BARE Re o ansposons A e T ansla ed and Replica ed
ia Dis inc RNA Pools
Wei Chang1, Ma ko Jääskeläinen1, Song-ping Li2¤, Alan H. Schulman1,3*
1 Ins i u e o Bio echnology, Viikki Biocen e , Uni e si y o Helsinki, Helsinki, Finland, 2 Genome-Scale Biology P og am, Uni e si y o Helsinki, Biomedicum,
Helsinki, Finland, 3 Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, Jokioinen, Finland
Abs ac
The eplica ion o Long Te minal Repea (LTR) e o ansposons, which can cons i u e o e 80% o highe plan
genomes, esembles ha o e o i uses. A majo ques ion o e o ansposons and e o i uses is how he wo
con lic ing oles o hei ansc ip s, in ansla ion and e e se ansc ip ion, a e balanced. He e, we show ha he
BARE e o ansposon, despi e i s o ganiza ion in o jus one open eading ame, p oduces h ee dis inc classes o
ansc ip s. One is capped, polyadenyla ed, and ansla ed, bu canno be copied in o cDNA. The second is no
capped o polyadenyla ed, bu is des ined o packaging and ul ima e e e se ansc ip ion. The hi d class is capped,
polyadenyla ed, and spliced o a o p oduc ion o a subgenomic RNA encoding only Gag, he p o ein o ming i us-
like pa icles. Mo eo e , he BARE2 sub amily, which canno syn hesize Gag and is pa asi ic on BARE1, does no
p oduce he spliced sub-genomic RNA o ansla ion bu does make he eplica ion compe en ansc ip s, which a e
packaged in o BARE1 pa icles. To ou knowledge, his is i s demons a ion o dis inc RNA pools o ansla ion and
ansc ip ion o any e o ansposon.
Ci a ion: Chang W, Jääskeläinen M, Li S-p, Schulman AH (2013) BARE Re o ansposons A e T ansla ed and Replica ed ia Dis inc RNA Pools. PLoS
ONE 8(8): e72270. doi:10.1371/jou nal.pone.0072270
Edi o : Chen Liang, Lady Da is Ins i u e o Medical Resea ch, Canada
Recei ed May 28, 2013; Accep ed July 14, 2013; Published Augus 6, 2013
Copy igh : © 2013 Chang e al. 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, 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 au ho and sou ce a e c edi ed.
Funding: This wo k was suppo ed by he Academy o Finland (h p://www.aka. i/ i/A/), P ojec 123074. Funding o open access cha ge: Uni e si y o
Helsinki. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing in e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: [email p o ec ed]
¤ Cu en add ess: Depa men o Pa hology, Uni e si y o Tu ku and Tu ku Uni e si y Hospi al, Tu ku, Finland
In oduc ion
Long e minal epea (LTR) e o ansposons o Class I
ansposable elemen s a e ubiqui ous in he euka yo es and
can comp ise o e 80% o he la ge genomes o plan s [1,2].
They p opaga e simila ly o he in acellula phase o
e o i uses: by a “copy and pas e” cycle o ansc ip ion o
genomic copies, ansla ion, packaging o ansc ip s in o i us-
like pa icles (VLPs) composed o Gag, e e se ansc ip ion,
and a ge ing o he cDNA copy o he nucleus o in eg a ion
in o he genome [3,4]. The li ecycle depends upon p o eins
encoded by he e o ansposon i sel .
The s uc u al Gag is o en in a sepa a e open eading ame
(ORF) om Pol, which encodes he enzymes e e se
ansc ip ase (RT), RNAse H (RH), aspa ic p o einase (PR),
and in eg ase (IN). The s oichiome y be ween gag and pol
p oduc s is c i ical o eplica ion because he assembly o he
VLP equi es excess Gag ela i e o he enzymes [5,6]. A
common s a egy among e o i uses and e o ansposons o
p oduce mo e Gag is -1 o +1 ansla ional ameshi ing
be ween gag and pol [6–8]. Howe e , he Copia supe amily o
e o ansposons [3] and also mos plan e o ansposons ha e
only a single ORF [9]. While al e na i e splicing in copia o
D osophila dele es i ually all o pol, 2950 n , gene a ing an
RNA dedica ed o Gag ansla ion [10], his has no been seen
o o he membe s o he supe amily. In some cases pos -
ansla ional p o ein deg ada ion se es o achie e a mola
excess o Gag [11].
Ano he majo conund um is ha e e se ansc ip ion o
e o ansposon and e o i us RNA, which des oys he RNA
empla e, con lic s wi h i s u he ansla ion [12]. Al e na i ely,
ins ead o a single pool o RNA, sepa a e popula ions may
se e each unc ion wi h no all RNA being seques e ed in o
capsids o e e se ansc ip ion. Among he e o i uses,
Mu ine Lukemia Vi us (MLV) may use dis inc RNA pools [13],
whe eas HIV-1 and -2 do no [14]. The ques ion has no been
in es iga ed o e o ansposons.
Al hough e o ansposons comp ise much o mos plan
genomes, he de ails o hei li ecycle ha e been in es iga ed
o only a ew. The BARE e o ansposon o supe amily Copia
accoun s o o e 10% o he ba ley genome [2,15]. BARE1
has no ameshi be ween gag and pol [16,17]. A a ian called
BARE2 canno exp ess Gag [16,18]. BARE1 and BARE2
p oduce mul iple classes o RNA ansc ip s om wo TATA
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boxes, o which only 15 o 25% a e polyadenyla ed [19].
Mo eo e , hose which a e polyadenyla ed lack he R domain
needed o e e se ansc ip ion. These obse a ions aise he
ques ions add essed he e: which BARE RNAs se e o
ansla ion, which ones a e packaged, and does BARE use an
al e na i e o ameshi ing o Gag p oduc ion. We we e able o
demons a e no only di e en RNA pools o ansla ion and
e e se ansc ip ion bu also a no el splicing pa e n o Gag
syn hesis.
Ma e ials and Me hods
Plan Ma e ials and RNA Isola ion
Ba ley (Ho deum ulga e L.) plan ma e ials and callus
cul u es used o RNA isola ion, as well as he me hods used o
isola e he RNA, a e desc ibed in he Ma e ials S1.
RNA End S uc u e
The p esence o a 5’ 7-me hylguanosine cap on BARE
ansc ip s was assayed by he p ocedu e called RLM-
media ed apid ampli ica ion o 5’ cDNA ends (5’ RLM-RACE)
using a ki (Fi s Choice®RLM-RACE, Ambion AM1700) wi h
small changes and a cus om adap e . To examine 3’
polyadenyla ion o poly ibosome-associa ed RNA, 3’ RLM-
RACE was ca ied ou . De ails o bo h me hods a e p esen ed
in he Ma e ials S1.
Analysis o BARE1 and BARE2 Exp ession Le els
To e alua e he ela i e exp ession le els o BARE1 and
BARE2, RT-PCR was ca ied ou using p ime AP4 (Table S1)
o p ime cDNA syn hesis and hen p ime s LS1 (Table S1) and
AP4 o he ampli ica ion eac ion. The p ime pai binds bo h o
BARE1 and BARE2, and ampli ies hem equally well [16]. The
wo e o ansposon amilies we e ampli ied om genomic DNA
using he same p ime pai .
Poly ibosome Isola ion and RT-PCR
Ba ley callus cul u es cells we e collec ed, ozen unde
liquid N2, and hen pul e ized wi h mo a and pes le unde
liquid N2. Poly ibosomes we e isola ed la gely as p e iously
desc ibed [20]. The p ocedu e is desc ibed in de ail in he
Ma e ials S1.
Splicing assays
Splicing analysis was made wi h 1µg o RNA ea ed wice
wi h DNase, e e se ansc ibed in o cDNA as using he BARE-
speci ic p ime 81567. The BARE ansc ip s we e ampli ied
ollowing cDNA p oduc ion using wo p ime s close o he
spliced egion, F1593 and F1594 (Table S1), and a PCR
p og am consis ing o 94oC o 5 min, 40 cycles o 94oC o 30
sec, 56oC o 1 min, and 72oC o 1 min, wi h a inal ex ension
a 72oC o 5 min. To in es iga e splicing in polyadenyla ed
RNA, p ime 81567 was used o ini ia e cDNA syn hesis,
ollowed by F1594 and AP4 o PCR ampli ica ion. Fo
de ec ion o capped RNA splicing, an RNA linke was i s
liga ed o dephospho yla ed and decapped RNA and hen
cDNA syn hesized as abo e. Two PCR eac ions we e
p epa ed by 5’ RACE using he linke and 81567 as he p ime
pai . One eac ion was ampli ied, he o he se ed as a con ol.
The second RACE eac ion used 1µl o ei he he i s PCR
p oduc o he con ol as he empla e and p ime s F1594 and
81567. Con ols p oduced no signal om he second
ampli ica ion. The decapped RNA ga e he same size p oduc
as did o al RNA RT-PCR using same p ime pai .
Resul s
BARE1 bu no BARE2 RNA Is Spliced
Ampli ica ion o BARE om o al RNA p oduced wo
p oduc s, one consis en wi h he size o genomic BARE copies
and he o he somewha smalle (Figu e 1). Ampli ica ions om
genomic DNA (Figu e 1B) p oduced no smalle p oduc . A
smalle p oduc was ampli ied om he RNAs o all issues
es ed, which we e callus and emb yo (Figu e 1C) as well as
lea and oo (da a summa ized in Table 1). A o al o 60 clones
we e sequenced om he mo e abundan , longe p oduc ;
BARE1 and BARE2 we e equally p esen among he
sequences. The wo la ge p oduc s seen in callus RNA (Figu e
1C) di e only by ampli ica ion om a seconda y PCR p iming
si e. All he sequences om he sho p oduc , howe e , we e
om BARE1 and con ained a dele ion a he beginning o p
domain, comp ising a segmen o 104 n lanked by GT and AG
espec i ely a he le and igh bo de s (Figu e 2A).
The p esence o he sho o m o BARE1 in he RNA bu no
he genome sugges ed ha i is a spliced ansc ip . The
BARE1 genomic sequences con ain conse ed CAG/GTAT
and CAG/GA mo i s espec i ely ma ching he 5’ and 3’
junc ions CAG/---/GA ha lank he segmen missing in he
mino cDNA sequence (Figu e 2A). These a e a e y good
ma ch o he consensus sequence AG/GT o he dono si e in
a genome-wide su ey o he model species B achypodium
dis achyon [21] and o consensus dono and accep o splice
si es, espec i ely C(A) AG/GTA and CAG/G, in A abidopsis
and ice [22,23]. The BARE1 junc ions a e also well iden i ied
by he Ne gene2 splice si e p edic o (h p://www.cbs.d u.dk/
se ices/Ne Gene2/) wi hin he A abidopsis genome and by
SpliceP edic o (h p://deepc2.psi.ias a e.edu/cgi-bin/sp.cgi)
agains maize and human genomes, suppo ing he
in e p e a ion ha he sho e BARE ansc ip is a splicing
p oduc .
No ably, he p edic ed splicing signals a e no ound in he
BARE2 genomic o RNA sequences (Figu e 2A). The a eas
immedia ely 5’ o he dono si e and 3’ o he accep o si e o
BARE1 a e also di e gen in BARE2, al hough he sequence o
he acul a i e in on is qui e simila in bo h. While he sho
o m comp ised a mino ac ion o o al RNA comp ising bo h
BARE1 and BARE2, abou 12.5% using he p ime s (Figu e
1A) ha ampli ied bo h, i ep esen ed ully hal o he BARE1-
speci ic p oduc ampli ied om polyadenyla ed RNA (Figu e
1C). The p edic ed and sequenced splice junc ion is 2 n
beyond he end o he gag coding domain [16,17], he eby
c ea ing a s op codon h ee amino acids beyond he end o
Gag (Figu e 2B,C,D) ollowed by many mo e wi hin pol.
Consequen ly, he spliced RNA can exp ess only Gag (Figu e
2D). The p edic ed molecula weigh o he Gag om he
BARE Re o ansposon T ansc ip ion o Replica ion
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Figu e 1. Splicing o BARE ansc ip s. A. Aga ose gel
elec opho esis o he RT-PCR ampli ica ion p oduc om
callus RNA using p ime s LS1 and AP4. The uppe band
(a ow, 1.3 kb) con ains p oduc s om bo h BARE1 and
BARE2 and is he same size as he ampli ica ion p oduc om
genomic DNA using he same p ime pai (B); he lowe , ain
band (A, a ow, 1.2 kb) is he spliced BARE1 o m and i is no
seen in he genomic DNA ampli ica ion. B. RT-PCR (+) om
o al RNA (p ime s LS1 and AP4); he lanes display eac ions
con aining e e se ansc ip ase (+), nega i e con ol lacking
e e se ansc ip ase (-), o genomic DNA ins ead o RNA (G)
as he posi i e con ol. C. De ec ion o splicing in emb yo, E,
and callus, C, o al and poly(A) RNA (labelled p (A)) using a
BARE1-speci ic p ime pai (81567, F1594). A ows indica e
he unspliced and spliced o ms. Size ma ke s (m), 100 bp
ladde , ma ked band is 1 kb (A, B) o 0.5 kb (C).
doi: 10.1371/jou nal.pone.0072270.g001
spliced RNA is 32.1 kDa, he same as p edic ed om he
elec opho e ic mobili y o Gag om VLPs [18].
In ons in plan s a e gene ally 15% mo e U- ich han he
lanking exons, while exons a e 15% mo e GC- ich han he
co esponding in on [24–26]. In BARE1, he 52 n lanking he
slicing signals a e 13.5% U s. 37.5% U o he 104 n in on,
making he in on 24% and 2.8- old mo e U- ich han he exon
segmen s. In BARE2, which does no splice, he in on egion
is only 16.7% and 1.9- old mo e U- ich han he su ounding
egion. Mo eo e , hese lanking exon egions in BARE1 a e
GC- ich, being 53.8% GC, 16.3% mo e GC han he in on,
whe eas he co esponding BARE2 segmen is 47.2% GC, only
a 7.6% di e ence wi h he su ounding egions. Bo h hese
measu es and he splice si e compa ison show ha he BARE1
in on con o ms o expec a ions o plan in on unc ionali y and
sugges ha he e has been selec i e p essu e on BARE1 o
splicing compa ed wi h BARE2.
T ansc ip s om BARE TATA1 a e uncapped, bu hose
om TATA2 a e capped
Because he wo RNA splice a ian s o BARE1 possess
di e en ansla ion capaci ies, we in es iga ed he ansc ip s
o ea u es associa ed wi h ansla ion. The RNA des ined o
ansla ion in euka yo ic cells commonly ecei es a 7-
me hylguanosine cap as pa o he ma u a ion p ocess [27],
al hough many plan i uses as well as HIV exploi cap-
independen ansla ion ins ead [28,29]. We ea lie showed [19]
ha BARE p oduces en classes o ansc ip s om wo TATA
boxes (Figu e 3A), i e each om TATA1 and TATA2. In o de
o in es iga e which migh be ansla ed, we i s looked a
hose which ha e 5’ caps.
Capped and uncapped RNAs we e dis inguished by
enzyma ic ea men be o e RNA-ligase-media ed (RLM) PCR,
espec i ely by py ophospha ase decapping and phospha ase
5’ dephospho yla ion. Py ophospha ase-media ed cap emo al
gene a es a 5’ phospha e in i s place, which will allow liga ion
o an RNA adap e and PCR. Uncapped 5’ ends can be liga ed
di ec ly wi hou py ophospha ase ea men .
The expe imen s we e ca ied ou wi h p ime s posi ioned
(Figu e 3A) so ha RNA p oduc s o bo h TATA1 and TATA2
could be de ec ed, bu only hose om in ac BARE elemen s
con aining in e nal domains and no om ead- h ough
Table 1. Summa y o BARE RNA species de ec ed.
TE S uc u e Occu ence
Sp Cap pA R C L E
BARE1 + + + - + + +
BARE1 - + + - + + +
BARE1 - - - + + + +
BARE2 - - - - + + +
BARE2 - + + - + + +
Abb e ia ions: TE, e o ansposon amily; Sp, ansc ip spliced; Cap, cap(Gppp)
p esen ; pA, polyadenyla ion; R, R-domain p esen ; C, callus; L, lea ; E, emb yo
Each able ow co esponds o one RNA ype ha ing he ea u es ma ked as
p esen (+) o absen (-)
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ansc ip s o solo LTRs. Capped RNAs ansc ibed om BARE
we e ound in all issues examined (Figu es 2, 4B). Con ol
eac ions lacking py ophospha ase ga e no PCR p oduc
(Figu e 3E). Sequenced PCR p oduc s showed ha capped
ansc ip s de i e only om TATA2; he longe p oduc s om
he emb yo (Figu e 3B) we e sequenced and a e non-speci ic.
The capped ansc ip s s a a n 1686-1689 (Z17327),
co esponding wi h he published RACE-PCR da a [19], which
could no dis inguish capped om uncapped RNA. No ably, he
5’ ends o he capped RNAs a e a posi ions shown ea lie o
be oo a downs eam o pe mi o ma ion o an R domain
needed o eplica ion by e e se ansc ip ion [19].
In a complemen a y expe imen , he uncapped ansc ip s,
which o igina e only om TATA1, we e cloned and sequenced
om o al RNA o emb yo and callus (Figu e 3C). The s a
si es o hese RNAs o callus a e a n 1351, 1379, and 1382
(numbe ing om Z17327). The wo majo bands om emb yo
issue ep esen BARE1 and BARE2 RNA and espec i ely
s a a n 1350 (numbe ing om Z17327) and 1682 (numbe ing
om AJ279072). All s a si es co esponded o hose ound in
he ea lie RACE-PCR da a o TATA1 in BARE1 and BARE2
[19].
Bo h Spliced and Unspliced Capped TATA2 RNAs A e
Polyadenyla ed
As desc ibed abo e, TATA2, bu no TATA1, ansc ip s om
bo h BARE1 and BARE2 a e capped; hose om BARE1 a e
spliced abou hal he ime and BARE2 ansc ip s a e no
spliced. Whe eas ansla ed cellula RNAs a e gene ally bo h
capped and polyadenyla ed, plan i al RNAs and mos
posi i e-s and RNA i uses a e ansla ed no only wi hou
caps bu also wi hou poly(A) ails [30]. To cla i y he si ua ion
o BARE, polyadenyla ed RNAs isola ed om lea and callus
we e subjec ed o RLM-PCR diagnos ic o he p esence o
caps and hen he PCR p oduc s sequenced. Caps we e
Figu e 2. Splicing o BARE1. A. Alignmen o a se o BARE genomic DNA sequences and cDNA clones showing he o ms wi h
he consensus splice junc ions (SD and SA) wi hin he gag domain. Nucleo ides shaded blue ma ch he consensus slice junc ions,
hose in ed do no . Genomic DNA sequences a e labelled as “G”, cDNA as “T”. Genomic sequences in he alignmen a e: G1,
AJ279072; G2, Z17327; G3, AY66155; G4, AY485643; G5, BQ900685. B. Schema ic diag am o BARE e o ansposons showing
he LTRs, encoded p o eins o he open eading ame, and he cDNA p iming si es (PBS, PPT), oge he wi h he posi ion o he
diagnos ic p ime s as a ows below. The in e ed iangle indica es he 8 bp dele ion o he s a codon in BARE2 ha elimina es
syn hesis o Gag; he ollowing ATG o he Pol domain is indica ed. C. Diag ams o he unspliced (1) and spliced (2) o ms o he
BARE1 ansc ip s as well as he ansla ed p oduc o he spliced o m (3). D. Concep ual ansla ion o he BARE1 ORF (Accession
Z17327) co e ing he Gag and pa o he PR egion o he unspliced (Gag) and spliced (Gag_S) ansc ip o ms. Amino acids
al e ed by he splice-induced ameshi a e shown in ed, he s op codon as *.
doi: 10.1371/jou nal.pone.0072270.g002
BARE Re o ansposon T ansc ip ion o Replica ion
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Figu e 3. Capping o BARE ansc ip s. A. Schema ic ep esen a ion o he BARE LTR and pa o he ORF. The black box
be ween gag and p ep esen s he speci ic dele ion in BARE2 (dele ion no o scale). The 5’ LTR is shown as a hick box, he egion
be ween he LTR and he s a codon o gag as a hin box. The posi ion o TATA1 (T1) and TATA2 (T2) a e ma ked wi h ben
a ows, hei posi ion and hose o he beginning and end o he LTR and he beginning o gag numbe ed acco ding o acc. Z17327.
P ime s used o PCR and making cDNA a e indica ed by a ows below. Wa y lines indica e he capped TATA2 and uncapped
TATA1 ansc ip s iden i ied by RLM-RACE PCR. B. RLM-RACE PCR analysis o he ansc ip ion s a si es o BARE om o al
RNA in di e en issues (C, callus; E, emb yo; R, oo ) ollowing phospha ase and py ophospha ase ea men o selec o capped
ansc ip s. (+) and (-) indica e he p esence o absence (con ol) o e e se ansc ip ase in he assay. The p oduc size (a ow, 244
bp) co esponds o ampli ica ion om he 5’ adap e p ime and E1625. C. De ec ion o uncapped BARE ansc ip s in emb yos and
callus o al RNA by RLM-RACE PCR wi hou phospha ase and py ophospha ase ea men . The la ge band (a ow) co esponds o
BARE1, he smalle (a ow) o BARE2. D. De ec ion o capped BARE poly(a) RNA (a ow) in callus (Cp(A)) and emb yo (Ep(A)) by
RLM-RACE PCR; RNA ea ed as in (B). The uppe band in emb yo is due o a seconda y p iming si e; he ampli ica ion gene a es
a p oduc o he same size o BARE1 and BARE2. 100 bp ladde s (m) a e shown. E. Con ol eac ions ea ed wi h phospha ase
bu no subsequen py ophospha ase.
doi: 10.1371/jou nal.pone.0072270.g003
BARE Re o ansposon T ansc ip ion o Replica ion
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Figu e 4. Spliced BARE RNA is associa ed wi h poly ibosomes. A. Diag am o he 3’ LTR, indica ing o e e ence he posi ion
o he wo TATA boxes TATA1 (T1) and TATA2 (T2); only hose in he 5’ LTR se e o ansc ibe he e o ansposon. The posi ions
o o wa d p ime s RLM1 and RLM2 o 3’ RLM-RACE a e shown. The wa y lines show, espec i ely, he app oxima e e mina ion
posi ions o he poly ibosome-associa ed poly(A) RNA. B. Supe na an (Sup) and poly ibosome pelle (Pel) ac ions om o al
callus RNA (To ) ul acen i uged on a 10-45% suc ose g adien . RNA bands a e labelled. C. Elec opho esis o RLM-RACE
eac ions om poly ibosome-associa ed callus and lea RNA ampli ying bo h BARE1 and BARE2. A 100 bp size ladde (m) is
shown, 500 bp ma ked. D. Ampli ica ion o BARE2 and BARE1 om poly ibosome-associa ed callus RNA (p ime s 1965 and 1966
o BARE2, p ime s Gag5 and AP4 o BARE1, Figu e 2). Nega i e con ols (-) o he p esence o genomic DNA con amina ion lack
e e se ansc ip ase; posi i e con ols, G, con ain genomic DNA. Ba s poin o 1000 bp and 500 bp size ma ke s, m. E. RT-PCR
assay (p ime s F1594 and F1593, Figu e 2, which a e no speci ic o BARE1) om he ac ions in (B). Fo size compa ison, PCR
om genomic DNA wi h he same p ime s is shown on he igh . Unspliced and spliced ansc ip s a e indica ed as U and S,
espec i ely. The RT-minus con ols ga e no ampli ica ion.
doi: 10.1371/jou nal.pone.0072270.g004
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p esen in bo h BARE1 and BARE2 p oduc s om he
polyadenyla ed RNA ac ion; hese ansc ip s s a only a e
TATA2 (Figu e 3D). In con ol expe imen s, he 5’ adap e s
we e di ec ly liga ed o polyadenyla ed RNA om emb yo and
callus. The eac ions yielded no p oduc , indica ing ha no
BARE RNA was simul aneously polyadenyla ed and uncapped.
Polyadenyla ed BARE RNA Is Poly ibosome-
Associa ed
Gi en he mul iple BARE1 and BARE2 RNA species, we
in es iga ed which pool is ansla ed by examining RNA in
poly ibosomal ansla ion complexes. As desc ibed abo e, he
BARE2 ansc ip s nei he exp ess Gag no a e spliced, aising
he ques ion o whe he hey a e none heless p esen among
he poly ibosomal RNAs. Poly ibosomes we e isola ed; he 28S
and 18S ibosomal RNAs we e e ec i ely concen a ed in o he
pelle (Figu e 4B). The supe na an e ained mainly he small
RNAs (mos ly RNAs), indica ing he p esence o he
poly ibosomes in he pelle . Capped RNA was de ec ed by 5’
RLM-RACE using a p ime ma ching bo h BARE1 and BARE2
(Figu e 4B). BARE1 and BARE2 RNAs we e dis inguished by
sequencing and ound in poly ibosomes o lea , callus and
emb yos, he h ee issues in es iga ed. Sequencing showed
he s a si es a n 1689 (accession Z17327) o BARE1 and
25n downs eam o TATA2 o BARE2 (AJ279072) as be o e
[19]. Fo compa ison, cloning and sequencing o he uncapped
RNA p oduc s in o al RNA e ealed ha TATA1 ansc ip s
s a a posi ion n 1351 in emb yos and 1412 in callus as
desc ibed ea lie [19].
To look a he ela i e abundance o BARE1 and BARE2
ansc ip s in he poly ibosomes, speci ic p ime pai s we e
used. The BARE2 poly ibosomal ansc ip s a e mo e abundan
han hose o BARE1 (Figu e 4D), co esponding o ea lie
esul s o he same ba ley cul i a (Bomi): BARE1 and TATA2
ansc ip s we e shown o ep esen espec i ely 32% and 6–
25% o he o al BARE pool [16,19]. Hence, he lack o a
ansla able gag domain does no appea o in e e e wi h he
BARE2 ansc ip s ei he being capped o o ming
poly ibosomes and sugges s ha BARE2 pol is ansla ed,
e en i gag canno be, by ei he ibosome scanning o in e nal
en y.
The p esence o he spliced BARE1 ansc ip ha codes
only o Gag aises he expec a ion ha i he spliced o m
con ibu es o p oduc ion o Gag i , oo, should be
polyadenyla ed and associa ed wi h poly ibosomes. The o al
RNA om callus was used as a empla e o RT-PCR using
p ime s nea he splice junc ion; bo h spliced and unspliced
o ms we e p esen (Figu e 3E). The pelle con aining he
poly ibosomes also con ained he spliced o m, a a p opo ion
o he o al BARE pool simila o ha expec ed. The da a show
ha he spliced o m is no di e en ially excluded om
poly ibosomes, al hough some o he unspliced o m emains in
he supe na an . In o de o examine poly ibosomal BARE RNA
o polyadenyla ion, o al RNA was i s isola ed om he
poly ibosomes and wo ounds o 3’ RLM-RACE ca ied ou ,
i s o selec o he 3’ LTR segmen o he LTR (Figu e 4A)
and hen o a poly(A) ail. The PCR p oduc s we e sequenced;
he polyadenyla ed, poly ibosomal BARE ansc ip s sha ed
hei 3’ e mini wi h hose in he polyadenyla ed BARE
popula ion in o al RNA [19].
Non-Polyadenyla ed BARE RNA Is Packaged in o VLPs
The TATA1 ansc ip s, as shown abo e, a e no capped,
spliced, o polyadenyla ed. Because only hey con ain he R
domain, i BARE is being eplica ed hen TATA1 ansc ip s
should be packaged in o VLPs. To in es iga e his, we isola ed
VLPs and examined he ends o RNAs associa ed wi h hem.
The RNA was isola ed om he pooled and pu i ied VLP
ac ions 9-11 desc ibed p e iously [18], and used as he
empla e o 3’ RLM-RACE (Figu e 5). The sequenced p oduc s
e eal ha only non-polyadenyla ed RNA is packaged in VLPs.
Sequences o he packaged RNAs ha e he p e iously
desc ibed end poin s ha a e expec ed o ansc ip s ini ia ed
by TATA1 and no TATA2 [19]. Fu he mo e, he RNA
sequences include TATA1 ansc ip s o BARE2, which is no
able o p oduce he Gag o he VLPs in o which i s ansc ip s
a e packaged. This clea ly demons a es he pa asi ism o
BARE2 on BARE1.
Discussion
Re o ansposon and e o i us ansc ip s se e wo dis inc
oles: as empla es o he p o eins needed o hei own
eplica ion; as genomic RNA, which is i s packaged in o
capsids comp ised o Gag, i s own ansla ion p oduc , and
hen la e des oyed du ing i s e e se ansc ip ion in o cDNA.
We ea lie showed ha e o ansposons BARE1 and i s
pa asi ic ela i e BARE2, which canno syn hesize i s own
capsid p o ein, p oduce wo se s o ansc ip s; one om each
o he wo TATA boxes in he LTR [19,31]. We also showed
ha only a mino i y o he BARE ansc ip s, 15 o 25%, we e
polyadenyla ed, al hough nei he was ansc ip p ocessing
u he examined no he eason o he incomple e
polyadenyla ion ound. He e, we ha e unco e ed a eplica ion
sys em whe eby BARE1 and BARE2 encode dis inc classes o
RNAs o se e he wo dispa a e unc ions, one o ansla ion
and he o he as he genomic RNA des ined o be e e se-
ansc ibed in o cDNA (Figu e 6). The esul s a e eminiscen o
he dis inc pools o ansla ion and e e se ansla ion
pu po edly o med by he MLV e o i us [13], a he han he
single pool o HIV [14].
The i s RNA pool, ansc ip s om TATA2 o bo h BARE1
and BARE2, is capped, polyadenyla ed, and poly ibosome-
associa ed. These ea u es indica e ha TATA2 RNA se es
o ansla ion o he p o ein p oduc s o BARE. Ea lie , we had
shown ha epo e gene exp ession d i en by he BARE LTR
is dependen on he p esence o TATA2 and no TATA1
[19,31], s ongly sugges ing ha all ansla ed BARE p o eins
a e de i ed om TATA2. Capping and polyadenyla ion ha e
no been well in es iga ed o LTR e o ansposons; Ty1 and
copia o Supe amily Copia a e ansla ed om capped RNA
[32,33] while Ide ix o supe amily Gypsy exploi s bo h cap-
dependen and independen mechanisms [34]. Re o i uses,
which a e likely de i ed om Gypsy e o ansposons [3],
p oduce capped ansc ip s bu appea o exploi bo h cap-
dependen and ‑independen ansla ion [35]. He e, he
BARE Re o ansposon T ansc ip ion o Replica ion
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p esence o na i e polyadenyla ed, capped TATA2 ansc ip s
in he poly ibosome “ ansla ome” is he clea es indica ion o
ac i e BARE1 ansla ion in he issues examined [36,37].
In e es ingly, he TATA2 ansc ip s o BARE2 a e also capped,
polyadenyla ed and poly ibosome-associa ed, e en i he
conse ed BARE2 dele ion abolishes he s a codon o he
ORF and he eby ansla ion o Gag [16]. Howe e , he
Figu e 5. BARE ansc ip s in VLPs. Elec opho esis o 3’
RLM-RACE PCR eac ions, pe o med on pu i ied VLP
ac ions 9-11 [18]. The ampli ied p oduc s (a ows, 220 bp and
180 bp) ep esen wo o he ansc ip g oups seen ea lie in
o al RNA, dis inc om hose in poly(A) [19]. Fo wa d nes ed
p ime s a e RLM1 and RLM2; p ime E2146, which ma ches
he liga ed linke sequence, was used as he e e se p ime .
100 bp ladde (m) is shown.
doi: 10.1371/jou nal.pone.0072270.g005
subsequen AUG s a codon a he end o gag could make he
es o BARE2 ansla able.
T ansla ion o e o ansposons and e o i uses aises a
majo challenge: balancing he s oichiome y o se e al gene
p oduc s exp essed by a single p omo e . The s uc u al Gag is
needed in g ea e abundance han he enzymes o pol;
al e a ion o he a io in e e es wi h e o i al in ec i i y [38] and
e o ansposon mobili y [39]. Mos e o i uses use -1
ameshi ing o yield wo eading ames, Gag and Gag-Pol
[35] and mos Supe amily Gypsy elemen s use +1
ameshi ing [9,40]. In con as , sequence analysis sugges s
ha he o e whelming majo i y o Copia e o ansposons such
as BARE encode Gag and Pol as one ORF [9], al hough Ty1
uses a +1 ameshi [41].
The p oblem o p oducing enough Gag is especially acu e o
BARE o se e al easons: BARE2 lacks i s own Gag [16], ye
BARE2 comp ises abou 68% o he BARE ansc ip s [16]; he
TATA2 ansc ip s, which a e he only ones ound in he
poly ibosomes, comp ise on a e age 15% o he ansc ip s.
Despi e he p esence o he la ge pool o uncapped TATA1
BARE ansc ip s, he BARE e o ansposons appea no o
exploi cap-independen ansla ion on hese ansc ip s as
does HIV [29] and many plan i uses [28]. Hence, BARE
ansc ip s able o exp ess Gag amoun o only 4.8% o he
o al. Fu he mo e, sequence analysis o BARE likewise had
shown a single ORF o Gag and Pol [17]. Howe e , he e we
show ha , o he BARE1 TATA2 p oduc s, abou hal a e
spliced so as o exp ess only Gag, e en i he spliced o m
ep esen s only 2.4% o he o al BARE ansc ip pool.
Immunoblo ing p oduces much s onge signals o Gag han
o IN om ba ley p o ein ex ac s, consis en wi h he ac ions
o a mechanism o inc ease he ela i e p opo ion o Gag [18].
These ac s oge he sugges ha splicing in BARE1 may
se e o inc ease he con en o Gag compa ed o Pol. Fo
copia, he spliced sub-genomic RNA also is p esen in abou
equal amoun s wi h he ull-leng h ansc ip [10] ye he Gag
p oduc is mo e abundan han Pol. The copia Gag RNA is
ansla ed abou en- old mo e e icien ly han he genomic
RNA [10]. The enhancemen in copia may be due o emo al o
a 2.9 kb o Pol sequence; in BARE, only 104 n is spliced ou o
c ea e a s op codon. The ole o his domain and he
ansla ional e iciency o he spliced BARE1 RNA a e cu en ly
unde in es iga ion.
Splicing o e o ansposon ansc ip s in plan s is known in a
ew o he cases. The en sub-genomic RNA in he e o i al-
like clade o supe amily Gypsy, which includes Bagy2, esul s
om splicing [42]. Two o he Gypsy elemen s, Og e and CRR,
also splice ansc ip s; Og e splices be ween ORFs [20]
whe eas CRR splices o emo e and c ea e wo ORFs. Fo
e o i uses such as MMTV, exp ession o Gag ac ually
equi es supp ession o splicing [43]. The BARE splicing
epo ed he e is he only case demons a ed in supe amily
Copia aside om ha o copia i sel and he only one whe eby
a small, al e na i e in on wi hin pa o he gag ORF esul s in
a nea ly ull-leng h sub-genomic RNA encoding jus a single
p o ein. I emains o be seen i he s a egy employed by copia
and BARE is gene al among ela ed e o ansposons.
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In con as o ansla ion, o eplica i e compe ence
e o ansposons mus a oid he packaging, e e se
ansc ip ion, and in eg a ion o spliced RNA. Se e al lines o
e idence suppo he iew ha BARE eplica es only TATA1
ansc ip s, which a e no spliced. Fi s , only TATA1 ansc ip s
con ain he R domain, which is necessa y o s and swi ching
du ing e e se ansc ip ion [19]. Second, DNA copies o he
spliced RNA, p oduced by TATA2, canno be ampli ied om
ba ley genomic DNA. Thi d, analyses o he BARE LTR
demons a ed ha TATA2 was su icien o gi e ull epo e
exp ession bu TATA1 alone ga e none [19,31]. The s onges
a gumen ha he uncapped, un-polyadenyla ed TATA1
ansc ip s se e as he sole empla es o BARE1 cDNA
syn hesis, in addi ion o he exclusi e occu ence o he R
domain in hem, is hei p esence (and he con as ing absence
o TATA2 ansc ip s) in VLPs. I is wi hin he VLPs ha
Figu e 6. Schema ic model o BARE RNA exp ession, ansla ion, and eplica ion. A. BARE e o ansposon, d awn o scale,
showing: 5’ LTR ( u quoise), including he posi ions o TATA1 (T1) and TATA2 (T2); un ansla ed leade (g ay box); gag (yellow),
encoding he capsid p o ein Gag; dele ion in BARE2 (black in e ed iangle), which abla es gag s a codon; pol (g een), encoding
aspa ic p o einase (PR), in eg ase (IN), and he e e se ansc ip ase – RNase H complex (RT-RH); he al e na i ely spliced in on
(dashed box), which gene a es a ameshi ha knocks ou pol exp ession in BARE1; 3’ un ansla ed egion (g ay box); 3’ LTR
( u quoise), including e mina ion si e o ansc ip s om TATA2 (S2) and om TATA1 (S1). B. T ansc ip s om BARE1, including
he al e na i ely spliced capped (Gppp) and polyadenyla ed (aaaa) RNA om TATA2 and he uncapped non-polyadenyla ed RNA
om TATA1, he la e which ha e he e minal epea s (R, u quoise boxes) needed o eplica ion in o cDNA. Unexp essed ORFs
a e shown as ha ched boxes labelled in g ay C. T ansc ip s om BARE2, including he TATA1 p oduc s and he capped and
polyadenyla ed TATA2 p oduc s, which canno exp ess gag. D. Mapping o he o ma ion o he ansla ion p oduc s om he
a ious RNAs, including: Gag om BARE1; he polyp o ein om BARE1, which is clea ed by PR in o unc ional uni s (GAG, yellow;
PR, iole ; RT-RH ( ed and b own). A schema ic ep esen a ion o he assembly o he componen s in o he i us-like pa icle (VLP)
is shown, in o which he TATA1 ansc ip s oge he wi h RT-RH and IN a e packaged.
doi: 10.1371/jou nal.pone.0072270.g006
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