1
Posp in o : FEBS Le e s Volume 585, Issue 19, 3 Oc obe 2011, Pages 2958–2964
A s uc u al insigh in o he C- e minal RNA ecogni ion mo i s o T-cell in acellula an igen-
1 p o ein
Edi ed by Michael Ibba
Ángeles A oca, An onio Díaz-Quin ana, I ene Díaz-Mo eno
Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa-CSIC, A da. Amé ico
Vespucio 49, Se illa 41092, Spain
Abs ac
T-cell in acellula an igen-1 (TIA-1) plays a pleio opic ole in cell homeos asis h ough he
egula ion o al e na i e p e-mRNA splicing and mRNA ansla ion by ecognising u idine- ich
sequences o RNAs. TIA-1 con ains h ee RNA ecogni ion mo i s (RRMs) and a glu amine- ich
domain. He e, we cha ac e ise i s C- e minal RRM2 and RRM3 domains. No ably, RRM3
con ains an ex a no el N- e minal α-helix (α1) which p o ec s i s single yp ophan om he
sol en exposu e, e en in he wo-domain RRM23 con ex . The α1 ha dly a ec s he he mal
s abili y o RRM3. On he con a y, RRM2 des abilises RRM3, indica ing ha bo h modules a e
umbling oge he , which may in luence he RNA binding ac i i y o TIA-1.
Highligh s
► The ex a N- e minal α-helix o TIA-1 RRM3 is o ien ed in he wo-domain RRM23 con ex .
► RRM3 is subs an ially des abilized by RRM2. ► TIA-1 RRM2 and RRM3 a e umbling
oge he , wi h implica ions in RNA binding.
Abb e ia ions
CD, ci cula dich oism; MD, molecula dynamics; NMR, nuclea magne ic esonance; PRD,
p ion- ela ed domain; RMSD, oo mean squa e de ia ions; RMSF, oo mean squa e
luc ua ions; RRM, RNA ecogni ion mo i ; SG, s ess g anules; TEV, obacco e ch i us; TIA-1,
T-cell in acellula an igen-1; XRD, X- ay di ac ion
Keywo ds
DNA–RNA binding p o ein (D/RBP); RNA me abolism; RNA ecogni ion mo i (RRM); P o-
apop o ic p o ein; T-cell- es ic ed in acellula an igen-1 (TIA-1)
1. In oduc ion
Human T-cell in acellula an igen-1 (TIA-1) is a DNA/RNA binding p o ein (D/RBP) in ol ed in
ansc ip ional and pos - ansc ip ional egula ion o euka yo ic gene exp ession. TIA-1
modula es gene ansc ip ion a es in he nucleus by binding DNA [1] and [2] and egula ing
he al e na i e p e-mRNA splicing o ca. 15% o human exons [3], [4], [5] and [6].
2
TIA-1 p o eins a he cy oplasm con ol he u no e o mRNAs and/o ep ess hei
ansla ion. Ou s anding TIA-1 a ge s encode umo nec osis ac o alpha (TNFα) and
cy och ome c (Cc), among o he s [7], [8], [9], [10] and [11]. These mRNAs equen ly bea
adenine- and u idine- ich elemen s (AREs) in hei 3′-un ansla ed egions (3′-UTRs) ha bind
ans-ac ing p o ein ac o s such as TIA-1 [10] and [12]. In esponse o en i onmen al s ess,
TIA-1 p omo es he assembly o a non-canonical p eini ia ion complex, in he cy oplasmic
s ess g anules (SG), ep essing he ansla ion o hese ARE-con aining mRNAs [13] and [14].
In addi ion, he con olled agg ega ion o TIA-1 depends on he TDP-43 p o ein [15].
The ole o TIA-1 in he con ol o cell dea h is o ou s anding in e es . In ac , TIA-1 seems o
ac as an apop osis-p omo ing ac o by con olling he al e na i e splicing o an mRNA
encoding he memb ane-bound o m o he Fas ecep o [6] and binding Cc-coding mRNA
wi hin SG, he eby p ecluding i s deg ada ion a p ocessing bodies [9]. In ac , mice lacking TIA
p o eins show highe a es o emb yonic le hali y [16] cell p oli e a ion and angiogenesis [17],
sugges ing ha TIA-1 ac s as a umou supp esso .
TIA-1 is a ubiqui ous 46-kDa mul i-domain D/RBP ha con ains h ee RNA ecogni ion mo i s
(RRMs), along wi h he p ion- ela ed domain (PRD), ich in glu amine, a he C- e minal end
(Fig. S1 a Supplemen a y Da a). Bo h PRD and he N- e minal RRM (RRM1) pa icipa e in he
ec ui men o U1snRNP [4]. Addi ionally, RRM1 associa es wi h single-s anded DNA
molecules (ssDNA) [1] bu no wi h ssRNA [18]. In i s u n, he second RRM domain (RRM2) is
necessa y o he RNA-binding ac i i y o TIA-1 by he u idine-speci ic sequence ecogni ion
mo i , whe eas he C- e minal RRM domain (RRM3) binds weakly o ssRNA [18], [19] and [20].
RRM domains adop a canonical β1α1β2β3α2β4 opology and con ain wo well-conse ed
consensus sequences, named RNP2 ([I/L/V]-[F/Y]-[I/L/V]-X-N-L) and RNP1 ([K/R]-G-[F/Y]-[G/A]-
[F/Y]-[I/L/V]-X-[F/Y]), placed a β1 and β3 s ands, espec i ely. The RNA binding si e includes
h ee a oma ic side-chains loca ed wi hin hese wo RNPs. Canonical RRM domains use he β-
shee su ace and one o wo addi ional loops o bind RNA a ge s. Howe e , se e al RRM-RNA
complexes bind RNA o p o eins in a di e en manne , owing o non-canonical seconda y
s uc u e ex ensions in he N- o C- e minal domain [21]. Recen ly, a new ype o RRMs –
named quasiRRMs (qRRMs) and iden i ied in he hnRNP-F – shows a comple ely di e en
mode o RNA ecogni ion in ol ing p o ein loops in RNA binding [22].
Gi en ha TIA-1 is implica ed in c i ical cellula e en s, explo ing he domain ea angemen o
TIA-1 p o ein along hei biophysical p ope ies would be highly aluable o unde s and he
pleio opic ole o TIA-1 in he con ol o cell homeos asis. No ably, he RRM2 s uc u e, as
well as he iden i ica ion o i s RNA a ge s, has al eady been sol ed by NMR and XRD [23] and
[24]. Wi hin his ame, his wo k sugges s ha bo h RRM2 and RRM3 domains o TIA-1, which
a e connec ed by a 22- esidue linke (Fig. S1 a Supplemen a y Da a), do no beha e
independen ly in solu ion. Fu he mo e, RRM3 shows an ex a N- e minal α-helix, which
p o ec s i s single yp ophan om he sol en exposu e, e en in he wo-domain RRM23 uni
con ex .
2. Ma e ials and me hods
3
2.1. Si e-di ec ed mu agenesis o TIA-1 RRM cons uc s
Isola ed RRM2 and RRM3 modules, along wi h RRM23 – he wo-domain in andem – we e
cons uc ed by PCR om he plasmid con aining ull-leng h TIA-1 [18], as desc ibed in
Supplemen a y Da a.
Design o he RRM2 mu an s eplacing yp ophans by phenylalanines, along wi h he RRM3-
Δα1 mu an , which lacks he N- e minal α-helix, was designed as explained in Supplemen a y
Da a.
2.2. P o ein exp ession and pu i ica ion o TIA-1 RRM cons uc s
Recombinan TIA-1 RRM domains, shown in Fig. S1 and p e iously cloned in pET11 ec o ,
we e exp essed in E. coli BL21 (DE3) cells as ollowed in Supplemen a y Da a.
Samples we e concen a ed o 0.1–0.4 mM in 20 mM po assium phospha e (pH 7.0) wi h 200
mM KCl, 150 mM MgCl2 and 1 mM DTT. P o ein concen a ion was de e mined using
spec opho ome y wi h p edic ed ex inc ion coe icien s. All molecula weigh s o he TIA-1
cons uc s used in his wo k we e e i ied by MALDI-TOF spec oscopy.
2.3. Ci cula dich oism spec oscopy
All ci cula dich oism (CD) spec a we e eco ded on a Jasco J-815 spec opola ime e ,
equipped wi h a Pel ie empe a u e-con ol sys em, using a 1-mm qua z cu e e. The
seconda y s uc u e analyses we e ca ied ou by eco ding hei a -UV CD spec a (190–250
nm) as explained in Supplemen a y Da a.
The mal un olding was moni o ed be ween 280 and 373 K ollowing a p o ocol desc ibed in
Supplemen a y Da a. Fo all hese assays, he TIA-1 species a 10 μM inal concen a ion we e
sol ed in o 20 mM sodium phospha e (pH 7.0) wi h 5 mM KCl and 5 mM MgCl2.
2.4. Fluo escence measu emen s
Emission spec a we e moni o ed using a Pe kin-Elme LS-5 luo ime e equipped wi h a
wa e - he mos a cell holde . Ten mic omola o isola ed RRM2 and RRM3 and 10 μM o
RRM23 we e sol ed in o 20 mM sodium phospha e (pH 7.0) wi h 200 mM KCl, 150 mM MgCl2
and 1 mM DTT. Then, hey we e incuba ed wi h inc easing amoun s o GdnHCl (1–6.5 M)
concen a ion o 1 h a 293 K be o e eco ding he spec a (see Supplemen a y Da a).
2.5. Modelling and molecula dynamics
Molecula dynamics (MD) compu a ions we e pe o med on he s uc u e o he RRM3 domain
o TIA-1 – along wi h he s uc u e o he RRM3-Δα1 mu an – which was modelled as
desc ibed in Supplemen a y Da a.
De ailed MD p o ocol is explained in Supplemen a y Da a.
3. Resul s
4
3.1. TIA-1 RRM3 shows an ex a N- e minal α-helix
The c ys allog aphic s uc u e o he human TIA-1 RRM2 [24] shows a quasi-canonical RRM
adop ing he well-desc ibed βαββαβ opology (Fig. S1 a Supplemen a y Da a). Howe e , i
shows a new loop connec ing wo β-s ands (β2 and β2′) ha plays a ole in binding u idine-
ich s e ches [23].
We ha e ob ained a homology model o he s uc u e o human TIA-1 RRM3 (Fig. S1).
Su p isingly, his model displays an ex a, sho α-helix a i s N- e minal end, named α1. The
sequence alignmen o RRM3 om di e en species indica es ha α1 is highly conse ed
among kingdoms (Fig. S1).
Ou homology model is in a good ag eemen wi h he seconda y s uc u e con en s o TIA-1
cons uc s. Fig. 1 shows he no malized a -UV CD spec a o RRM2, RRM3 and bo h C- e minal
RRM domains in andem (RRM23). No ably, he di e en domains only show mino di e ences
in hei global seconda y s uc u es, as summa ised in Table S1 a Supplemen a y Da a. In ac ,
RRM3 and RRM23 domains show helical composi ions signi ican ly la ge han ha o RRM2.
This inding can be a ibu ed o he end o he N- e minal amino acid sequence o RRM3 o
o m a helix. Such kind o e-a angemen may also be p esen in he wo-domain cons uc .
To co obo a e he amino-acid sequence in ol ed in he N- e minal α-helix o ma ion, he
mu an RRM3-Δα1 was designed. Indeed, he dele ion o h ee well-conse ed esidues placed
a N- e minal α1-helix (Glu193–Val194–Val195 s e ch) is su icien o dec ease he α-helix
con en o RRM3 down o a alue simila o ha ound o RRM2 (Table S1).
To es he s uc u al model u he , we subjec ed RRM3 and RRM3-Δα1 o MD calcula ions.
Resul s a e summa ised in Fig. 2. A 298 K, oo mean squa e de ia ions (RMSD, Fig. S2) om
he ene gy-minimised WT and Δα1 domain co es a e 1.6 Å and 1.3 Å, espec i ely. The la ges
a omic luc ua ions – measu ed as oo mean squa e luc ua ions (RMSF) – map a he end
e minus linke , loop egions wi hin he RRM domain, and he C- e minus (Fig. 2A). No ably, α1
helix emains unal e ed and keeps i s posi ion along he ajec o ies eckoned a 298 and 398 K
(Fig. 2B). Analysis o he ajec o ies showed ha s and β4, which lies nea α1, is he less
s able egula elemen o he domain. In ac , his s and is los du ing he simula ions
pe o med a 398 K (Fig. 2B).
3.2. TIA-1 RRM3 is des abilised by RRM2
Gi en ha TIA-1 RRM3 is ex ended by helix α1 a i s N- e minus (Fig. S1 and Table S1), we
es ed whe he his ac a ec s he he mal s abili y o he domain. CD spec oscopy indica es
ha he loss o α1 has no consequences on p o ein he mal s abili y. Ac ually, RRM3 and
RRM3-Δα1 show iden ical alues o he midpoin mel ing empe a u e (Tm) (Fig. 3A and B).
In e ac ions be ween neighbou ing RNA binding domains wi hin an RBP in luence hei
s uc u e and s abili y [25]. To es a pu a i e in e ac ion be ween RRM3 and he adjacen
RRM2 o TIA-1, we ha e pe o med he mal un olding s udies on he single modules and he
wo-domain cons uc . CD da a shows ha he Tm o isola ed RRM2 (333.5 ± 1.05 K; Fig. 3C) is
subs an ially lowe han he one o RRM3 (Fig. 3A o 3B). No ably, he un olding cu e o
RRM23 (326.8 ± 0.4 K; Fig. 3D) canno be ep oduced by lineal combina ion o he cu es o
5
he indi idual domains, as migh be in he absence o in e ac ions be ween hem. In ac , he
espec i e Tm alues o RRM2 and RRM3 a e ca. 6 and 22 K highe han ha o RRM23. This
clea ly indica es ha in e -domain in e ac ions a e aking place [25]. In iguingly, such an
in e ac ion subs an ially des abilises RRM3.
3.3. The N- e minal α-helix o TIA-1 RRM3 pa ially p o ec s i s yp ophan om exposu e o
sol en in he p esence o RRM2
Fig. 4A shows he luo escence emission spec a o TIA-1 cons uc s. Whe eas RRM2 and wo
C- e minal domains in andem RRM23 ha e he maximum luo escence in ensi y a 354 and
352 nm espec i ely, he isola ed RRM3 shows a maximum a 308 nm. This indica es ha
yp ophan esidues a e well-exposed o he aqueous sol en in he na i e s uc u e o RRM2
and RRM23, which ha e ou and i e yp ophans, espec i ely. Howe e , he single
yp ophan placed a RRM3 – T p272 – was bu ied in he p o ein co e. These esul s a e
consis en wi h he human TIA-1 RRM2 s uc u e [23] and he homology model buil o TIA-1
RRM3 he ein p oposed. Ac ually, Fig. S1 shows how T p272 ancho s he end o s and β4 o
he domain co e o med by helices α1 and α3.
Fo he RRM3-Δα1 mu an , he luo escence maximum shi s o 325 nm, indica ing ha α1 is
pa ially occluding T p272 (Fig. 4A). Al hough T p272 is pa ially bu ied in bo h, RRM3 and
RRM3-Δα1, he a oma ic esidue is sligh ly mo e sol a ed in RRM3-Δα1 mu an han in he
wild- ype species. In ac , he side-chain o his esidue shows 8.9 Å2 exposed su ace in RRM3,
whe eas i inc eases up o 11.8 Å2 in he RRM3-Δα1. Fig. 2C displays he adial dis ibu ion
unc ions o wa e molecules a ound T p272. A well-s uc u ed i s sol a ion sphe e is
obse ed o his esidue in RRM3 and RRM3-Δα1 s uc u es, con aining, on a e age, 3.2 and
4.7 wa e molecules, espec i ely.
On he o he hand, GndCl-induced ansi ion cu es o he RRM3 and he mu an RRM3-Δα1
show no di e ences in he midpoin GndCl concen a ion (Cm) o un olding ansi ions, by
eco ding he luo escence in ensi y a 350 nm (Fig. 4B and C). Ac ually, Cm alues a e
iden ical: 4.54 M ± 0.02 M and 4.52 ± 0.02 M o RRM3 and RRM3-Δα1 species, espec i ely.
To co obo a e whe he he ela i e o ien a ion be ween he addi ional α-helix o RRM3 and
T p272 emains unchanged in he wo-domain cons uc , we designed an RRM23 mu an in
which ou ou o i s i e yp ophans – T p80, T p147, T p160 and T p170 – we e eplaced by
phenylalanines, ollowing he same s a egy as be o e [26]. Consequen ly, only T p272 loca ed
a RRM3 emains a he RRM23 mu an , named RRM23-T p272. As a esul , he luo escence
emission spec um o he RRM23-T p272 almos o e laps wi h ha o he RRM3 (Fig. 4A). In
ac , bo h he 308-nm maximum and he in ensi y in luo escence signal o RRM23-T p272 a e
close o he ones obse ed o he isola ed RRM3. This indica es ha T p272 emains bu ied
in o he co e o RRM23 cons uc as pa o he TIA-1 p o ein. In e es ingly, yp ophan-by-
phenylalanine subs i u ions p esen in RRM23-T p272 mu an ha e no signi ican e ec s on i s
seconda y s uc u e wi h ega d he RRM23 (Table S1). Mo eo e , he RRM23-T p272 mu an
beha es as RRM23 in e ms o s abili y wi h a Tm alue o 325.1 ± 2.4 K (Fig. 3D and E).

6
As expec ed om a wo-domain cons uc , chemical dena u aliza ion pe o med by he
addi ion o inc easing GndCl concen a ion (1–6.5 M) esul s in a sigmoidal ansi ion cu e
wi h a Cm signi ican ly la ge han ha obse ed o he single RRM3 (5.50 ± 0.01 M),
sugges ing ha andem domains a e mo e esis an o dena u a ion by chemical agen s.
4. Discussion
Ou esul s sugges he p esence o an ex a N- e minal α-helix – named α1 – as pa o he
RRM3 domain o TIA-1. This α1 a he N-end is su p isingly well-conse ed among kingdoms, so
i migh be pe o ming a common unc ion. α1-ex ended RRM3 does no ma ch wi h he
canonical opology epo ed o classical RRMs al hough changes in he numbe and o de o
he seconda y s uc u e elemen s in RRM domains ha e been ex ensi ely epo ed. Fo
ins ance, he C- e minal RRM o La and U1A snRNP p o eins is ex ended by an α-helix ha lies
on he β-shee su ace [27] and [28]. Simila ly i happens o he Cs F-64 p o ein, which also
has ano he sho α-helix a he N- e minus, in addi ion o ha a he C- e minal end [29].
No ewo hy, his helix is adjacen o he i s β s and so i shows a di e en o ien a ion in
compa ison o ha o α1 in TIA-1 RRM3. In addi ion, he middle RRM domains o PTB p o ein
show a β-shee cons i u ed by i e β-s ands [30]. These changes in he RRM opology a e
in ol ed in RNA–p o ein and/o p o ein–p o ein in e ac ions.
O in e es is he ole ha his no el helix α1 o RRM3 plays in he con ex o he ull-leng h
TIA-1 p o ein. The mal dena u a ion s udies on RRM3 wild- ype and on he mu an lacking he
helix α1 (RRM3-Δα1) e eal ha α1 is no clue o p o ein s abili y. Mo eo e , T p272 placed a
RRM3-β4 seems o be pa ially bu ied by he p o ein co e – mainly by α3 – as well as by α1, as
shown in Fig. 5A. Indeed, when RRM3 looses α1, he luo escence measu emen s and MD
simula ions indica e ha T p272 is less p o ec ed om sol en exposu e. I is wo h o
men ion ha he ela i e o ien a ion be ween α1 and he domain co e con aining T p272
emains unchanged in he wo-domain RRM23 cons uc , as he luo escence spec um o
RRM23-T p272 almos o e laps wi h ha o RRM3.
The p esence o hyd ophobic in e ac ions be ween amino acids pai s Leu189–Leu255 and
Val194–Ala252 (Fig. 5A), which a e bu ying T p272 o exposed su ace, seems o s abilise he
in e ac ion among he seconda y s uc u e elemen s α1, α3 and β4. Ac ually, α1 is well-
compac ed on o he domain co e, as in e ed om he space- illing ep esen a ion (Fig. 5B).
Recen s uc u al s udies show ha no only he β-shee su ace bu also he loops and o he
ex a non-canonical seconda y elemen s can be c ucial o nucleic acid o p o ein ecogni ion
[23]. The human U11/U12-65 K p o ein o he spliceosome con ains an ex ension in he N-
e minal ail which suppo s he app op ia e olding o he p o ein and o ien a ion o RNA-
binding elemen s [31]. Wi hin his ame, he o ien a ion o he well-conse ed ex a helix α1
o RRM3 in he ull-leng h TIA-1 con ex and he ac ha T p272 has emained unchanged
om kingdoms du ing e olu ion could play he key o unde s and he ole o TIA-1 in he
RNA/p o ein ecogni ion e en s.
The analysis o RRM s uc u es sol ed o da e shows ha wo consecu i e RRMs sepa a ed by
a linke can in e ac wi h each o he o ming a compac uni , which can be induced by he
p esence o RNA/DNA, bu also occu s in he absence o i [32]. In his s udy, he he mal
un olding analyses e eal ha RRM3 is he mos s able RRM domain o TIA-1 p o ein. The
7
un olding s udies o isola ed C- e minal RRM domains and he andem RRM23 ha e e ealed
subs an ial di e ences in e ms o he mal s abili y o RRM3 when RRM2 is pa o he same
cons uc . In ac , RRM3 dec eases i s he mal s abili y in ca. 20 K in he RRM23 andem. These
da a help o explain ha bo h single domains RRM2 and RRM3 o human TIA-1 do no ope a e
independen ly in solu ion. The e o e, i is emp ing o specula e ha his modula in e ac ion
be ween RRM2 and RRM3 migh be ocused in he RNA binding ac i i y o TIA-1 simila ly o
wha p e iously obse ed o RRM3–RRM4 domains o PTB p o ein [32] and he wo N-
e minal RRM domains o he homologous yeas Pub1 p o ein [33], in o de o c ea e a high-
a ini y RNA-binding uni . Las ly, con ac s be ween RRM2 and RRM3 in TIA-1 may be key o
s abilise a sui able con o ma ion ha can adap o he changes in he di ec ion o he RNA
chain inside he highly s uc u ed 5′ splice si e o exon 6 o RNA encoding he Fas ecep o
and/o 3′-UTRs, as was p oposed o he cen al K-Homology domains o KH- ype splicing
egula o y p o ein (KSRP) [25]. S ill, addi ional s uc u al s udies will help us o unde s and he
p ope ies o he no el ex a helix α1 in RRM3 and he ea angemen be ween bo h C-
e minal RRM domains o TIA-1 wi hin RNA/DNA binding con ex .
Acknowledgemen s
The au ho s wish o hank he Andalusian Go e nmen (P07-CVI-02896 and BIO198) o
inancial suppo . The plasmid con aining he TIA-1 ull-leng h p o ein was kindly p o ided by
D . M. Go ospe (Na ional Ins i u es o Heal h, Bal imo e, USA) and D . P. Ande son (Ha a d
Medical School, Bos on, USA). We a e g a e ul o P o . Miguel A. De la Rosa o c i ical eading
o he manusc ip .
8
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16
Figu e 3

17
Figu e 4
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Figu e 5