Template switching can create complex LTR retrotransposon insertions in Triticeae genomes

BioMed Cen al Page 1 o 5 (page numbe no o ci a ion pu poses) BMC Genomics Open Access Resea ch a icle Templa e swi ching can c ea e complex LTR e o ansposon inse ions in T i iceae genomes F ançois Sabo 1 and Alan H Schulman*1,2 Add ess: 1MTT/BI Plan Genomics Labo a o y, Ins i u e o Bio echnology, Viikki Biocen e , Uni e si y o Helsinki, P.O. Box 56, FIN-00014 Uni e si y o Helsinki, Finland and 2Plan Genomics, Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, Mylly ie 10, FIN-31600 Jokioinen, Finland Email: F ançois Sabo - ancois[email p o ec ed]; Alan H Schulman* - [email p o ec ed] * Co esponding au ho Abs ac Backg ound: The LTR (long e minal epea ) e o ansposons o highe plan s a e eplica ed by a mu agenic li e cycle con aining ansc ip ion and e e se ansc ip ion s eps. The DNA copies a e o en subjec o ecombina ion once in eg a ed in o he genome. Complex elemen s, whe e wo elemen s sha e an LTR, a e no uncommon. They a e hough o esul om he e ologous ecombina ion be ween wo adjacen elemen s ha occu s ollowing hei in eg a ion. Resul s: He e, we p esen e idence o ano he po en ial mechanism o he c ea ion o complex elemen s, in ol ing abno mal empla e swi ching du ing e e se ansc ip ion. The empla e swi ching c ea es a la ge, complex daugh e elemen , o med by he usion o wo pa en sequences, which is hen inse ed in o he genome. Conclusion: Those complex elemen s a e pa o he genome s uc u e o plan s in he Poaceae, especially in he T i iceae, bu no o A abidopsis. Hence, e o ansposon dynamics shaping he genome a e lineage-speci ic. Backg ound Long Te minal Repea (LTR) e o ansposons a e Class I ansposable elemen s ha eplica e by a "Copy-and- Pas e" mechanism, called e o ansposi ion, which is qui e simila o len i i us (such as he HIV) eplica ion. Highe plan genomes, especially o he g asses (such as maize, whea and ba ley), ha bo a la ge numbe o hese elemen s, which o m he as majo i y o he nuclea DNA. Re o ansposi ion in ol es a e e se ansc ip ion s ep, whe e cDNA is syn hesized om an RNA empla e. Re e se ansc ip ion is ca alyzed by e e se ansc ip ase, which is gene ally encoded by he e o ansposon being copied, and he cDNA is inse ed in o a new genomic loca- ion by he in eg ase, which is also sel -encoded [1]. A canonical e o ansposon inse ion comp ises wo LTRs and an in e nal domain con aining he coding domain o in eg ase, e e se ansc ip ase, a p o einase, he s uc u al p o ein GAG, and he signals o e e se ansc ip ion. Many composi e s uc u al pa e ns de i ed om canoni- cal LTR e o ansposon inse ions we e p e iously iden i- ied in BACs and o he s long genomic sequences om a ious plan s (Figu e 1; [2-7] and e e ences wi hin). These appea p ima ily as nes ed inse ions o one e oe- lemen in o ano he . The nes s can comp ise mo e han h ee o ou laye s a anged in a "Russian doll" ashion. In some cases, he nes ed e oelemen s a e solo LTRs a he han elemen s con aining wo LTRs and a cen al domain. The solo LTRs a e hough o a ise om non- Published: 24 July 2007 BMC Genomics 2007, 8:247 doi:10.1186/1471-2164-8-247 Recei ed: 3 Ap il 2007 Accep ed: 24 July 2007 This a icle is a ailable om: h p://www.biomedcen al.com/1471-2164/8/247 © 2007 Sabo and Schulman; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. BMC Genomics 2007, 8:247 h p://www.biomedcen al.com/1471-2164/8/247 Page 2 o 5 (page numbe no o ci a ion pu poses) ecip ocal ecombina ion be ween he LTRs o a single ele- men (Figu e 1, second case; [2]). In addi ion o he nes s, some complex inse ions a e cha ac e ized by a hi d LTR sha ed be ween wo po en- ially comple e elemen s (Figu e 1, bo om, "Complex inse ion"). Unde s anding he mechanism h ough which his class o e o ansposon complex is de i ed is equi ed o a ull ision o genome e olu ion. Au ho s ha e p e iously desc ibed hem as me ely he esul o ecombina ion be ween he p oximal LTRs o wo adja- cen e oelemen s, leading o he elimina ion o he in e - ening genomic sequence (Figu e 2; om [2,8]). He e we will desc ibe ano he possible o igin o hese complex inse ions, an abno mal empla e-swi ching du ing he e e se ansc ip ion s ep. Resul s and discussion In he genome o A abidopsis haliana, a e ho ough anal- yses, De os e al. iden i ied no complex elemen s o he han hose o igina ing om ecombina ion be ween wo e oelemen s [2]. Fo hese, he wo ou e mos LTRs di e om each o he by no being de i ed om he same e e se ansc ip ion and in eg a ion. This has wo s uc- u al consequences. Fi s , a ecombina ion be ween he 3' LTR om one elemen and he 5' om ano he , closely ela ed one on he same s and (Figu e 2) gi es ise o a hi d, in e nal LTR. This LTR is a chime a o he wo LTRs in ol ed in he ecombina ion. A second consequence, because he wo elemen s in ol ed come om wo inde- penden inse ion e en s ha gene a ed wo di e en a - ge -si e duplica ions (TSDs), is ha he esul ing complex does no ha bo lanking TSDs. By hese measu es, he as majo i y o he complex elemen s al eady iden i ied a ose om unequal and he e ologous ecombina ion be ween adjacen and independen inse ions [2,8]. Ne e heless, a ca e ul analysis o he complex inse ions o a ailable la ge genomic sequences om he T i iceae has e ealed ha he e is ano he g oup o complex ele- men s. Accession AF497474 (Aegilops auschii, [3]) con- ains an Angela-like (Copia) complex in posi ion 11808– 29240 ( e e se o ien a ion, nes ed wi h a Sab ina Gypsy in o wa d o ien a ion). Accession AY368673 (B genome o T i icum u gidum, [4]) also includes an Angela-like com- plex (posi ion 218046–233487, in e e se o ien a ion; Figu e 3). These wo complex inse ions ha bo ea u es ha a e no consis en wi h an o igin h ough ecombina- ion. Fi s , hey ha e lanking 5 bp di ec epea s (TATAA and GCCGG, espec i ely), a leng h cha ac e is ic o TSDs o Copia elemen s. In addi ion, hei wo ex e nal LTRs o he se o h ee a e highly homologous (Figu e 4, do -plo analysis om he AF497474 sequence; he sequence alignmen s o he LTRs a e p o ided in he addi- ional ile 1). The p esence o lanking TSDs suppo s hei o igin om a single in eg a ion e en . The high simila i y be ween he ou side LTRs is consis en wi h hei o igin, u he mo e, om a single cycle o e e se ansc ip ion. Based on hese obse a ions, we checked wi hin o he Poaceae sequences o he occu ence o such complex s uc u es. We ca ied ou an ab ini io scan o he ice pseudomolecules and all he a ailable genomic sequences om maize, using he LTR_STRUC so wa e [9] o de ec- ion o comple e LTR e o ansposons. This so wa e de ec s only comple e elemen s, based on he p esence o bo h wo LTRs and he TSD mo i s lanking hem. Ou o 4704 iden i ied po en ial LTR e o ansposons, we we e Fo ma ion o a complex s uc u e by DNA ecombina ionFigu e 2 Fo ma ion o a complex s uc u e by DNA ecombi- na ion. A he e ologous ecombina ion be ween LTRs om wo closely inse ed elemen s occu s, elimina ing he in e nal sequence. Legends a e he same as in Figu e 1 ( om [2]). Pa e n o inse ions gene ally encoun e ed in he long sequences analyzed om he T i iceae and closely ela ed speciesFigu e 1 Pa e n o inse ions gene ally encoun e ed in he long sequences analyzed om he T i iceae and closely ela ed species. Thin black lines ep esen he hos DNA, hick colo ed lines elemen s' in e nal sequences, ec- angles he LTRs, small boxes wi hin he ec angle he R egion, and he iangles he TSD ( a ge -si e duplica ion). Rec angles and lines om he same colo s de i e om he same elemen . Dashed ea u es may o may no be p esen . No mal inse ion, wi h TSD Solo-LTR, w/ o w/o TSD Nes ed inse ion in LTR, an i-sense Nes ed inse ion in LTR, sense Nes ed inse ion in co e, an i-sense Nes ed inse ion in co e, sense Complex inse ion, no TSD ? Nes ed Solo-LTR in co e, sense Nes ed Solo-LTR in co e, sense Nes ed Solo-LTR in LTR, an i-sense Nes ed Solo-LTR in LTR, sense Nes ed inse ion in Solo-LTR, sense Nes ed inse ion in Solo-LTR, an i-sense BMC Genomics 2007, 8:247 h p://www.biomedcen al.com/1471-2164/8/247 Page 3 o 5 (page numbe no o ci a ion pu poses) able o clea ly iden i y 2 new complex s uc u es ha bo - ing he diagnos ic ea u es: an in e nal LTR, 2 comple e co e sequences, lanking TSDs and simila i y be ween he ou e mos LTRs. The i s elemen is loca ed on ch omo- some 5 o ice, in posi ion 14011139–14022766 (TIGR pseudomolecule), in he o wa d o ien a ion. This ele- men is a membe o he Squiq sub amily, wi h CAAAC as he TSD sequence. The second de ec ed complex is a membe o he Opie amily in he maize BAC AY078063 [10], in posi ion 57992–74088, e e se o ien a ion, wi h GCATG as he TSDs ( he de ailed alignmen s o LTRs as he do e images o hose complexes a e p o ided in addi ional ile 2). A model ha can explain complex inse ions such as hese in ol es abno mal empla e swi ching as a pa o e e se ansc ip ion. Immedia ely p eceding e e se ansc ip- ion, he RNA ma ix o ms a loop, using he high homol- ogy be ween he wo R egions (5' and 3') wi hin he LTRs o buckle he wo ends o a single empla e oge he . This allows he (-)-s and cDNA, which o he wise canno p o- ceed once i eaches he 5' end o he RNA empla e, o jump ac oss o he 3' end and con inue. The jump is called empla e swi ching. The p ocess leads o pe ec iden i y be ween he 5' and he 3' LTR o he newly syn hesized elemen , because he R and U3 segmen s o he 5' LTR and he U5 segmen o 3' LTR in he RNA a e copied in o bo h LTRs o he cDNA. The cDNA is ul ima ely inse ed in o a new genomic loca ion by he in eg ase. The enzyme liga es he cDNA o one s and o he asymme ic double- s and b eak in he hos DNA, which is o med concomi- an ly wi h he liga ion. The epai ing o his b eak leads o he TSD (Figu e 5A; e iewed in [1]). (A) No mal in a-s and pai ing o e e se ansc ip ion (middle) and inse ion (bo om) o an LTR e o ansposonFigu e 5 (A) No mal in a-s and pai ing o e e se ansc ip ion (middle) and inse ion (bo om) o an LTR e o ansposon. (B) P oposed o ma ion o a empla e-swi ching complex. In e -s and pai ing (middle) occu s be ween wo di e en RNAs, and he esul ing inse ion (bo om) ha bo s TSDs as well as homologies be ween he wo ex e nal LTRs. AB A emis [16] iew o Angela complex on he AY368673 sequence ( om [7])Figu e 3 A emis [16] iew o Angela complex on he AY368673 sequence ( om [7]). The LTRs a e shown in da k blue, he pu a i e polyp o eins in g een, and he whole elemen sequence in ligh blue. The a ows indica e he di ec ion o he inse - ion. Do e analysis o he h ee LTRs om he Angela complex on he AF497474 whea sequenceFigu e 4 Do e analysis o he h ee LTRs om he Angela complex on he AF497474 whea sequence. Diagonal lines ep esen he simila i ies be ween he sequences. The longe and mo e solid he line is, he s onge he simila i y is. The LTRs a e labeled acco ding o hei posi ion in he elemen . LTR-3 LTR-1 LTR-2 LTR-3 LTR-1 LTR-2 BMC Genomics 2007, 8:247 h p://www.biomedcen al.com/1471-2164/8/247 Page 4 o 5 (page numbe no o ci a ion pu poses) E o s in empla e choice du ing he e e se ansc ip ion can occu anywhe e along he sequence. The g owing cDNA can jump o he o he packaged empla e ins ead o o he o he end o he empla e i is al eady on. Gene ally, because he wo packaged empla es a e almos iden ical (de i ed om he same e o ansposon o e o i us RNA), he phenomenon is unde ec able because he e a e no majo modi ica ions o he esul ing cDNA. Howe e , i wo di e en RNAs a e packaged in he same i us-like pa icle, a jump o he o he empla e du ing e e se an- sc ip ion leads o abno mal o new elemen s, opening a new mode o LTR e o ansposon e olu ion. The VejuL [11] and BARE2 [8] elemen s appea o ha e been o med in his way. I RNAs om wo sligh ly di e en indi idual LTR e o- ansposons a e co-packaged, he s and swi ch could occu also be ween he wo R egions. This would lead o o ma ion o a he e odime (Figu e 5B) a he han a no - mal monome (Figu e 5A). The esul ing cDNA would cons i u e a chime ic complex be ween he wo elemen s, and possess chime ic LTRs. The p ocess o e e se an- sc ip ion desc ibed abo e ende s he ex e nal LTRs iden- ical. Thei 3' ends would be he e o e also iden ical and could se e as subs a es o he same ype o in eg ase. Thus, a chime ic complex elemen ne e heless would be in eg a ed ia s anda d in eg ase ca alysis, leading o a new genomic inse ion ha bo ing TSDs on ei he side (Figu e 5B). The dime iza ion could occu be ween he wo packaged RNAs om highly simila elemen s, such as closely ela ed membe s o he same e o ansposon am- ily, leading o a complex ha bo ing h ee iden ical LTRs in e spe sed be ween wo simila in e nal egions. Mo eo- e , because he LTRs would be comple e and no comp o- mised by he e oduplex o ma ion, each o hem would be able o p omo e he exp ession o i s co esponding downs eam elemen . Thus, he wo o iginal elemen s could be exp essed as no mal and indi idual copies and e en p opaga e h ough he genome as sepa a e elemen s. Conclusion Only one empla e-swi ching complex could be iden i ied on he 350 Mb sequence o he ice genome, one on he a ailable maize sequences, and none in he A abidopsis genome. On he ~7 Mb o sequences cu en ly a ailable o he T i iceae (whea , ba ley, and ela ed species), wo empla e-swi ching complexes we e iden i ied ou o he 20 ecombinan complex elemen s ecognized (Table 1). Al hough such chime ic complexes, o med by e e se ansc ip ion, o m a ela i ely mino sha e o he genome when compa ed o hose o med by pos -inse ional ecombina ion, hey appea none heless o be mo e abun- dan in he T i iceae genomes han elsewhe e. The genome o diploid ba ley is oughly 5 × 109 bp and ha o hexaploid b ead whea abou 16 × 109 bp. I he obse ed equency o wo o hese complexes in he a ailable sequences holds h oughou he ba ley and whea genomes, he wo ce eals should ha bo ca. 6000 com- plexes o med by e e se ansc ip ion. Fo ma ion o hese complexes is ano he mani es a ion, oge he wi h low eplica ion ideli y and ansduc ion o genomic sequences, o he luid and lexible na u e o e o anspo- si ion. Fu he mo e, he complex elemen s epo ed he e may poin o mechanis ic di e ences be ween plan spe- cies, in iew o he di e ences in hei abundance be ween he species we we e able o examine. The model we p opose is consis en bo h wi h he a aila- ble da a and wi h he es ablished de ails o he e o ans- poson li e cycle. A di ec demons a ion o he mechanism would en ail isola ion o i us-like pa icles con aining wo pai ed RNAs (Figu e 5B) and demons a ing he RNA s uc u e. This, howe e , awai s bo h an e icien sys em o p oduc ion o packaged complexes (pe haps by o e - exp ession o a e o ansposon wi h a endency o o m complexes) and a means o dis inguishing he numbe o mRNAs p esen wi hin he buckle. Me hods All cu en ly public a ailable T i iceae (whea and ba ley) BACs we e e-analyzed as in [7]. The upda ed anno a ions we e used o analyze he inse ion complexes. The o iginal analyses o AF497474 om Aegilops auschii, AF368673 om T i icum u gidum and AY078063 om Zea mays we e pe o med espec i ely by [3,4], and [10]. The sequences o he ice pseudomolecules (~367 Mb) we e downloaded om he TIGR websi e [12]. The scanned maize sequences ep esen he whole la ge sequences a ailable o maize in he public da abase, i.e., excluding he ace iles and he gene-only sequences. They we e downloaded om he NCBI websi e [13] and ep esen ~1 650 Mb. Table 1: Numbe o inse ions in ~7 Mb o T i iceae la ge-inse sequences Type o e en Numbe o e en s LTR e o ansposon inse ions 400 Copia inse ions 137 Gypsy inse ions 245 LARD inse ions 9 TRIM inse ions 9 Solo-LTR o ma ions 70 O he ecombina ion e en s 220 Recombinan Complexes 20 Templa e Swi ching Complexes 2 LINE inse ions 61 DNA T ansposon inse ions 118 Da a in i alics we e p oduced om he e-anno a ed sequences o [7]. The ecombina ion e en s ep esen all he dele ions, inse ions, and emodeling e en s de ec able in he elemen s. Publish wi h BioMed Cen al and e e y scien is can ead you wo k ee o cha ge "BioMed Cen al will be he mos signi ican de elopmen o dissemina ing he esul s o biomedical esea ch in ou li e ime." Si Paul Nu se, Cance Resea ch UK You esea ch pape s will be: a ailable ee o cha ge o he en i e biomedical communi y pee e iewed and published immedia ely upon accep ance ci ed in PubMed and a chi ed on PubMed Cen al you s — you keep he copy igh Submi you manusc ip he e: h p://www.biomedcen al.com/in o/publishing_ad .asp BioMedcen al BMC Genomics 2007, 8:247 h p://www.biomedcen al.com/1471-2164/8/247 Page 5 o 5 (page numbe no o ci a ion pu poses) The ab ini io iden i ica ion o LTR e o ansposons wi hin he ice and maize sequences was pe o med by he LTR_STRUC so wa e [9] using s anda d speci ica ions. All o he 4072 po en ial complex elemen s ou pu by his p og am we e i s sc eened by a home-made Py hon sc ip acco ding o hei size, and he 1416 candida es mee ing he c i e ion o >10 kb leng h we e hen manually checked using Do e [12] o he p esence o he in e nal LTR. The LTR s. LTR analyses we e pe o med using Do e [12], and he a ge -si e duplica ion we e manually e i ied. The LTR alignmen s we e e i ied using Clus alX [15], a e manual edi ing as necessa y (see Supplemen al da a). Abb e ia ions LTR, long e minal epea TSD, a ge -si e duplica ion. Au ho s' con ibu ions FS designed and pe o med he esea ch and w o e he manusc ip . AHS di ec ed he esea ch as well as edi ed and con ibu ed o he manusc ip . Bo h au ho s ead and app o ed he inal manusc ip . Addi ional ma e ial Acknowledgemen s The au ho s hank Jaakko Tanskanen o his help wi h he Py hon sc ip s. FS was suppo ed by a ellowship om CIMO and by a Uni e si y o Helsinki Pos doc o al Fellowship. Expe imen s desc ibed he e we e ca ied ou unde a g an om Academy o Finland, P ojec 106949. Re e ences 1. Sabo F, Schulman AH: Pa asi ism and he e o ansposon li e cycle in plan s: A hi chhike 's guide o he genome. He edi y 2006, 97:381-388. 2. De os KM, B own JKM, Benne zen JL: Genome size educ ion h ough illegi ima e ecombina ion coun e ac s genome expansion in A abidopsis. Genome Res 2002, 12:1075-1079. 3. Ande son OD, Rausch C, Moulle O, Lagudah ES: The whea D- genome HMW-glu enin locus: BAC sequencing, gene dis i- bu ion, and e o ansposon clus e s. Func In Genomics 2003, 3:56-68. 4. Kong X-Y, Gu YQ, You FM, Dubco sky J, Ande son OD: Dynamics o he e olu ion o o hologous and pa alogous po ions o a complex locus egion in wo genomes o allopolyploid whea . Plan Mol Biol 2004, 54:55-69. 5. Chan e N, Cenci A, Sabo F, Ande son OD, Dubco sky J: Sequencing o he T i icum monococcum Ha dness locus e eals good mic ocolinea i y wi h ice. Mol Gene Genomics 2004, 271:377-386. 6. Chan e N, Salse J, Sabo F, Rahman S, Bellec A, Laubin B, Dubois I, Dossa C, Sou dille P, Joud ie P, Gau ie MF, Ca olico L, Becke M, Aubou g S, Weissenbach J, Caboche M, Be na d M, Le oy P, Chal- houb B: Molecula basis o e olu iona y e en s ha shaped he Ha dness, Ha locus in diploid and polyploidy whea spe- cies, T i icum and Aegilops. The Plan Cell 2005, 17:1033-1045. 7. Sabo F, Guyo R, Wicke T, Chan e N, Salse J, Laubin B, Le oy P, Sou dille P, Chalhoub B, Be na d M: Upda ing ansposable ele- men anno a ions om la ge whea genomic sequences e eals di e se ac i i ies and gene associa ion o elemen s. Mol Gene Genomics 2005, 274:119-132. 8. Vicien CM, Kalenda R, Schulman AH: Va iabili y, ecombina ion and mosaic e olu ion o he ba ley BARE-1 e o ansposon. J Mol E ol 2005, 61:275-291. 9. McCa hy EM, McDonald JF: LTR_STRUC: a no el sea ch and iden i ica ion p og am o LTR e o ansposons. Bioin o ma - ics 2003, 19:362-367. 10. S am M, Belele C, Ramak ishna W, Do weile JE, Benne zen JL, Chan- dle VL: The egula o y egions equi ed o B' pa amu a ion and exp ession a e loca ed a ups eam o he maize b1 ansc ibed sequences. Gene ics 2002, 162:917-930. 11. Sabo F, Sou dille P, Be na d M: Ad en o a new e o anspo- son s uc u e: he long o m o he Veju elemen s. Gene ica 2005, 125:325-335. 12. TIGR homepage [h p://www. ig .o g] 13. NCBI homepage [h p://www.ncbi.nlm.nih.go ] 14. Sonnhamme EL, Du bin R: A do -ma ix p og am wi h dynamic h eshold con ol sui ed o genomic DNA and p o ein sequence analysis. Gene 1995, 167:GC1-10. 15. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: imp o ing he sensi i i y o p og essi e mul iple sequence alignmen h ough sequence weigh ing, posi ion-speci ic gap penal ies and weigh ma ix choice. Nucleic Acids Res 1997, 22:4673-4680. 16. A emis a Sange Ins i u e [h p://www.sange .ac.uk/So wa e/ A emis/] Addi ional ile 1 Clus alX alignmen s o he LTRs om he wo Angela complexes om T i iceae. Click he e o ile [h p://www.biomedcen al.com/con en /supplemen a y/1471- 2164-8-247-S1.pd ] Addi ional ile 2 Do e and Clus alX alignmen s o he LTRs om he Rice and Maize complexes. Click he e o ile [h p://www.biomedcen al.com/con en /supplemen a y/1471- 2164-8-247-S2.pd ]