expression panel) i.2 eeinaayi frgltr ein nthe in regions regulatory of EQ analysis Deletion 2. Fig. 3 9 UTR. ( A 9 T sacnrl( control a as UTR .CTpoen(pe ae)admN (lower mRNA and panel) (upper protein CAT ). D L osrc,the construct, ALD ESAG9-EQ 6 h oe n pe ausfo two from values upper and lower the D B L etradtobiological two and vector ALD otenbo nlssof analysis blot Northern ) D 6 3 L 3 ALD 9 .%ad77 and 2.5% T erse CAT repressed UTR D D ESAG9-EQ 9 L;unmodified ALD; ESAG9 L;Fg 2A,B). Fig. ALD; T oto.The control. UTR 6 tnaderror standard 9 .brucei T. T was UTR 6 intergenic .%of 1.7% 6 ESAG9- 3 CAT 3.2%, 9 CAT UTR CAT Journal of Cell Science Fg A,smlrt hto h intact the of that to similar 2A), (Fig. xrsinlvlfloe h aeteda h atr of RNA pattern level the the the (45% construct as deletion construct, nt trend 350 deletion the same that with each such the expression, for followed protein level that expression shows the containing 2B vector Fig. control of 32% the only for was expression seen protein CAT that since construct, nt 300 uat r hw nsplmnaymtra i.S2. 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CAT 9 T osrc)o eas oeo h sequence the of some because or construct) UTR T ‘e UTR ESAG9-EQ xrsin uhta h ee fCAT of level the that such expression, 3 etodsrcue eeae o h full-length the ESAG9-EQ for generated structures Centroid ESAG9-EQ package software sfold.wadsworth.org/cgi-bin/index.pl) i.3 N eodr tutr rdcin for predictions structure ESAG9-EQ secondary RNA 3. Fig. 9 9 3 T (B). UTR T ihteecpino h removed the of exception the with UTR 9 ESAG9-EQ D T.Hratr h eunecontained sequence the Hereafter, UTR. 9 D T eunewsaaye by analysed was sequence UTR )wsteeoetasetdinto transfected therefore was ’) eeomna oto 2297 control developmental osrc leitdsm fthe of some alleviated construct S n a sdt rdc h tutr fthe of structure the predict to used was rna euaoyeeet(‘e’). element regulatory 3 3 3 9 9 9 T A n the and (A) UTR Ensemble the are Shown UTR. T sequences. UTR 3 9 9 T eunewssimilar was sequence UTR T oeta regulatory potential UTR 9 T construct). UTR ESAG9-EQ The S od(http:// fold e S D fold, Journal of Cell Science o10 to xrsini sup-ie forms? ‘stumpy-like’ in expression olfrteaayi fgn xrsinatvto nstumpy-like in transcript, useful the activation by a expression driven gene enriched provided forms of it analysis stumpy the Hence, for 2012). tool reporter another to Matthews, a and of to differentiation (MacGregor expression enhances the linked activates and gene also it and arrest formation, forms, stumpy growth procyclic for proxy cell limited promotes a Although provides 1997). al., analogue et this Vassella 2012; Matthews, and permeable MacGregor cell the 8-pCPT-2 using positive analogue ‘stumpy-like’ cAMP for become by positive earlier to potential generated cells lines (developmental the inducing reporter monomorphic examine forms the to in regulation stumpy decided both We of in regulation). regulation) combination activation negative a (developmental and/or forms from slender result stumpy in bloodstream repression could in trypanosomes genes of form expression developmental The 3 ESAG9-EQ the Is expression. repress not did 3 another into gene element reporter of the repression in the expression 4B). to (Fig. contributed levels element regulatory protein as trend same (CAT the observed followed also was levels expression reporter overall 3 D of the aldolase and truncated repression the structure, of overall its remainder conserve the 3 to of structure predicted the position into a inserted at was 3 aldolase element truncated regulatory nt 34 26 53 was protein 2298 L I90 eI ALD h eut o h aiu 3 various the for results The the cells monomorphic in that indicated experiments These 6 .%frteintact the for 1.7% m 8-pCPT-2 M ora fCl cec 2 (10) 126 Science Cell of Journal 6 %wt epc othe to respect with 6% 6 ESAG9-EQ .%o the of 5.2% 9 9 T gnrtn osrc ‘CAT construct (generating UTR ESAG9-EQ 9 T epnil o h oto fgene of control the for responsible UTR OM-APfr4 or r hw in shown are hours 48 for -O-Me-cAMP 9 T rmacntttvl xrse gene expressed constitutively a from UTR ESAG9-EQ 9 OM-AP(amne l,2006; al., et (Laxman -O-Me-cAMP 3 9 9 T osrcsepsd rnot, or exposed, constructs UTR T.Hwvr neto fthis of insertion However, UTR. 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Omega was Clustal al., or et Multi- 2.1) sequences (Larkin version (http://sfold.wadsworth.org/cgi-bin/index.pl). tool, DNA Alignment Sequence settings of default alignment the the package with structure software web-based secondary available RNA freely the of prediction basic prediction For structure RNA and Bioinformatics 8-(4-Chlorophenylthio)-2 8-pCPT-2 anel .M,vnDusn .J,Jaok . mt,K . azl,R M., R. Maizels, A., K. Smith, L., Jeacock, J., F. Deursen, van M., E. Barnwell, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126011/-/DC1 online available material Supplementary release. immediate for G0700029 PMC number in [grant Deposited (UK) studentship S.M.]. Council a Wellcome to Research and the P.S.]; Medical and the from K.M. from to Evolution 095831MA grant and number [grant the Programme Infection Trust support Trust to Immunity, Award Wellcome for Strategic a a Centre K.M.]; to by 088293MA number supported [grant was work This Funding P.S. S.M., analyses; manuscript. structural the data; RNA wrote out the K.M. carried analysed and K.M. K.M. and and S.M. S.M. P.S., experiments; the performed S.M. the contributions and Author advice for MacGregor analysis. statistical Paula for and Dr parasites pleomorphic form assistance, stumpy generating for technical Wilson Julie thank We Acknowledgements utr sdfrRAetato n hnnrhr ltaayi a described (as remaining analysis the blot and northern assay then ELISA and CAT extraction a in RNA above). use for for used samples culture lysate cell generate sMEdfeecs{FD;ie h ecnaedfeec nME fntv and native of MFEs in difference percentage retains expressed the that were i.e. SSE Results {MFEDs; in 2004). differences al., algorithm MFE et as NDR (Simmonds the frequencies submission dinucleotide using (MFEDs) parallel in scrambled by expectation biases sequences calculated null were control the 1999) 100 from Krogh, of and (Simmonds, differences (Workman package MFE Z-scores SSE sequence, and the each within 2005) For Zuker, 2012). and (Markham UNAfold using cabe eune acltda [(MFE as calculated sequence, scrambled dniiscnevdpie n nardbssi iiu nryfold energy detection minimum site 2008). in that energy/covariant al., et package bases minimum (Gruber SSE ALIFOLD combined unpaired in the a implemented identify and method in and to STRUCTUREDIST 2012), paired method (Simmonds, by conserved grammar 1999), model structure (Edgar, structure identifies Hein, context-free RNA MUSCLE an RNA and stochastic of program supportive (Knudsen for sites a the co-variant conserved by approaches PFOLD, phylogenetically aligned used Alternative sequences 2004) 1999)}. nucleotide values control in of Krogh, distribution prediction the in and sequence native the (Workman of position (the Z-scores as pCPT-2 isle nDS DS)t ocnrto f1 Mwsaddt h cell the to added was mM 10 of concentration a ( to culture (DMSO) DMSO in dissolved rpnsm development. trypanosome xrsino oi eepout nbodtemadpoylcTyaooabrucei. Trypanosoma from procyclic and Parasitol. product bloodstream Biochem. in products Mol. gene gene toxic of expression-site-associated expression VSG a forms. bloodstream of brucei K. Trypanosoma release Matthews, and extracellular A. Acosta-Serrano, 9 , OM-AP ilgLf cec nttt,Bee,Germany) Bremen, Institute, Science Life Biolog -O-Me-cAMP; 20) niei aito n lei exclusion. allelic and variation Antigenic (2002). 9 5 OM-APtreatment -O-Me-cAMP 6 10 5 D el/lcnetain oafnlcnetaino 100 of concentration final a to concentration) cells/ml L elln n rMroCaeTpigfor Chase-Topping Margo Dr and line cell ALD .brucei T. 9 -O-methyladenosine-3 85 ˚ o.Bohm Parasitol. Biochem. Mol. o 8hus lo ahcluewsue to used was culture each of ml 5 hours, 48 for C 99-112. , 19) hne nplsm rflsaccompany profiles polysome in Changes (1998). eeomna oto 2303 control developmental .Cl Sci. Cell J. 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