Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal ...

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Multiple domains of EBER 1, an Epstein-Barr virus
noncoding RNA, recruit human ribosomal protein L22
VICTOR FOK,1,2,4 RACHEL M. MITTON-FRY,1,4 ANGIE GRECH,3 and JOAN A. STEITZ1,2
1DepartmentofMolecularBiophysicsandBiochemistryand2HowardHughesMedicalInstitute,YaleUniversitySchoolofMedicine,
NewHaven,Connecticut06536,USA
3McKinseyandCompany,Inc.,NewYork,NewYork10022,USA
ABSTRACT
EBER1,asmallnoncodingviralRNAabundantlyexpressedinallcellstransformedbyEpstein-Barrvirus(EBV),hasbeenshown
to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and
recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding
protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of
amixtureofrL22andMBP-L22indicatesthatthesethreeshiftscontainone,two,orthreeL22proteinsperEBER1molecule.
EMSAs performed withEBER 1deletionconstructs and EBER1stem–loops insertedinto a nonbinding RNA,HSUR3, identify
stem–loopsI,III,andIVasL22bindingsites.TheexistenceofmultipleL22bindingsitesonEBER1insidecellsisdemonstrated
byin vivo UVcross-linking.Our resultsare discussedwith respect tothe function ofEBER1in EBV-infectedhuman Bcells.
Keywords: Epstein-Barr virus; EBER 1; ribosomal protein L22; electrophoretic mobility-shift assay; in vivo UV cross-linking;
RNA–protein interaction
INTRODUCTION genesdeletedwasshowntoexhibit100-foldlowerefficiency
in malignant transformation of resting B cells compared
Epstein-Barr virus (EBV) is a member of the human
withitswild-typecounterpart(Yajimaetal.2005),suggest-
gherpesvirusfamilythatinfects>90%ofadultsworldwide
ing a role for EBERs in cellular transformation. This
and maintains a lifelong presence. EBV is the causative
confirmsearlierreportsthatexpressionofEBERsalonepro-
agentofinfectiousmononucleosis(Henleetal.1968)andis
motesthetumorogenicphenotypeasassessedbycellgrowth
associated with Burkitt lymphoma (BL) (Henle and Henle
insoftagarandtumorgenicityinnudemice(Komanoetal.
1966),nasopharyngealcarcinoma(zurHausenetal.1970),
1999;Rufetal.2000;Yamamotoetal.2000).However,the
and other human tumors including Hodgkin’s disease
molecular mechanisms underlying the function(s) of the
(Johansson et al. 1970, 1975) and AIDS-associated lym-
EBERs in the viral life cycle remain unclear.
phoma (Subar et al. 1988; Haluska et al. 1989). While
EBER 1 and EBER 2 (167 and 172 nt, respectively) have
most of the EBV genome is silent in EBV-transformed
considerablesecondarystructureconsistingofseveralstable
B-lymphocytes, two genes encoding the small RNAs EBER
stem–loops (Glickman et al. 1988). They contain 39 termi-
1 and EBER 2 (Epstein-Barr encoded RNAs) are actively
nal polyuridylate stretches that interact stably with the
transcribed by RNA polymerase III (z106 copies per cell)
La antigen (Lerner et al. 1981). In addition to La, EBER 1
(Lerner et al. 1981; van Santen et al. 1981; Arrand et al.
(Fig. 1A) binds the human ribosomal protein L22 tightly
1989). Recently, a recombinant EBV with the two EBER
(Toczyski and Steitz 1991) and the interferon-inducible
protein kinase R (PKR) more weakly (Clarke et al. 1990,
1991; Clemens et al. 1994). L22 is a 14.8-kDa protein con-
4Theseauthorscontributedequallytothiswork.
stituent of the large ribosomal subunit, which is not
Reprint requests to: Joan A. Steitz, Department of Molecular Bio-
physics and Biochemistry and Howard Hughes Medical Institute, Yale conserved in prokaryotes (Nakao et al. 2004) and does
University School of Medicine, New Haven, CT 06536, USA; e-mail: not share RNA binding motifs with any other known
[email protected];fax:[phone].
protein(http://us.expasy.org/prosite).Inaddition,L22asso-
Articlepublishedonlineaheadofprint.Articleandpublicationdateareat
http://www.rnajournal.org/cgi/doi/10.1261/rna.2339606. ciates with human telomerase (Le et al. 2000), the small
872 RNA(2006),12:872–882.PublishedbyColdSpringHarborLaboratoryPress.Copyright"2006RNASociety.
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L22 bindsmultipledomains ofEBER1
50% of the L22 protein associates with
this RNA (Toczyski et al. 1994). Pre-
vious studies concluded that L22 binds
EBER1viastem–loopIII(Toczyskiand
Steitz1993;Fig.1A),butthatadditional
binding site(s) may also exist (Toczyski
and Steitz 1993; Dobbelstein and Shenk
1995). SELEX (systematic evolution of
ligands by exponential enrichment)
experiments established a generalized
RNA motif (Fig. 1D) for L22 binding
(Dobbelstein and Shenk 1995). Both
stem–loops III and IV of EBER 1 fit
this motif, thereby implicating stem–
loopIVasanadditionalbindingsitefor
L22 on EBER 1.
To investigate the molecular interac-
tions involved in RNP formation by
EBER 1 and L22, we carried out more
extensive binding studies using un-
tagged and MBP-tagged L22 protein.
EMSAs (electrophoretic mobility-shift
assays) performed with wild-type EBER
1 and these recombinant proteins re-
vealed three specific mobility shifts that
FIGURE1. L22interactionswithEBER1andHSUR3inEMSAs.(A)Secondarystructureof
aredependentonproteinconcentration
wild-type EBER 1 as determined previously (Glickman et al. 1988). Bold roman numerals
and differ in the ratios of protein/RNA
denotethestem–loops.(B)SecondarystructureofHSUR3fromHerpesvirussaimiri(Leeetal.
1988). The arrow indicates where stem–loops I,II, III, or IVwere inserted into the RNA to in the shifted complexes. Examination
generatetheHSUR3chimerasanalyzedinFigure5.(C)EMSAsperformedwith5nMbody- of EBER 1 deletion constructs and
labeled HSUR 3 (lanes 1–6) or EBER 1 (lanes 7–11) and increasing concentrations of
chimericRNAmoleculescontainingiso-
recombinantL22protein.EBER1,whichalonemigratesasaclosedoublet,formsthreedistinct
shiftswithL22,whileHSUR3shiftsonlyatproteinconcentrations$3mM.Anasterisk(*) latedEBER1stem–loopsinEMSAsiden-
indicates an additional, nonspecific shift that appears at very high L22 concentrations. Note tified three L22 binding sites. These
thatthefreeHSUR3containsaslower-migratingbandevenwithoutL22.(D)Thegeneralized include the above-mentioned stem–loops
RNAconsensusmotifproposedtomediateinteractionbetweenL22andRNA(adaptedfrom
IIIandIV,andapreviouslyundescribed
DobbelsteinandShenk1995).
site involving stem–loop I. In addition,
we demonstrate the existence of multi-
viral RNA HVP-1 in Herpesvirus papio–infected baboon pleL22bindingsitesonEBER1insidecellsbyaUVcross-
cells (Toczyski and Steitz 1991), the 39X region within the linkingassay.UnderstandinghowL22associateswithEBER
39-untranslated region (UTR) of the human Hepatitis C 1 may help elucidate the contribution of the EBER RNPs
virus(HCV)genomicRNA(Woodetal.2001),poly(ADP- to the EBV life cycle.
ribose)polymeraseinDrosophilamelanogaster(Koyamaetal.
1999),andtheHerpessimplex–infectedcellproteins(ICP)4
RESULTS
and ICP 22 in human cells (Leopardi and Roizman 1996;
Leopardietal.1997).However,thefunctionofL22bothin
Stem–loops I, III, and IV are all binding sites for rL22
the ribosome and in these several RNP complexes remains
unknown. EMSAwas usedtostudytheinvitro interaction ofhuman
In uninfected human B cells, L22 is located in the ribosomal protein L22 with EBER 1 in comparison to an
nucleoli and cytoplasm, whereas following EBV infection, unrelated RNA derived from Herpesvirus saimiri, HSUR 3
L22 is found in the nucleoplasm as well (Toczyski et al. (Lee et al. 1988). Increasing concentrations of untagged
1994).InsituhybridizationindicatesthatEBERsarelargely recombinant L22 (rL22) were incubated with either 5 nM
nucleoplasmic(HoweandSteitz1986;Barlettaetal.1993), 32P-labeled full-length EBER 1 (Fig. 1A) or HSUR 3 (Fig.
suggesting that L22 relocalization results from its associa- 1B). Three distinct protein-dependent mobility shifts were
tion with EBER 1 in vivo (Toczyski et al. 1994). In trans- observed for EBER 1 (Fig. 1C, lanes 8–11), suggesting that
formed B cells, EBER 1 is quantitatively bound by L22 the dominant species present at 1 mM rL22 is the triply
(>95%) (Toczyski and Steitz 1993), whereas only 30%– boundRNA. Anadditional diffuseshift canalsobeseenat
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Fok etal.
3 mM rL22. In contrast, HSUR 3 begins to shift at protein previously indicated that stem–loop III of EBER 1 binds
concentrationsonlyatorabovethislevel(Fig.1C,lanes2– L22 (Toczyski and Steitz 1993), and SELEX and EMSAs
6). Collectively, these data argue that EBER 1 shifts 1, 2, (Dobbelstein and Shenk 1995) suggested stem–loop IV as
and3(Fig.1C,lanes8–11)representspecificrL22–EBER1 an additional L22 protein binding site. To verify these and
complexes, whereas the additional diffuse shift most likely identify other RNA elements within EBER 1 important for
arises from nonspecific electrostatic association with the binding L22, we performed a series of mobility-shift ex-
RNA at high protein concentrations. periments using EBER 1 deletion constructs in which each
The three distinct mobility shifts (Fig. 1C) suggest that of the stem–loops was deleted independently (Fig. 2B–E).
rL22 forms at least three distinct complexes withwild-type Interestingly, single deletion of three of the stem–loops
EBER1.Oneinterpretationisthatwild-typeEBER1possesses of EBER 1 pictured in Figure 1A abolished or greatly
three unique L22 binding sites. However, it cannot be hindered the formation of the three specific shifts. These
assumed that each shift represents a unique molecular were stem–loop I (EBER 1 Dstlp I, Dnts 14–33; Fig. 2B),
complex;asingleshiftcouldcontainheterogeneousmixtures stem–loopIII(EBER1DstlpIII,Dnts60–83;Fig.2D),and
ofdifferentcomplexesthattravelwiththesamemobilityin stem–loop IV (EBER 1 Dstlp IV, Dnts 87–123; Fig. 2E). In
native polyacrylamide gels. Immunoprecipitation studies contrast, deletion of stem–loop II from EBER 1 (EBER 1
FIGURE2. EMSAsperformedwithEBER1deletionconstructsidentifystem–loopsI,III,andIVasbindingsitesforL22onEBER1.Predicted
secondary structures of the EBER 1 deletion RNAs tested (top) and EMSAs performed with 5 nM RNA (body-labeled) and increasing
concentrationsofL22(bottom).(A)Full-lengthEBER1,(B)EBER1DstlpI(Dnts14–33),(C)EBER1DstlpII(Dnts45–58),(D)EBER1DstlpIII
(Dnts60–83),(E)EBER1DstlpIV(Dnts87–123).Thedeletionmutantsarenumberedaccordingtothefull-lengthRNA.Anasterisk(*)indicates
anadditional,nonspecificshiftthatonlyappearsatL22concentrations$3mM.
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L22 bindsmultipledomains ofEBER1
DstlpII,Dnts45–58;Fig.2C)producedanRNAwithrL22- presence of either rL22 or MBP-L22 alone, three specific
bindingbehaviorqualitativelyidenticaltothatofwild-type concentration-dependentbandsappeared(Fig.3,lanes2–5
EBER1(Fig.2A).Thesedifferencesarebestexemplifiedby or9–11,respectively).WhenEBER1DstlpIIwasincubated
examinationofEBER1shiftsinthepresenceof1mMrL22. withamixtureofthetwoproteins(Fig.3,lanes6–8),these
Themajorityofthesignalfromfull-length(Fig.2A,lane4) individual shifts were observed, as well as three distinct
and Dstlp II RNAs (Fig. 2C, lane 5) is found in shift 3. In intermediate shifts. The formation of a band intermediate
contrast, the bulk of the signal from EBER 1 Dstlp I (Fig. to a shift seen with each protein alone would suggest that
2B,lane4)orEBER1DstlpIII(Fig.2D,lane5)isfoundin two (or more) molecules of protein bind.
shift1,whilethatfromEBER1DstlpIV(Fig.2E,lane5)is The simplest interpretation of the above mixing exper-
found in shift 2. iment(Fig.3)isthatproteinshifts1,2,and3containone,
These results corroborate the previous findings that two, and three of the relevant L22 molecules bound to
stem–loops III and IV are essential for full rL22 binding EBER 1, respectively, while the intermediate shifts contain
toEBER1andsuggestanadditional,previouslyundescribed both rL22 and MBP-L22 molecules simultaneously bound
protein binding site in stem–loop I. It was surprising, to EBER 1. Mixed shift 1 represents EBER 1 bound to two
however, that deletion of stem–loop III did not appear to L22 moieties (one molecule of rL22 and one molecule of
retard the formation of the first protein shift. In fact, the MBP-L22), while mixed shifts 2 and 3 represent EBER 1
first protein shift was observed even at the lowest concen- bound to three L22 moieties (two rL22 and one MBP-L22
tration(0.1mM)shownforallsingle-loopdeletionconstructs, moleculesinmixedshift2,andonerL22andtwoMBP-L22
suggesting malleability in the rL22–EBER 1 interaction. molecules in mixed shift 3). Although both mixed shifts
2and3containthreeproteinmolecules,thelattermigrates
significantly more slowly, consistent with its proposed
EBER 1 binds three protein molecules simultaneously
greater bulk.
The protein-concentration dependence of the appearance
of the mobility shifts, in concert with the presence of
EBER 1 fragments bind rL22
binding sites on stem–loops I, III, and IV, suggests that
EBER 1 can bind multiple copies of rL22 simultaneously. To probe the rL22-EBER 1 interactions further, we per-
Todeterminetheprotein/RNAstoichiometryforshifts1–3, formed mobility-shift assays with several EBER 1 frag-
binding studies were performed using a mixture of rL22 ments. EBER 1 nts 45–124, which comprises stem–loops
(15 kDa) and MBP-L22 fusion protein (56 kDa) (Fig. 3). II–IV of EBER 1, formed two shifts with rL22. These shifts
EBER 1 Dstlp II was substituted for full-length EBER 1, as appeared at similar rL22 concentrations to the first two
it exhibited very similar binding behavior with both rL22 shifts of full-length EBER 1. Shortening stem–loop IV
(Fig. 2A,C) and MBP-L22 (data not shown). However, the reducedtheassociationof theRNA fragmentwiththefirst
increasedresolutionwiththissmallerRNAbetweenbound protein molecule and abrogated the second shift (data not
speciesgreatlyfacilitatedinterpretationoftheresults.Inthe shown),supportingtheconclusionthatstem–loopIVisan
rL22-binding site. Identical results were seen upon sub-
stituting theloopofstem–loop II, whichwasshown above
not to be essential for protein binding, with a GAAA
tetraloop(datanotshown).Ourrationalewasthatasmaller
construct comprised of just stem–loops II and III would
usethisGAAAtetraloopinstem–loopIItopromoteproper
RNA folding. Again, this construct showed only one shift
with rL22 (Fig. 4B), but with slightly reduced affinity for
rL22. This suggests that elements in the base of stem–loop
IV might be necessary either for direct binding to rL22 or
for correct presentation of stem–loop III to the rL22-
binding surface. Similar results were seen upon removal
of stem–loop II entirely (Fig. 4C).
FIGURE 3. Shifts 2 and 3 contain multiple L22 molecules. EMSAs AnotherseriesofEBER1fragmentsremovedstem–loops
performedwith5nMbody-labeledEBER1DstlpIIandtheindicated I, II, and IV from the backbone of the RNA molecule.
concentrationsofrL22and/orMBP-L22fusionprotein.EBER1Dstlp
Unexpectedly, EBER I Dstlps I, II, and IV formed two
II was chosen for the mixing experiment, as it exhibits L22-binding
behaviorsimilartofull-lengthEBER1butbetterseparationbetween RNA–protein complexes in mobility-shift assays (Fig. 4D).
protein-bound species. Mixed shift 1 is presumed to comprise one This behavior may be explained by the presence of two
rL22andoneMBP-L22moleculeboundtotheRNA,withtworL22
energetically comparable RNA conformers, one of which
andoneMBP-L22moleculesinmixedshift2,andonerL22andtwo
containsanovelbindingsiteforL22(seeDiscussion).Short-
MBP-L22moleculesinmixedshift3.Anasterisk(*)indicatesaminor
multiplyshiftedspecies. ening the terminal RNA stem abolished the second shift
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FIGURE 4. EMSAs investigating the interaction of L22 with EBER 1 fragments. Predicted secondary structures of EBER 1 truncations and
EMSAsperformedwith5nMRNAandincreasingconcentrationsofL22areshown.(A)EBER1nts45–124,(B)EBER1nts45–84,GAAA,(C)
EBER1nts59–87,(D)EBER1DstlpsI,II,andIV,and(E)EBER135–135DstlpsIIandIV.Thedeletionmutantsarenumberedaccordingtothe
full-lengthRNA.EMSAswereperformedwithbody-labeledRNAs,withtheexceptionoftheconstructsinAandE,whichwere59-endlabeled.An
asterisk(*)indicatesanadditional,nonspecificshiftthatonlyappearsatveryhighL22concentrations.AsstlpIIwasshownnottobeinvolvedin
L22binding,itsloopwasreplacedwithaGAAAtetralooptopromotecorrectfoldingoftheconstructstudiedinB.
without affecting the binding affinity of the first shift (Fig. HSUR3eachproducedasingle-shiftedcomplexwithrL22.
4E; data not shown). The same amounts (1 and 4 mM) of rL22 did not yield
aspecificshiftwithHSUR3alone(Fig.5,lanes3,4)orwith
Stem–loops I, II, and IV can individually bind rL22
the HSUR 3 stem–loop II chimera (data not shown). We
AlthoughthedatainFigures2and4arguethatstem–loops concludethatstem–loopsIandIVcanindependentlyserve
I,III,andIVarenecessaryforL22bindingtoEBER1,they as binding sites for rL22. Interestingly, the HSUR 3 stem–
donotaddresswhethertheseregionsaresufficient.Because loop III (nts 59–84) chimera consistently yielded two
stem–loop III alone binds poorly to rL22 even at 10 mM differently migrating forms of the free RNA (Fig. 5, lane 8)
concentration (data not shown) and decreases in binding inmultipleRNApreparationspurifiedfrompolyacrylamide
affinitywereobservedwhenstem–loopIVwasdeleted(Fig. gels, arguing that it adopts two major conformations (see
4,cf.AandB/C),wereasonedthatthestem–loopsofEBER Discussion). Increasingamounts ofrL22shiftedbothRNA
1 might require the context of a larger RNA molecule to conformersintospecificcomplexes(Fig.5,lanes9–11)but
fold properly. We therefore performed binding assays on with more background than the profiles of the HSUR 3
the individual EBER 1 stem–loops inserted (see Fig. 1B for stem–loop I or IV chimeras. The diminution of the free
siteofinsertion)intothenonbindingHSUR3moleculeto HSUR 3 stem–loop III band even at 0.4 mM rL22 (Fig. 5,
form HSUR 3 stem–loop chimeras. lanes 9–11) argues that stem–loop III is an L22-binding
TheinsertionofEBER1stem–loopI(nts14–33)(Fig.5, site.Takentogether,thesedatasupporttheideathatEBER
lanes 6,7) or IV (nts 87–123) (Fig. 5, lanes 14,15) into 1 stem–loops I, III, and IV can individually bind L22.
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L22 bindsmultipledomains ofEBER1
DISCUSSION
In vitro binding experiments presented here demonstrate
that wild-type EBER 1 forms three distinct mobility shifts
with rL22 or MPB-L22 that are protein-concentration
dependent (Fig. 1; data not shown). The association is
direct since it occurs in the absence of any other viral or
host proteins and does not require post-translational
modifications. The three distinct RNP complexes observed
differinthetotalnumberofboundL22proteinmolecules;
shifts1,2,and3appeartocontainone,two,andthreeL22
molecules bound to EBER 1, respectively (Fig. 3). Because
thedeletionofEBER1stem–loopI,III,orIVgreatlyaffects
theformationofathirdcomplex(Fig.2),thesethreestem–
FIGURE 5. L22 interacts with HSUR 3 chimeras containing in- loopsappeartocomprisethethreebindingsitesforL22in
dividual EBER 1 stem–loop sequences. EMSAs were performed with
vitro. Indeed, our data agree with previous observations
5 nM HSUR 3 without an insert (lanes 1–3) and HSUR 3 chimeras
containingeitherstem–loopI(lanes4–7),stem–loopIII(lanes8–11), that stem–loops III and IV are L22 binding sites (Toczyski
or stem–loop IV (lanes 12–15) in the presence of the indicated andSteitz1993;DobbelsteinandShenk1995).Insertionof
concentrations of rL22 protein. Note that HSUR 3 stem–loop III
stem–loop I into HSUR 3 confers binding activity on this
containstwoformsofthefreeRNA;theirrespectiveshiftsaremarked
unrelatednonbindingRNA,confirmingthatstem–loopIis
withsingle(*)anddouble(**)asterisks.The**shiftsometimes,but
notalways,appearsasadoublet. abindingsiteforL22(Fig.5).Finally,invivocross-linking
experiments show that there are also multiple L22-binding
sites on EBER 1 inside cells (Fig. 6).
EBER 1 binds multiple L22 Proteins in vivo
HavingestablishedthatEBER1containsthreeL22-binding
Can stem–loops be assigned to particular shifts?
sitesinvitro,wewishedtodeterminewhethermultipleL22
proteins likewise interact with EBER 1 inside transformed With the wild-type EBER 1 molecule, we estimate the
cells where other EBER 1-binding proteins could poten- apparent binding constants (K ) for the first, second, and
d
tiallycompete.WeusedapowerfulinvivoUVcross-linking third shifts to be 0.1, 0.3, and 1 mM, respectively. In an
method (Cook et al. 2004) in which anti-L22 antibody was attempttoassigntheseshiftstoparticularbindingsites,we
used to immunoprecipitate cross-linked EBER 1–L22 com- first examined fragments of EBER 1. We constructed two
plexes under semidenaturing conditions. When a DNA small stem–loop III-containing fragments, EBER 1 nts 45–
construct expressing wild-type EBER 1 and EBER 2 from 84, GAAA (Fig. 4B) and EBER 1 nts 59–87 (Fig. 4C), and
their native promoters was transfected
intoHEK293cells,EBER1wasdetected
in the anti-L22 precipitate in a cross-
linking-dependent manner (Fig. 6, top,
cf. lanes 8,18 and 7,17).
IfEBER1bindsmultiplecopiesofthe
L22 protein in vivo, deletion of any one
binding site from the RNA should not
abolishprecipitationafterUVcross-link-
ing. Indeed, upon expression in HEK
293 cells and UV cross-linking, EBER 1
Dstlp I (Fig. 6, top, lane 10), EBER 1
FIGURE6. InvivointeractionsbetweenL22andEBER1deletionconstructs.HEK293cells
Dstlp III (Fig. 6, top, lane 12), and
weretransfectedwiththefollowingEBER1andEBER2expressionconstructs:wild-typeEBER
EBER 1 Dstlp IV (Fig. 6, top, lane 20) 1 (lanes 1,2,7,8,13,14,17,18), Dstlp I (lanes 3,4,9,10), Dstlp III (lanes 5,6,11,12), or Dstlp IV
weredetectedintheanti-L22precipitate. (lanes15,16,19,20).Intactcellswereirradiatedwith254-nmUVlighttoinducecross-linking
The specificity of these interactions is between protein and RNA in vivo (even-numbered lanes); untreated cells served as negative
controls (odd-numbered lanes). Whole-cell lysates were subjected to immunoprecipitation
demonstrated by the absence of EBER 2
with anti-L22 antibody under denaturing conditions. Precipitated RNAs were released from
in the precipitates (Fig. 6, bottom). We theantibodybyproteinaseKdigestionandwereresolvedinan8%ureapolyacrylamidegel.A
concludethatmultipleL22moleculesare totalof0.5%oftheinputand100%oftheprecipitateswereanalyzed,andEBER1sequences
weredetectedbyNorthernblotting(top).ThelowexpressionlevelsofEBER1DstlpI(lanes
boundtoEBER1insidecells,asinvitro,
3,4)andDstlpIII(lanes5,6)areduetothedisruptionofBoxAorBoxBoftheEBER1RNA
but we cannot distinguish whether two
polymeraseIIIpromoter,respectively.Tocontrolfornonspecificcross-linkingandimmuno-
or more L22-binding sites exist. precipitation,themembraneswerestrippedandreprobedforEBER2sequences(bottom).
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estimatedthatstem–loopIIIhasaK of1mM.However,in tionofanystem–loopfromtherestofthemolecule(Fig.5)
d
amoleculethatcontainsstem–loopsII,III,andIV(EBER1 negatively affects the interaction between L22 and EBER 1
nts 45–124;Fig. 4A) the first and second shifts haveK sof stem–loops. Therefore, we conclude from our results only
d
0.1 and 1 mM, respectively. These affinities suggest that that wild-type EBER 1 has three in vitro L22-binding sites,
shift 1 with EBER 1 nts 45–124 corresponds primarily to whoserelativeaffinities remain unknown. It is quite possi-
L22 binding at stem–loop IV, with an additional L22 ble that each shift represents a mixture with L22 bound to
molecule at stem–loop III to form shift 2. Alternatively, several of its binding sites.
additionalsequencesfromstem–loopIVmightallowstem–
loop III to bind L22 with a higher affinity, perhaps by What molecular features does L22 recognize?
facilitating proper RNA folding. In this case, shift 1 on
SELEX experiments predicted a consensus RNA structure
EBER 1 nts 45–124 would correspond primarily to L22
that enables specific interactions between L22 and RNA
binding at stem–loop III.
(Dobbelstein and Shenk 1995; Fig. 1D). The domains of
We used M-fold (Zucker 2003) to predict RNA second-
EBER 1 that fit this motif best are stem–loops III and IV.
ary structures for wild-type EBER 1 and all of the deletion
Sincestem–loopIIIwasalreadyestablishedasabindingsite
constructRNAsusedinFigures2and4.M-foldconfirmed
(Toczyski and Steitz 1993), Dobbelstein and Shenk (1995)
thestructuresshowninallcasesexceptforEBER1DstlpsI,
proposed stem–loop IV as the additional binding site on
II, and IV (Fig. 4D). Here, an alternate structure of
EBER 1. EMSAs performed with isolated stem–loop IV in
comparable predicted stability can form by refolding nts
theabsenceofcompetitorRNAindeeddemonstratedweak
127–138tocreateahairpinloopwitha4-bpstemclosedby
bindingthatcouldbecompetedbytheadditionofisolated
GC and a U in the 39 position of the loop, conforming to
stem–loop III (Dobbelstein and Shenk 1995).
theconsensusmotifforL22binding(Fig.1D).L22binding
Herewehaveidentifiedstem–loopIofEBER1asathird
both to stem–loop III and to this novel stem–loop in the
binding site for L22. This assignment is supported by
molecule would then explain the two shifts seen in the
a comparison of the EBER 1 stem–loop I to a sequence
EMSA profile of this molecule (Fig. 4D). Moreover, this
from the 39X sense strand of HCV genomic RNA that is
structure would not form with EBER 1 35–135 Dstlps II
reported to bind L22 (Wood et al. 2001); nt 19–25 within
and IV (Fig. 4E), explaining loss of the second shift.
the 39X sense strand are identical to nt 13–19 in EBER 1
Because individual stem–loops of EBER 1 may require
stem–loopI(Woodetal.2001).Yet,stem–loopIofEBER1
the context of a larger RNA molecule to fold properly, we
doesnotfitthepredictedconsensussequence(Dobbelstein
also inserted them into a nonspecific RNA, HSUR 3.
andShenk1995)entirely(seeFig.1A,D);itmaintainsaGC
Curiously,theHSUR3stlpIIIchimeraassumedtwomajor
base pairat theapicalend,withtheG59andtheC39,but
conformations (Fig. 5, lane 8), again predicted by M-fold
lacks the U residue in the 39 position of the loop. A
(data not shown). While the two folds are dramatically
commonfeatureisthatthestemsofbothstem–loopsIand
different, they both retain stem–loop III. Thus, both con-
III consist of 6 canonical bp, with no bulged residues. The
formers are shifted (Fig. 5, lanes 9–11) and the estimated
identification of more recognition sites for L22, including
K s, based on the reduction of the free RNA signals, are
d that in the large subunit of the ribosome, would provide
<1 mM. The estimated K for the HSUR 3 stlp IV chimera
d additional insights into the consensus-binding sequence
(whose predicted structure is consistent with M-fold) is
and structure.
2mM(Fig.5).However,forHSUR3stlpI,M-foldpredicts
fourisoenergeticstructures,threeofwhichlackstem–loopI.
Significance of the association between EBER 1
Its apparent Kd of >4 mM (Fig. 5, lanes 4–7) is therefore
and ribosomal protein L22
not meaningful. Thus, we can only conclude that, when
inserted into HSUR 3, stem–loop III exhibits stronger L22 ThehighabundanceofEBER1andEBER2inEBV-infected
bindingthanstem–loopIV.Itisstrikingthattheestimated cells,togetherwiththeirevolutionaryconservation(Arrand
K sfortheindividualstem–loopchimeras(Fig.5)areallat and Rymo 1982; Howe and Shu 1988), strongly suggests
d
least fourfold lower than when the same stem–loops are that these RNAs contribute importantly to the EBV life
part of EBER 1 (Figs. 1C, 2A). cycle.TheassociationbetweenEBER1andribosomalprotein
Given the results with both the EBER 1 fragments and L22 immediately suggests that the EBERs might modulate
with the HSUR 3-EBER 1 chimeras, can we assign the translation. Based on the estimate that the intracellular
various shifts to L22 interactions with specific stem–loops? concentration ofL22(z50mM)is10-foldthat ofEBER1,
Figure2showsthattheindividualdeletionofstem–loopsI, nearly all of EBER 1 is predicted to be bound by multiple
III, or IV surprisingly has no effect on the appearance of molecules of L22 in vivo. This is confirmed by the in vivo
shift1.Thus,itisimpossibletodeducewhichstem–loopis cross-linkingdatainFigure6.SincedepletionofL22fromthe
thestrongestbindingsiteforL22inthecontextofthefull- ribosomesofEBV-transformedcellswasnotexperimentally
length RNA. Together our results indicate that any major observed (Toczyski et al. 1994), L22 levels may be selec-
(Fig. 4) or minor (Fig. 2) deletion of EBER 1 or the isola- tively up-regulated in EBV-infected cells.
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L22 bindsmultipledomains ofEBER1
ItisalsopossiblethatthesequesteringofL22byEBER1 above.EBER135–135DstlpII,IVwasgeneratedbyPCRampli-
is not important, but rather that the RNP consisting of fication using EBER 1 Dstlps (I, II, and IV) plasmid as the DNA
EBER1,L22,andadditionalhostproteinsfulfillssomespe- template and the following primers: 59 primer-GGGAATTCTAA
cificfunctioninEBV-transformedcells.EBER1hasbeenshown TACGACTCACTATAGGAGACGUGUGUGGCUGU, whichcon-
tains anEcoRI restriction sitefollowed byaT7RNApolymerase
tointeractstablywiththeLaantigen(Lerneretal.1981)and
promoter,and39primer-CGCCGGATCCTTTAAAGACGGCAGA
PKR (Clarke et al. 1991; Sharp et al. 1993; Clemens et al.
CACCACC,whichcontainsDraIandBamHIrestrictionsites.The
1994;Eliaetal.1996;ClemensandElia1997;Vuyisichetal.
resulting product was cloned into a pUC19 plasmid using the
2002).Laissufficientlyabundant(z23107molecules/cell)
EcoRIandBamHIrestrictionsites.EBER145–124wasgenerated
that binding by EBERs (z106 molecules/cell) should not
by PCR amplification using wild-type EBER 1 plasmid and the
sequester La away from other roles in the cell (Wolin and followingprimers:59primer-GGGAATTCTAATACGACTCACTA
Cedervall 2002). On the other hand, PKR is much less TAGUGGCUGUAGCCACCC, which contains an EcoRI restric-
abundant(ClemensandElia1997)anditsbindingbyEBER tion site, followed by a T7 RNA polymerase promoter, and 39
1 could block its ability to inhibit protein synthesis in primer-CGCCAAGCTTTAAAGCAGAGUCUGGGAAGAC,which
infectedcells (Clarke et al. 1991; Sharp etal. 1993; Clemens containsDraIandHindIIIrestrictionsites.Theresultantproduct
et al. 1994).Recently,this model has beenextended to sug- was cloned into a pUC19 plasmid using the EcoRI and HindIII
restriction sites. For EBER 1 nts 45–84, GAAA and EBER 1 nts
gest that L22 may exert a protective effect against the
59–87,DNAtemplatescontainingaT7RNApolymerasepromoter
transforming potential of EBER 1 in vivo by making EBER
followed by the relevant sequence were synthesized by the Keck
1 unavailable for binding to PKR (Elia et al. 2004). This
Facility at Yale University and purified on a 15% denaturing
hypothesis rests on the idea that L22 and PKR compete for
polyacrylamidegel.Stem–loopswereaddedtoHSUR3byligating
a common binding site on EBER 1, namely stem–loop IV
twoDNAinserts(59and39fragments)intotheHSUR3construct
(Dobbelstein and Shenk 1995; Vuyisich et al. 2002), while usingtheEcoRIandSmaIrestrictionsites.TheHerpesvirussaimiri
otherstudies,incontrast(Rufetal.2005;Wangetal.2005), HSUR 3 plasmid was previously constructed (Myer et al. 1992)
do not find evidence for interactions between EBER 1 and andwasusedasPCRtemplate.The59fragmentsofHSUR3stlpI,
PKR. Clearly, there is much more to be learned concerning HSUR 3 stlp III, and HSUR 3 stlp IV were generated by PCR
the role of the EBER 1 RNP in EBV-transformed cells. amplificationwith59-CCTCTAGGGCTTGGGTTGTTAATCTCCTA,
59-TGTACCCGGGACGGCTTGGGTTGTTAATCTCCTA, and
59-CAACCACAGACACCGTCCTCCTTGGGTTGTTGTTAATCTCCTA,
respectively, and T7 primer. The 39 fragments of HSUR 3 stlp I,
MATERIALS AND METHODS HSUR 3 stlp III, and HSUR 3 stlp IV were generated by PCR
amplification with 59-TTTTGCTAGGGAATAATTTTTGAAGGC
Plasmid construction TCTGG,59-AGTCCCGGGTGGAATAATTTTTGAAGGCTCTGG,
and 59-TCTTCCCAGACTCTGCTTTAATAATTTTTGAAGGCTC
Wild-type EBER 1 was previously cloned into a pUC119 vector TGG, respectively, andSP6 primer.
(Toczyski and Steitz 1993). This gene fragment (225 nt) was For the in vivo UV cross-linking experiments, EBER 1 and its
removed from the pUC119 vector and placed into a pUC19 deletionvariantswereexpressedusingtheirendogenouspromoters
vector. Briefly, the EBER 1 gene (167 nt), preceded by an EcoRI from a plasmid encoding both EBER 1 and EBER 2. p73EBERs
restriction site and a T7 RNA polymerase promoter (23 nt) and was constructed by ligating a 1.1-kb DNA fragment from the
followed by a HindIII restriction enzyme site, was excised from EcoRIJfragmentofEBVintopSP73(Promega)usingtheSacIand
the pUC119 vector by cleavage with the enzymes EcoRI and BamHIrestrictionsites.Stem–loopsweredeletedfromEBER1by
HindIIIandinsertedintotheEcoRIandHindIIIsitesofpUC19. ligating two DNA inserts (59 and 39 fragments) into p73EBERs
All EBER 1 stem–loop deletion plasmids were synthesized using usingtheEcoRIandBamHIrestrictionsites.The59fragmentsof
the QuikChange Site-Directed mutagenesis kit (Stratagene) Dstlp I, Dstlp III, and Dstlp IV were generated by PCR amplifi-
according to the manufacturer’s protocol. Wild-type EBER 1 cation with 59-AGGAGACGTGTGTGGCTG, 59-TGAGGACG
plasmid was used as the DNA template in all cases, unless GTGTCTGTGG,and59-TCTGCCGTCTTCGGTCAA,respectively,
otherwise specified. The following 59 oligonucleotides were used and an SP6 primer. The 39 fragments of Dstlp I, Dstlp III, and
tocreatethecorrespondingdeletionconstructs(with39oligonu- Dstlp IV were generated by PCR amplification with 59-GCG
cleotides complementary to their respective 59 oligonucleotides): TAGGTCCTGAGGTTCA, 59-GTGGCTACAGCCACACAC, and
EBER 1 Dstlp I, 59-AGGACCTACGCTGAGGAGACGTGTGTGG 59-CACCACCCGGGACTTGTACC,respectively,andaT7primer.
CTG;EBER1DstlpII,59-GCTAGGGAGGAGACGTGTCCGTCC DNAsequences ofallplasmidswereverifiedbytheKeckFacility
CGGGTACAAGT;EBER1DstlpIII,59-CGTGTGTGGCTGTAGC at YaleUniversity.
CACCGTGAGGACGGTGTCTGTG.EBER1DstlpIVwasconstructed
in two rounds. The first round removed nt 106–123 using
59-GGACGGTGTCTGTGGTTGTCTGCCGTCTTCGGTC.Thesec- Protein expression and purification
ond round used this EBER 1 plasmid as the DNA template and
removed nt 87–105 using 59-CAAGTCCCGGGTGGTGTCTGCC OverexpressionofrecombinantL22(rL22)andMBP-L22proteins
GTCTTCGGTC.EBER1Dstlps(I,II,andIV)usedEBER1Dstlp followedasimilarprocedure. Atotalof0.4–1.2LofLBmedium
IVplasmidastheDNAtemplateandremovedstem–loopsIandII containing 50 mg/mL ampicillin and 34 mg/mL chloramphenicol
sequentially using the corresponding oligonucleotides described wereinoculatedwith0.5–1mLofanovernightcultureofRosetta
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Fok etal.
(DE3)pLysScells(Novagen)harboringanL22expressionplasmid. linearized with DraI, which yields a 39 end identical to that of
Cells were grown at 37°C for 3.5–5 h to an A of 0.6–0.7, and EBER1.HSUR3andHSUR3chimeraswerelinearizedwithSmaI
595
IPTG was added to a final concentration of 1 mM. Protein andPmlI,respectively,resultingintheadditionofaGnucleotide
expression was allowed for 2 h at 37°C. Cells were harvested by at the 39 end. To obtain unlabeled RNAs, in vitro transcription
centrifugation andresuspended atA of30–60in50mMTris- was performed overnight at 37°C in a final volume of 100 mL
595
HClatpH8.0/10%sucroseforrL22orincolumnbuffer(20mM containing 40 mM Tris-HCl (pH 7.5), 15 mM MgCl , 4 mM
2
Tris-HCl at pH 7.4, 200 mM NaCl, 1 mM EDTA, and 10% spermidine, 0.05% Triton X-100, 10 mM DTT, 2.5 mM NTPs,
glycerol) for MBP-L22 proteins before being stored at "80°C. 0.05 mg/mL linearized plasmid or DNA template, and 0.1 mg/mL
Frozen cells were thawed in an ice-water bath. All subsequent T7 RNA polymerase. The RNA transcripts were purified on 8%
stepswerecarriedoutat4°C.Proteaseinhibitorcocktail(Calbio- denaturing polyacrylamide gels. Gel slices were eluted overnight
chem) and a final concentration of 1–4 mM EDTA were added inTEbuffer(10mMTris-HClatpH7.5,1mMEDTA)containing
to the thawed cells. Lysed cells were cleared by centrifugation at 1% SDS, and then phenol-extracted (pH 4.5), chloroform-
100,000g for 1 h at 4°C. Both L22 proteins were purified as extracted twice, and ethanol-precipitated in 0.3 M NaOAc (pH
described below to 90%–95% homogeneity as judged by SDS- 4.5). Precipitated RNAs were washed with 70% ethanol and
PAGE. The calculated extinction coefficients (e ) for rL22, dissolved in water. The RNA concentration was determined by
280
1209 M"1 cm"1, and for MBP-L22, 76,810 M"1cm"1, were used measuring theUV absorbanceat 260nm.A totalof50 rmolsof
to determine proteinconcentrations. each transcript were treated for 1 h at 37°C with 0.5 U/mL calf
intestinal alkaline phosphatase (Roche) to remove the 59 tri-
phosphate. The RNA was extracted with phenol (pH 4.5),
Purification of recombinant L22 protein
followedbyachloroformextractionandanethanolprecipitation.
It was resuspended in water and 59 phosphorylated by adding
Plasmid pET11a-L22 constructed by PCR amplification of the
10 U of T4 polynucleotide kinase (PNK), 1 3 T4 PNK buffer
L22-encoding sequence and ligation into the pET11a vector was
(Roche), and 150 mCi of [g-32P]ATP for 45 min at 37°C.
usedforexpressionofrL22.TheDNAsequencewasconfirmedby
Radiolabeled transcripts were purified by separation on denatur-
the Keck Facility. Cleared cell lysates were adjusted to Buffer A
ingpolyacrylamidegels,elutionfromgelslices,andprecipitation
(50 mM HEPES at pH 7.9, 4 mM EDTA, and 10% glycerol)
asdescribed above. Thepelleted RNAswere dissolvedin water.
containing 200mMNaClandloadedontoa1-mLSP-Sepharose
To produce [a-32P]UTP body-labeled RNAs, in vitro tran-
FastFlowcolumnequilibratedwiththesamebuffer.Thecolumn
scription wasperformedfor 90minat37°Cinafinal volumeof
was washed with 1 mL Buffer A containing 200 mM NaCl and
10 mL containing 40 mM Tris-HCl (pH 7.5), 6 mM MgCl ,
then 10 mL Buffer A containing 400 mM NaCl. rL22 was eluted 2
2 mM spermidine, 10 mM DTT, 0.5 mM ATP and CTP, 0.5 or
in 6 mL Buffer A containing 900 mM NaCl. The purified rL22
1.0mMGTP,0.1or0.05mMUTP,20or40mCiof[a-32P]UTP
protein was dialyzed twice against 25 mM HEPES (pH 7.9),
(400 Ci/mmol), 0.1 mg/mL linearized plasmid or 6 ng/mL DNA
50 mMNaCl, 2 mMDTT,and 10%glycerol for 4–12h.
template, and 0.1 mg/mL T7 RNA polymerase. RNAs were
purified asdescribedabove.
Cloning and purification of MBP-L22 fusion protein
Electrophoretic mobility-shift assays
TheL22encodingsequencewasPCRamplifiedfromtheGST-L22
plasmid (Toczyski and Steitz 1993) using a 59 primer-GCGGA The 59-end-labeled or [a-32P]UTP body-labeled RNAs (5 nM)
ATTCGCCCCCGTGAAGAAGCTGTGanda39primer-TTTCAC wereheatedat95°Cfor3min,followedbysnapcoolingonicefor
CGTCATCACCGAA. The resulting PCR product was restriction 3min.Bindingreactions(10mL)containingtheindicatedamounts
digestedwithEcoRIandSalIenzymesandligatedintopMAL-c2X of protein were incubated for 30 min on ice in RNA-binding
vector(NEB)cutwiththesameenzymes.TheMBP-L22sequence buffercontaining37.5mMHEPES(pH7.9),75mMNaCl,5mM
wasconfirmed byDNAsequencing (Keck Facility). MgCl , 8% glycerol, and 1 mg Escherichia coli tRNA. Prior to
2
Before centrifugation, thawed cells were sonicated on ice for loading, 2 mL of 50% glycerol were added to each sample. The
10sectwice,20-secapart,withaBransonSonifieratsetting3and sampleswereloadedonapre-electrophoresed(200V,30min),8%
100% duty cycle. The soluble lysates were diluted with 20 mL nativegelin1XTBEbuffer(100mMTris,100mMborate,2mM
column buffer and then loaded onto a 4-mL amylose column EDTA).Electrophoresiswascarriedoutat200Vfor3–6hat4°C.
(NEB)equilibratedwithcolumnbuffer.Thecolumnwaswashed
with60mLcolumnbufferandtheboundMBP-L22waselutedin
UV cross-linking
6 mL column buffer containing 10 mM maltose. The purified
MBP-L22proteinwasdialyzedtwiceagainst25mMHEPES(pH
To detect in vivo cross-linking between L22 and RNA, 20 mg of
7.5), 50 mM NaCl, 2 mM DTT, and 10% glycerol for 4–8 h. A
p73EBERs or its deletion variants were transfected into HEK293
fraction of the MBP-L22 preparation may have been inactive,
cellsat40%–60%confluencyina10cm-diameterplateusingthe
since rL22 exhibits about threefold higher binding affinity to
TransIT293reagent(Mirus)accordingtothemanufacturer’spro-
EBER1 ascompared withMBP-L22.
tocol.After48h,thecellswerewashedtwicewithcoldphosphate-
buffered saline and resuspended in 3 mL of phosphate-buffered
In vitro transcription saline. Cells were irradiated (or not irradiated as a negative
control) on ice with 254-nm UV light at a distance of 4 cm for
RNAs for binding studies were prepared by in vitro run-off 4.5minandcollectedbyscrapingandcentrifugationinEppendorf
transcription. Wild-type or deletion EBER 1 plasmids were tubes. Cell lysates were prepared, and immunoprecipitation with
880 RNA, Vol.12, No.5
JOBNAME:RNA12#52006PAGE:10OUTPUT:MondayApril315:40:282006
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L22 bindsmultipledomains ofEBER1
anti-L22 antibody (Toczyski and Steitz 1993) was carried out as Haluska,F.G.,Russo,G.,Kant,J.,Andreef,M.,andCroce,C.M.1989.
described (Cook et al. 2004). Precipitated RNAs were released Molecular resemblance of an AIDS-associated lymphoma and
from the antibody by proteinase K digestion and resolved on an endemicBurkittlymphomas:Implicationsfortheirpathogenesis.
Proc.Natl.Acad.Sci.86:8907–8911.
8%ureapolyacrylamidegel.EBER1andEBER2sequenceswere
Henle,G.andHenle,W.1966.Immunofluorescence incellsderived
detected by Northern blotting using [a-32P]-labeled DNA oligo-
fromBurkitt’slymphoma.J.Bacteriol.91:1248–1256.
nucleotide probes, EBER 1R152 (59-CCAGCTGGTACTTGACC Henle,G.,Henle,W.,andDiehl,V.1968.RelationofBurkitt’stumor-
GAAGAC)andEBER2R134(59-ATTAGAGAATCCTGACTTGCA associated herpes-type virus to infectious mononucleosis. Proc.
AATGCTCT). Natl.Acad.Sci.59:94–101.
Howe, J.G. and Shu, M.D. 1988. Isolation and characterization of
the genes for two small RNAs of herpesvirus papio and their
ACKNOWLEDGMENTS comparisonwithEpstein-Barrvirus-encodedEBERRNAs.J.Virol.
62:2790–2798.
We thank Kazio Tycowski and Nikolay Kolev for comments on Howe, J.G. and Steitz, J.A. 1986. Localization of Epstein-Barr virus-
the manuscript and Enrique De La Cruz, Robert P. Rambo, and encodedsmallRNAsbyinsituhybridization.Proc.Natl.Acad.Sci.
TimothyC.Taylorfortheircontributionstoearlierphasesofthis 83:9006–9010.
Johansson,B.,Klein,G.,Henle,W.,andHenle,G.1970.Epstein-Barr
study. R.M.M-F. is a fellow of the Jane Coffin Childs Fund for
virus(EBV)-associatedantibodypatternsinmalignantlymphoma
Medical Research. This work was supported by grant CA16038
andleukemia.I.Hodgkin’sdisease.Int.J.Cancer6:450–462.
from the NIH. J.A.S. is an investigator of the Howard Hughes
Johansson, B., Killander, D., Holm, G., Mellstedt, H., Henle, G.,
Medical Institute. Henle, W., Klein, G., and Soderberg, G. 1975. Epstein-Barr virus
(EBV)-associatedantibodypatternsinrelationtothedeficiencyof
Received December 20, 2005; acceptedJanuary 25, 2006. cell-mediatedimmunityinpatientswithHodgkin’sdisease.IARC
Sci.Publ.11:237–247.
Komano,J.,Maruo,S.,Kurozumi,K.,Oda,T.,andTakada,K.1999.
Oncogenic role of Epstein-Barr virus-encoded RNAs in Burkitt’s
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