of January 1, 2019.This information is current as

(CD154) mRNARegulating the Stability of CD40 LigandTract-Binding Protein Is Involved in A Complex Containing Polypyrimidine

Lori R. CoveyPenelope A. Kosinski, Jennifer Laughlin, Karnail Singh and

http://www.jimmunol.org/content/170/2/979doi: 10.4049/jimmunol.170.2.979

2003; 170:979-988; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/170/2/979.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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A Complex Containing Polypyrimidine Tract-Binding ProteinIs Involved in Regulating the Stability of CD40 Ligand(CD154) mRNA1

Penelope A. Kosinski, Jennifer Laughlin, Karnail Singh, and Lori R. Covey2

CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of“regulated instability” of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late timesof activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3� untranslated region of theCD154 message. We have undertaken experiments to extend these findings and to analyze thecis-acting elements andtrans-actingfactors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-richmotif. However, our current study shows that when this site was deleted additional complex binding was observed upstream anddownstream of the minimal binding region. Only after deletion of an extended region (termed�1515) was complex bindingcompletely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions boundrelated but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein.Deletion of the�1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally,using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclearribonucleoprotein I) as a candidate RNA-binding component of complex I. The Journal of Immunology, 2003, 170: 979–988.

I nteraction between CD40 expressed on B cells, macrophages,and other APCs with its ligand CD40 ligand (CD40L3; alsodesignated CD154) expressed on activated CD4� T cells has

been shown to be a critical parameter in eliciting both humoral andcell-mediated immune responses. The absolute requirement forCD40 signaling in a wide range of immune responses has beendemonstrated in animal models lacking either functional CD40 (1)or CD154 (2) and in humans suffering from both X-linked andCD40-associated hyper-IgM syndrome (hyper-IgM X-1 and hy-per-IgM 3, respectively) (3–9). In these cases, there is clear ab-sence of isotype switching in B cells, a lack of germinal centers,and deficient primary and secondary responses to thymus-depen-dent Ags. In addition to its role in the humoral immune response,a number of studies have revealed the importance of CD40 sig-naling in other immune functions including 1) the rescue fromapoptosis of both Ag-induced and naive B cells, 2) the induction ofperipheral tolerance, 3) the selection of self-reactive T cells, and 4)the activation of cellular immunity primarily through the interac-tion of activated CD4� T cells with CD40-expressing macro-phages (reviewed in Refs. 10–12).

Early studies examining the kinetics of CD154 expressionshowed rapid and transient surface expression on activated T cells(13, 14). The transient nature of CD154 expression on APC-acti-vated T cells corresponded to the internalization of CD154 byCD40-expressing APCs through receptor-mediated endocytosis(15). However, multiple studies showed that the time course andextent of CD154 expression depended on the type of stimuli aswell as on costimulatory interactions provided by B cells or APCs(14–24). Specifically, studies from our laboratory and others re-vealed that CD154 is expressed on purified T cells for an extendedperiod of time in response to anti-CD3 signaling either with orwithout costimulatory signals (18, 25–27) or in the presence ofdifferent cytokines (28, 29). Engagement of CD154 with CD40 onAg-selected B cells initially induces B cells to proliferate and un-dergo differentiation into Ab-secreting cells (reviewed in Ref. 10).However, it has been shown that continued CD40 signaling inactivated B cells prevents terminal differentiation as measured bya decrease in both secretory Ig and the transcriptional regulator Blymphocyte-induced maturation protein 1 (30). Together, thesestudies suggest that CD154 expression is critical for regulating theimmune response at early and late times after activation. Further-more, it is clear that CD154 expression is induced by the integra-tion of signals from multiple pathways that ultimately regulate thefunctional response of the CD4� T cell at a specific time in theongoing immune response.

Although CD154 is regulated both temporally and with respectto cell type, the underlying mechanisms responsible for this controlare just beginning to be defined.CD154 gene transcription is in-duced by TCR signaling and expression is enhanced in response tocostimulatory signals (31–33). Transcriptional regulation appearsto be dependent on NF-AT factor binding sites located in a region5� of the TATA box and start sites (34–36) and a T cell-dependentenhancer element has been located 3� of the coding region that isNF-�B dependent (35). In addition to transcriptional regulation,we and others have shown that posttranscriptional regulation playsa critical role in modulating the steady-state level of CD154

Department of Cell Biology and Neuroscience, Rutgers, State University of NewJersey, Piscataway, NJ 08854

Received for publication September 16, 2002. Accepted for publication November13, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Charles and Joanna Busch BiomedicalFoundation (Rutgers University) and the American Heart Association (Grant0051487T) to L.R.C.2 Address correspondence and reprint requests to Dr. Lori R. Covey, Department ofCell Biology and Neuroscience, Rutgers, State University of New Jersey, 604 AllisonRoad, Piscataway, NJ 08854. E-mail address: covey@biology.rutgers.edu3 Abbreviations used in this paper: CD40L, CD40 ligand; UTR, untranslated region;PTB, polypyrimidine tract-binding protein; NF90, nuclear factor 90; DRB, 5,6-di-chloro-�-D-ribofuranosylbenzimidazole; hnRNPI, heterogeneous nuclear ribonucleo-protein; YB-1, Y box-binding protein 1.

The Journal of Immunology

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mRNA (25, 37, 38). Specifically, CD154 transcripts are degradedin an activation-dependent manner (25). At early times after CD3-dependent signaling, CD154 mRNA is rapidly degraded (t1/2 � 40min) which is a feature shared by numerous cytokine, growth fac-tor, and cell cycle protein mRNAs including TNF-� (reviewed inRef. 39). However, unlike the TNF-� (25, 40) or c-myc (40, 41)transcript which remains unstable in activated T cells, the stabilityof the CD154 mRNA shows an unusual pattern of “regulated in-stability” that corresponds to a 3- to 4-fold increase in stabilityafter extended CD3 activation (25). The stability of the CD154transcript is only marginally increased by costimulatory signals butis highly stabilized by activators of protein kinase C (25, 37, 38).In a separate study, it was demonstrated that human endothelialcells enhance the stability of CD154 message in PHA-stimulatedhuman T cells through an LFA3-dependent mechanism (42), sup-porting the premise that posttranscriptional regulation is an impor-tant factor in CD154 expression.

To extend our initial observations, we used the Jurkat D1.1 Tcell line, a line that constitutively expresses CD154 mRNA with arelatively stable half-life (t1/2 � 2.3 h), to identify a putative sta-bility complex (termed complex I) (43). This complex bound spe-cifically to a highly pyrimidine-rich region in the 3� untranslatedregion (UTR) and contained a poly(CU) RNA-binding protein thatcross-linked only under conditions where the CD154 mRNA washighly stabilized. This was most clearly seen in comparing thebinding patterns of extracts from resting, 2-h, 24-h, and 48-h anti-CD3-activated CD4� T cells. With these extracts, complex for-mation and cross-linking were only observed in T cells stimulatedfor 48 h and, to a much lesser extent, in cells stimulated for 24 h.This work strongly suggested that complex I is directly involved inregulating the variable decay rate of CD154 mRNA during T cellactivation.

In our current study, we have extended our analysis of the “sta-bility” phase of CD154 mRNA decay and identified three differentbinding regions within the pyrimidine-rich region. We have iden-tified one protein, polypyrimidine tract-binding protein (PTB) as acomponent of complex I. Finally, we have shown both by in vitroand in vivo assays, that complex I binding results in an increase instability of the CD154 transcript.

Materials and MethodsCells and Abs

The Jurkat D1.1 T cell line have been previously described (17, 44). Rabbitanti-human PTB antisera was a gift from Dr. J. Patton (Vanderbilt Uni-versity, Nashville, TN). Anti-Y box-binding protein (YB) antisera was agift from Dr. W. Reynolds (Sidney Kimmel Cancer Center, La Jolla, CA)and the anti-nuclear factor 90 (NF90) antisera was provided by Dr. P. Kao(Stanford University, Palo Alto, CA).

Protein extracts

One to 2 � 107 D1.1 cells were harvested, washed with PBS (pH 7.4), andlysed with 200 �l of lysis buffer (0.2% Nonidet P-40, 40 mM KCl, 10 mMHEPES (pH 7.9), 3 mM MgCl2, 5% glycerol, 1 mM DTT, 8 ng/ml apro-tinin, and 0.5 mM PMSF) for 5 min. Lysates were centrifuged at 16,000 �g for 2 min and supernatants were stored at �80°C. The protein concen-tration of extracts was determined by the Bradford assay (Bio-Rad,Hercules, CA).

Deletion mutagenesis

The �E1�–E5�, �1506, and the �1515 mutations were generated using theQuick Change Mutagenesis kit (Stratagene, La Jolla, CA). For �E1�–E5�,the BamHI-BamHI fragment of CD154 containing the entire 3� UTR anddownstream sequences was used as a template and amplified with primers5� mutE1E5 (5�-cctctttcaatctctctctctccatctcctctagtctcttccctcccccagtctctcttctc-3�)and 3� mutE1E5 (5�-gagaagagagactagggggagggaaagagactagagagagatggagagagagagattgaaagag-3�). To generate the �1506, the above procedure wasused except the �E1�–E5� plasmid and primers �1377–1506 (5�-cgccac

cctctcggacagttattcattctccccctttctaacacacacacacacacacac-3�) and �1506–1377(5�-gtgtgtgtgtgtgtgtgtgttagaaagggggagaatgaataactgtccgagagggtggcg-3�) wereused. To generate the �1515 mutation, the �1506 template was used withprimers mut1348–517 (5�-ggagaaccgaaacccccccccccccccccacacacacacacacacacacacacacacac-3�) and mut1517–1348 (5�-gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgggtgggtgggtgggtgtttcggttctcc-3�). All reactions were conducted according to themanufacturer’s protocols.

RNA probes

The XbaI-E1�, E1�–E5�, E1�-BsrI and HaeIII-DraI templates were gener-ated as previously described (43). The T7-XbaI-E1� template was PCRamplified with the following primers: 5�-cgtaatacgactcactatagggctagaacgtctaacacagtggaga-3� (forward) and 5�-tgaaagagagagatggagagagagagagagatt-3� (reverse). The T7-E5�-HaeIII template was synthesized using thefollowing primers: 5�-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3�(forward) and 5�-agagaactgactagcaacggcctg a-3� (reverse). The XbaI-HaeIII, �1506, and �1515 templates were synthesized using the corre-sponding CD154 3� UTR template (see above) with the following primers:XbaI-HaeIII, 5�-cgtaatacgacgcactatagggctagaacgtctaacacagtggaga-3� (for-ward), and 5�-agagaactgactagcaacggcctga-3� (reverse). PCR were preparedin 10 mM Tris (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2,0.2 mM dNTPs, 200 ng of each primer, 1 ng of DNA template, and 2.5 UTaq polymerase (Promega, Madison, WI). Cycling parameters were as fol-lows: 1 cycle of 3 min at 94°C, 30 cycles of 94°C for 30 s, 55°C for 10 s,and 72°C for 45 s. The XbaI-HaeIII-poly(T), E1�-BsrI-poly(T), and �1515-poly(T) templates were synthesized as above except the 3� primer con-tained a 60 nt poly(T) sequence.

RNA probes were synthesized using 0.5 �g of the PCR DNA template;0.4 mM each of rGTP, rATP, and rCTP; 0.04 mM rUTP; 30 mM DTT; 20U of RNasin; 1� T7 transcription buffer (40 mM Tris-HCl (pH 7.9); 6 mMMgCl2; 2 mM spermidine; and 10 mM NaCl); 25–40 �Ci of [�-32P]rUTP;and 4.25 U of T7 RNA polymerase (Promega) at 37°C for 1 h. Reactionswere treated with DNase RQ1 for 15 min and centrifuged through G25columns (Amersham Biosciences, Piscataway, NJ) to remove unincorpo-rated nucleotides.

EMSA

Twenty-microliter reactions were prepared in EMSA binding buffer (40mM KCl, 10 mM HEPES (pH 7.9), 3 mM MgCl2, 1 mM DTT, and 5%glycerol), 4 ng of Escherichia coli tRNA, 5–10 �g of D1.1 extract, and 4 �104 cpm of in vitro-synthesized RNA probe. In supershift experiments,1-�l Abs at a concentration of 1 �g/�l was added to the reaction for 1.5 hbefore addition of the probe. Following a 30-min incubation at room tem-perature, 2 �l of a RNase mix of either 40 U of RNase T1 and 100 pg ofRNase A or 40 U of RNase T1, 100 pg of RNase A, and 0.015 U of RNaseV1 was added, and the reactions were incubated for an additional 40 minat 37°C. After addition of 100 �g of heparin, the reactions were incubatedfor 10 min on ice and separated on a 7% native acrylamide gel in 0.25�Tris-borate-EDTA at 200 V for 2–4 h. In competition experiments, unla-beled RNA synthesized in vitro was diluted 1/25 in binding buffer (referredto as 1�). Increasing amounts of unlabeled transcript was added to thereaction before addition of the probe. In experiments using depleted ex-tracts, 1.3 mg of D1.1 extract in 100 �l was incubated with 1–2 �l of 1�g/�l Abs overnight at 4°C followed by addition of protein A-Sepharose.The extracts were centrifuged and the supernatant was depleted two addi-tional times.

In vitro decay assays

Capped RNA transcripts were synthesized in vitro as described above with624 �M Cap analog (Amersham Biosciences). Reactions were incubated at37°C for 20 min and stopped with 100 �l ULB (7 M urea, 2% SDS, 0.35M NaCl, 10 mM EDTA, and 10 mM Tris (pH 7.5)). Samples were elec-trophoresed through a 5% acrylamide (29/1)/7 M urea gel and the appro-priate bands were excised from the gel and eluted with 150 �l of elutionbuffer (20 mM Tris (pH 7.5), 0.5 M NaOAc, 10 mM EDTA, and 1% SDS)at 65°C. Following precipitation, the pellets were resuspended in diethylpyrocarbonate ddH2O.

Twenty-five-microliter reactions containing 50 –100 �g of extract, 1 �104 cpm in vitro-synthesized capped RNA, 1� IVD-1 buffer (100 mMKOAc, 2 mM MgOAc, 2 mM DTT, 10 mM creatine phosphate, 1 mMATP, 0.4 mM GTP, 10 mM Tris (pH 7.5), and 0.1 mM spermine) wereincubated at 37°C for indicated time points. To monitor degradation of theRNA within the reaction, 150 �l of ULB buffer containing 1 � 102 cpm ofkinased oligonucleotide was added to the reaction. The reaction was ex-tracted with phenol/chloroform, ethanol precipitated, and resuspended with22 �l of 80% formamide/dye. The samples were boiled and 10 �l of eachreaction was electrophoresed through a 5% acrylamide (29/1)/7 M urea gel

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at 15 W. The gel was dried at 80°C and exposed on Kodak film (Kodak,Rochester, NY) using intensifying screens.

Plasmid constructs

The pcDEF3 vector was a gift from Dr. J. Langer (University of Medicineand Dentistry of New Jersey, Piscataway, NJ) and contains the humanelongation factor 1� promoter. The poly(A)� pcDEF3 was constructed bydigesting with NotI and BbsI to remove the bovine papilloma virus poly(A)site. The CD154 cDNA (1–1816 bp) was cloned into the poly(A)� pcDEF3construct. A 2.3-kb BamHI-BamHI fragment containing part of the 3� UTRand 1.5 kb of genomic sequence was subcloned from a phage clone con-taining the 3� region of the CD154 gene. A similar construct was made thatcontained the �1515 deletion. The mutDef3 and mut�1515Def3 constructswere engineered using the Erase-a-base kit (Promega) which introduced a388-bp deletion into the coding region of the CD154 cDNA. This codingregion replaced the wild-type coding region in both wtDef3 and�1515Def3.

Stable transfection

Jurkat D1.1 cells (5 � 106) were resuspended in 0.4 ml of RPMI 1640 and25 �g of linearized construct DNA was introduced by electroporation us-ing 250 mV, 960 �F. The pulsed cells were stored on ice for 10 min andincubated at 37°C in 5% CO2 for 36–48 h. To select for stable transfec-tants, cells were resuspended in medium containing 1 mg/ml G418.

5,6-dichloro-�-D-ribofuranosylbenzimidazole (DRB) treatmentand RNA isolation

Briefly, 2.5 � 107 cells were resuspended in 5 ml of complete RPMI 1640and 1 ml was removed for RNA extraction. DRB (40 �g/ml) was added tothe remaining 4 ml of cell suspension and 1-ml aliquots were removed atallotted time points. RNA was extracted using TRIzol (Life Technologies,Rockville, MD).

RT-PCR to analyze mRNA decay

Reverse transcription was conducted in a 30-�l volume with 3 �g of RNA,50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 300U of Moloney murine leukemia virus-RT, 60 U of RNasin, 0.3 mM dNTPs,and 500 ng of oligo(dT)15 primer. The reaction was incubated at 37°C for1 h followed by 10 min at 68°C.

PCR was performed using 2.5 �l of cDNA in a 40-�l reaction volumecontaining 10 mM Tris (pH 8.8), 1.5 mM MgCl2, 25 mM KCl, 0.25 mMdNTPs, 2.5 U of Taq, (Promega) with 200 ng of CD40L 5� primer (5�-AAACATACAACCAAACTTCTCCCC-3�) and CD40L 3� primer (5�-CTGTGCTGTATTATGAAGACTCCC-3�) plus 10 ng of the 5� and 3�GAPDH primers (5�-GTCTTCACCACCATGGAGAAGGCT-3� and 5�-CATGCCAGTGAGCTTCCCGTTCA-3�, respectively). Hot start cyclingconditions were 5 min at 92°C, then 25 cycles of 57°C for 1 min, 72°C for 1.5min, and 92°C for 1 min, followed by 1 cycle of 72°C for 5 min. Ten micro-liters of each reaction was electrophoresed through a 1.5% agarose/Tris-bo-rate-EDTA gel and bands were quantitated using the Kodak 1D analysis soft-ware.

UV cross-linking assay

Thirty-microliter reactions containing 500 ng of yeast tRNA, 40 mM KCl,10 mM HEPES (pH 7.9), 3 mM MgCl2, 1.0 mM DTT, 5% glycerol, 60 Uof RNasin, 20 �g of extract, and 2 � 104 cpm of in vitro-synthesized RNAprobe were incubated for 15 min at room temperature. Proteins were UVcross-linked to probes by exposing the reactions with 254 nm UV light for15 min at 0°C. RNase mix (2 �l; 40 U of RNase T1 and 100 pg of RNaseA or 40 U of RNase T1, and 1 �g of RNase A) was added to the reactionand incubated for 30 min at 37°C. Samples were boiled for 5 min andelectrophoresed through a 5% stacking gel/12% separating gel for 4.5 h at30 mA. The gel was fixed in 10% acetic acid, dried, and visualized byautoradiography.

ResultsThe region defined by �1515 completely lacks complex binding

Our previous work demonstrated that complex I binding to theCD154 transcript directly corresponds to an increase in stability ofthe message at extended times of T cell activation. Furthermore,we localized the complex I minimal binding site to a 63-nt stretchwithin the XbaI-HaeIII region in the 3� UTR (termed E1�–E5�)(Fig. 1A). To extend these findings and determine whether com-

FIGURE 1. Analysis of the complex I cis-acting binding element in theCD154 3� UTR. A, Schematic of the CD154 mRNA with the 5� UTR, codingregion, and the 988-nt 3� UTR indicated. The complete sequence of the XbaIto HaeIII complex I binding site is shown with the minimal binding region,E1�–E5�, underlined and the poly(C) tract and the CA repeats shown in bold.B, EMSA RNA was conducted without extract (lanes 6–10) or with 10 �g ofJurkat D1.1 total cell extract (lanes 11–15) with 4 � 104 cpm of either probeXbaI-HaeIII (lanes 6 and 11), E1�–E5� (lanes 7 and 12), �E1�–E5� (lanes 8and 13), �1506 (lanes 9 and 14), and �1515 (lanes 10 and 15). The individualprobes are shown in lanes 1–5. Below the figure is a schematic of the in vitrosynthesized probes used in RNA-binding studies: The E1�–E5� probe containsonly the minimal binding region, the XbaI-HaeIII probe contains all sequencesbetween the XbaI and HaeIII sites, the �E1�–E5� probe lacks the minimalbinding region, the �1506 mutant contains the XbaI-HaeIII sequence with adeletion of the region between nt 1377 and 1506, and the �1515 probe consistsof the XbaI-HaeIII region with a deletion of the sequence between 1349and 1515.

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plex I binding was required for CD154 mRNA stability, wedesigned experiments to directly test the effect of deleting theE1�–E5� region on CD154 mRNA decay. To this end, probeswere generated that either retained (XbaI-HaeIII) or lacked(�E1�–E5�), the minimal binding sequence within the contextof the XbaI to HaeIII sequence and conducted RNA-bindingassays to detect complex I binding. Surprisingly, complexformation was obtained with the �E1�-E5� probe lacking theminimal binding site (Fig. 1B, lane 13) as well as with thecontrol probes (Fig. 1B, lanes 11 and 12). In an attempt tocomprehensively define the region of complex I binding, wesynthesized two additional templates that lacked sequencesbetween 1377 and 1506 and 1349 and 1515 (designated �1506and �1515, respectively). These regions were chosen based onthe fact that they lacked most (�1506) and all (�1515) of theCU-rich sequences in the XbaI-HaeIII region (see Fig. 1). Usingthe �1506 probe, we observed a slower migrating complex (Fig.1B, lane 14), whereas no complex formed with the �1515 probe(Fig. 1B, lane 15). Because of the absence of complex forma-tion with the �1515 probe, RNA containing this deletion wasused in all subsequent experiments designed to define therelationship of complex I binding to CD154 mRNA stability.

Identification of multiple complex binding sites within the XbaI-HaeIII region

We were intrigued by the finding that complex I bindingoccurred in the absence of the minimal E1�–E5� binding site.This result suggested that either complex I was able to form onsequences outside this region or that bringing two noncontigu-ous regions together through deletion created a new, but phys-iologically irrelevant binding site. To distinguish between thesetwo possibilities, RNA-binding experiments were conductedwith probes synthesized from templates that included sequencesupstream and downstream of the E1�–E5� region (probes XbaI-E1� and E5�–HaeIII). We found that a complex migrating withthe same mobility as complex I formed with the upstreamXbaI-E1� probe and a faint, slower migrating complex bound tothe downstream E5�-HaeIII probe (Fig. 2, arrows). To deter-mine whether these two complexes were related to each otherand/or to complex I, we set up RNA-binding experiments withcapped and labeled XbaI-E1�, E1�–E5�, and E5�-HaeIII probesand competed complex formation with increasing concentra-tions of unlabeled self- and non-self RNA. As shown in Fig. 3,all three complexes were competed by cold self-RNA and to alarge extent by non-self RNA as well (A–C, lanes 4 –15). Incontrast, there was very little competition for any of the threecomplexes with the irrelevant HaeIII-DraI RNA that has beenpreviously shown to lack complex I binding (43) (Fig. 3, lanes16 –19). A consistent finding was that complexes were bettercompeted by cold self than by non-self RNA, suggesting thatthe complexes were similar but not identical. This was partic-ularly true for the slower migrating complex which bound to theE5�-HaeIII probe. Here, the complex was fully competed by50-fold addition of competitor self-RNA whereas the samecomplex required the addition of 100-fold cold Xba-E1� andE1�–E5� RNA to be fully competed (Fig. 3C, lanes 4 –15).These data strongly suggest that the three complexes bindingwithin the XbaI-HaeIII region are related to each other andsupport the idea that at least three independent complex bindingsites are adjacently located in the CD154 3� UTR.

The complex I element confers stability on the CD154 transcriptin vitro

To begin to decipher the role complex I plays in CD154 mRNAdecay, we conducted in vitro decay assays using transcripts con-taining the XbaI-HaeIII region of the 3� UTR with or without the1349–1515 sequences. In vitro-transcribed, -capped, and -polyad-enylated XbaI-HaeIII (309 nt) and �1515 (135 nt) transcripts wereincubated with D1.1 total extract over a 1-h time course. As shownin Fig. 4, the �1515 transcript was less stable than the XbaI-HaeIIIwild-type transcript during the first 15 min of incubation, with�25% loss during this time interval. In contrast, between 15 and60 min, the �1515 transcript was significantly more stable than theXbaI-HaeIII transcript. To ensure that the smaller size of the�1515 transcript was not a factor in its instability during the first15 min, we tested the E1�-BsrI transcript, a 79-nt RNA containingthe E1�–E5� minimal binding site, and found it significantly morestable than either the XbaI-HaeIII or the �1515 transcript. One

FIGURE 2. Complex formation occurs on at least three distinct siteswithin the XbaI-HaeIII region. RNA-binding assays were conducted withthe in vitro-synthesized probes that span the E1�–E5� (lanes 1–3), XbaI-E1�(lanes 4–6), and the E5�-HaeIII (lanes 7–9) regions. The probes wereincubated with RNase in the presence (lanes 3, 6, and 9 (E)) or absence(lanes 2, 5, and 8 (R)) of 5 �g of D1.1 total extract. Lanes 1, 4, and 7 showthe probes alone (P) in the absence of RNase and extract. The sequence ofthe individual probes is shown below.

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interpretation of these results is that complex I is acting as a sta-bility factor by protecting a site that is a “hot spot” for nucleaseattack.

The complex I element confers stability on the CD154 transcriptin vivo

Our in vitro results suggested that complex I binding was involvedin regulating the decay pathway of CD154 mRNA. However, toaddress questions about the in vivo significance of complex Ibinding within the context of the complete CD154 mRNA, weintroduced constructs containing the full-length CD154 cDNA,or the same region with the �1515 deletion, into Jurkat T cells.The CD154-specific sequences were subcloned into the pcDEF3vector (designated wtDEF3 and �1515DEF3) in which tran-scription is under the control of the human elongation factor 1�promoter and is active in Jurkat T cells independent of cellactivation (B. Barnhart and L. R. Covey, unpublished observa-tions). To ensure that regulatory elements within the vectorwere not influencing the decay rate of the transcribed RNA, thebovine papilloma virus poly(A) signals were removed from thepcDEF3 construct and replaced with the CD154 poly(A) siteplus 1000 bp of downstream sequence (Fig. 5A). Additionally,since one specific objective was to introduce the constructs intothe CD154� D1.1 Jurkat T cell line, the constructs wereengineered with a 388-bp deletion in the coding region thatallows discrimination between transcripts originating from the

construct and endogenously expressed CD154 transcripts. Thenew constructs were termed mutDef3 and mut�1515Def3 andwere introduced by stable transfection into the Jurkat D1.1 cellline. Individual mutDef3 and mut�1515Def3 subclones werescreened by RNase protection assays to identify those express-ing the construct wild-type and �1515 RNAs at similar levels(data not shown). Furthermore, preliminary PCR were con-ducted with varying amounts of cDNA to ensure that amplifi-cation was taking place within a linear range of the assay (datanot shown). RNA decay was measured by incubating the cellswith the RNA polymerase II inhibitor DRB over a 6-h timecourse.

RNA was isolated and RT-PCR was conducted using primersthat hybridized to either side of the coding region deletion and

FIGURE 3. Complexes formed within the XbaI-HaeIII region are re-lated to each other. Competition assays were established with in vitro-synthesized, -labeled, and -capped RNA probes in the presence of increas-ing amounts of unlabeled and capped competitor RNA. Lanes 1-3 show thedifferent labeled probes alone, with RNase in the absence of extract, andwith RNase plus extract, respectively. Unlabeled transcript was either un-diluted or diluted 1/25 in binding buffer and added to the different fractionssuch that the amount of cold probe increased from 1� to 25�, 50�, and100�. Competition of the different labeled probes was conducted in thepresence of increasing concentrations of cold XbaI-E1� (lanes 4–7), coldE1�–E5� (lanes 8–11), cold E5�-HaeIII (lanes 12–15), and cold irrelevantHaeIII-DraI RNA (lanes 16–19).

FIGURE 4. Complex I binding stabilizes the CD154 mRNA in vitro. A,In vitro RNA decay reactions were conducted with uniformly labeled,capped, and polyadenylated XbaI-HaeIII (lanes 1–4), �1515 (lanes 5–8),and E1�-BsrI (lanes 9–12) probes and 50 �g of extract from Jurkat D1.1cells. Percentage of the remaining RNA was determined after 60 min bycomparing the band intensity to the intensity of the input RNA signal(designated as 100%). Internal control bands refer to a labeled oligonucle-otide that was included in the stop buffer to normalize for RNA extractionsand gel loading. B, The semilog graph depicts the averaged values andSEM for nine (�1515 and E1�-BsrI) and three (XbaI-HaeIII) independentexperiments.

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gave an 839-bp and 451-bp fragment for the endogenous and con-struct-specific transcripts, respectively. To control for equal RNAamounts and loading errors, a 400-bp GAPDH fragment was am-plified in all samples. As shown in Fig. 5, B and C, the mutDef3

transcript was more stable than the mut�1515Def3 transcript overthe time course of DRB inhibition. This difference in stability wasdirectly related to the �1515 deletion since the two transcriptswere identical except for this deletion. The difference in decay ratebetween the two transcripts was greatest at the early time pointsafter transcription inhibition, which corresponds to what was ob-served in vitro. Also, since the level of construct-specific expres-sion was very similar between the two cell lines, specific kinetics

FIGURE 6. Identification of a 55-kDa protein that cross-links to thecomplex 1 binding sites within the XbaI-HaeIII region. A, UV cross-linking assays were conducted without (lanes 1–3) and with (lanes 4–6) 10�g of Jurkat D1.1 total cell extract and 1 � 104 cpm of uniformly labeledXbaI-HaeIII (lanes 1 and 4), E1�–E5� (lanes 2 and 5), or �1515 (lanes 3and 6) transcripts with 40 U of RNase T1 and 1 �g of RNase A. Positionand molecular mass (kDa) of protein standards are indicated. The arrownotes the 55-kDa protein. B, UV cross-linking was conducted as in A withuniformly labeled E1�–E5� (lanes 1–3), XbaI-E1� (lanes 4–6), and E5�-HaeIII (lanes 7–9) RNA. Arrow indicates position of �55-kDa protein thatis present with all three probes.

FIGURE 5. The complex I binding region increases the stability of theCD154 mRNA in vivo. A, Schematic of CD154 full-length and �1515regions, including the 388-bp coding region deletion, subcloned into thepcDEF3 vector to give the mutDef3 and mut�1515Def3 constructs. Theinsert contains the 1800-bp CD154 cDNA sequence plus a 1000-bpgenomic sequence which includes the CD154 poly(A) addition site and 950bp of additional downstream sequence. The �1515 construct lacks the 166bp denoted by an inverted triangle. The arrows indicate the relative locationof the primers used to amplify the construct-specific transcripts. B, Shownis a representative PCR experiment which analyzed the decay pattern ofCD154 mRNA in DRB-treated Jurkat D1.1 subclones containing either themutDef3 or mut�1515Def3 constructs. RT-PCR were conducted withRNA from cells incubated with DRB for 0 h (lanes 1 and 6), 1 h (lanes 2and 7), 2 h (lanes 3 and 8), 4 h (lanes 4 and 9), and 6 h (lanes 5 and 10)at a concentration of cDNA previously determined to be in the linear rangeof amplification. Primers specific to CD154 were used to amplify the frag-ment spanning the coding region deletion shown in A and GAPDH-specificprimers were also included in the reaction to control for differences in loading.C, The semilog graph represents the results of in vivo DRB experiments usingthe Jurkat D1.1 cells expressing either the mutDef3 (f) or mut�1515Def3 (F)constructs. The fraction of mRNA remaining at each time point was quanti-tated and was normalized relative to the GAPDH band. The values representthe average of three independent experiments SEM.

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of transcript decay in each line was not caused by measurabledifferences in the substrate to nuclease ratio. These data identifycomplex I binding as being a factor in regulating CD154 transcriptstability in vivo.

A 55-kDa protein binds to three independent sites within theCD154 3� UTR

To begin to identify proteins that were part of the stability com-plex, we initiated UV cross-linking studies using our differentRNA probes and Jurkat D1.1 extract. We previously demonstratedthat a 90-kDa protein cross-linked to the minimal binding site inboth Jurkat and late-activated CD4� T cell extracts (43). However,we consistently observed nonspecific bands in these binding reac-tions, indicating that the RNase treatment after UV exposure wastoo mild. To view the cross-linking in the absence of the nonspe-cific bands, we instituted a more stringent RNase A treatmentwhich included a 10,000-fold increase after UV exposure. Usingthese conditions, we no longer observed a 90-kDa protein butfound that a protein of �55 kDa bound to the XbaI-HaeIII andE1�–E5� probes but not to the �1515 probe (Fig. 6A, lanes 4–6).We also observed two faint proteins or degradation products of�40 and 25 kDa that were also absent with the �1515 probe. Allthree bands were competed with an excess of poly(dCT) and coldE1�–E5� RNA but were not competed with the nonspecific com-petitor (data not shown).

To identify RNA-binding proteins that bound to the 3� and 5�regions within XbaI-HaeIII, UV cross-linking experiments wereconducted with the XbaI-E1�, E1�–E5�, and E5�-HaeIII probes.Under these conditions, a 55-kDa protein specifically cross-linkedto all three probes (Fig. 6B), a finding that extended our earlierresults showing that highly related complexes formed on all threesubregions within the XbaI-HaeIII region.

PTB (heterogeneous nuclear ribonucleoprotein I (hnRNPI) bindsto the CD154 stability region in the 3� UTR

Before initiating protein purification experiments to identify the55-kDa protein, we wanted to establish whether it was a knownpoly(CU)-binding protein. Two poly(CU)-binding proteins thatwere approximately the correct size were the human YB-1 (45)and the PTB (also called hnRNPI) (46–48). It has previously beenreported that YB-1 binds to a pyrimidine-rich element that is in-volved in c-Jun N-terminal-mediated stability of the IL-2 mRNAin activated Jurkat T cells (41). Furthermore, PTB binds to pyri-midine-rich stretches in RNA and appears to have multiple func-tions in splicing as well as in other posttranscriptional processes(reviewed in Ref. 49). Thus, both proteins appeared to be candi-dates for having a role in CD154 mRNA stability.

RNA-binding experiments were conducted with the E1�–E5�probe and total D1.1 extract or extract that had undergone threerounds of depletion with either Abs against PTB, YB-1, or NF90(a dsRNA-binding protein initially identified in T cells (50)). Ourresults demonstrated that compared with the undepleted extract,there was no effect on the intensity of binding with extract depletedof NF90 and a modest decrease in binding with the YB-1-depletedextract (Fig. 7A). In contrast, we observed a marked decrease incomplex binding with extract that was depleted of PTB. WeFIGURE 7. Identification of PTB as the 55-kDa protein that binds to the

complex I binding site. A,. RNA EMSA was conducted with the uniformlylabeled E1�–E5� probe and 5 �g of either Jurkat D1.1 total extract (lane 3)or extract 3� depleted with either anti-PTB antisera (lane 4), anti-YB-1antisera (lane 5), or anti-NF90 antisera (lane 6). Lane 1 shows probe aloneand lane 2 shows probe with RNase in the absence of extract. B, Westernblot analysis of 50 �g of D1.1 extract before and after 3� depletion withspecific Abs. The large dark band in the YB-1- and PTB-depleted extractsis the Ig H chain which runs at a similar molecular mass as YB-1. C,

EMSA was conducted with the uniformly labeled E1�-BsrI probe and 5 �gof Jurkat D1.1 total extract (lanes 3–5). One microliter of 1 �g/�l anti-YB-1 (lane 4) and anti-PTB (lane 5) was added to the reaction beforeincubation with the probe. Lane 1 is probe in the absence of extract andRNase and lane 2 is probe with RNase in the absence of extract.

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checked the depleted extract for the presence of the different pro-teins and were unable to detect either PTB or NF90 by Westernblot analysis, demonstrating that the depletion had been complete.Unfortunately, we could not conclusively determine whether YB-1was completely removed from the depleted extract since its mo-lecular mass is similar to that of the Ig H chain (Fig. 7B). However,the depletion studies were suggestive that PTB was the RNA-binding component of complex I.

To further test this possibility, we conducted RNA-bindingexperiments using D1.1 extract with the E1�-BsrI probe in thepresence or absence of specific Abs (Fig. 7C). Here, the probemigrates approximately at the same position as complex Ialthough the control shown in Fig. 7C, lane 2, confirms thatcomplex formation only occurs in the presence of extract. UsingAbs to YB-1 and PTB, we found that only anti-PTB Abssignificantly interfered with RNA binding (Fig. 7C, lanes 3–5).This finding supports our results with the depleted extracts andstrongly suggests that PTB binds to the complex I-binding motifin the CD154 3� UTR.

DiscussionIn this report, we have extended our previous studies of CD154mRNA stability by presenting data on the nature of both thebinding site and protein composition of complex I. Previously,we defined the E1� to E5� minimal binding site for complex I asa 63-nt region located in the 3� UTR at nt 1411–1473. In ourpresent study, we were surprised to find that the E1�–E5�sequence could be deleted from the XbaI-HaeIII region andcomplex I still formed on the remaining sequences. This obser-vation led to the identification of two additional CU-rich bind-ing regions within the XbaI-HaeIII region. All three regionswere found by competition experiments to bind related com-plexes although the relationship between the different com-plexes has not been fully determined. The similarity of thecomplexes is reinforced by the fact that all three have as anRNA-binding component a 55-kDa protein whose identificationis consistent with PTB, or hnRNP-I. It is possible that the largerE5�-HaeIII complex is comprised of complex I plus additionalproteins as suggested from the faint band that migrates with thesame mobility as complex I. Additional experiments are inprogress to address this possibility.

Results from our in vitro decay assays suggest that complex Ibinding plays a role in regulating the turnover of CD154 mRNA.An interesting observation from these experiments is that both theXbaI-HaeIII and �1515 transcripts decay with unique second or-der kinetics throughout the 60-min time course. The rapid decay ofthe �1515 transcript in the first 15 min, vs relative stability of theXbaI-HaeIII during this interval, suggests that complex I facilitatesthe formation of a nuclease-resistant structure. However, once thecomplex is removed there is rapid degradation of the XbaI-HaeIIIbut not the �1515 RNA. This observation supports the idea that amajor factor driving the rapid kinetics of the XbaI-HaeIII RNA arethe sequences bound by complex I. That the E1�-BsrI transcript ishighly stable under the same conditions would implicate sequencesoutside E1�-BsrI, but within the adjacent CU-rich sequences, fortargeting nuclease activity. This type of model is reminiscent ofrecent examples of endonuclease-catalyzed decay which is regu-lated by RNA-binding proteins interacting with cis-acting mRNAstability elements. For example, the vigilin protein binds to thevitellogenin mRNA 3� UTR and inhibits transcript cleavage by thepolysomal ribonuclease 1 endonuclease (51). Also, it has been re-cently shown that the � complex, which forms on a specific site inthe 3� UTR of �-globin and confers transcript stability, masks a

unique cleavage site that is recognized by an erythroid-enrichedendonuclease (termed ErEN) (52, 53). Although we did not ob-serve specific cleavage intermediates under our in vitro conditions,it is possible that slowing the reaction rate would allow for detec-tion of these species. Alternatively, once complex I is removed,exonuclease may act more aggressively on the XbaI-HaeIII tran-script vs the �1515 transcript. Further experimentation will allowus to test these different models.

In accordance with our in vitro data, our in vivo resultssupport a role for complex I binding notably in the early hoursof the time course. The delay in the overall decay of both themutDef3 or mut�1515Def3 RNA, compared with the smallertranscripts in vitro, could be explained by the significantlydifferent sizes of the transcripts as well as a cell environmentthat may limit exposure to nuclease. Based on our in vitrofindings, it is quite possible that by removing complex I fromthe transfected cell instead of removing its binding site from thetranscript would measurably affect the decay rate of the CD154transcript in vivo.

Our finding that PTB is a major component of complex I is inagreement with others showing that PTB carries out diversefunctions in posttranscriptional processes (reviewed in Ref. 49).PTB is known to be a homodimer that belongs to a family ofRNA-binding proteins characterized by possession of at leastone RNA recognition motif (54) and was originally identified asa nuclear-localized splicing factor that binds to pyrimidine-richtracts within pre-mRNAs of a large number of genes (Refs. 46,47, and 55; reviewed in Ref. 49). PTB is also implicated in thecontrol of polyadenylation (56, 57), mRNA localization (58),and activates internal ribosome entry site-driven translation inpicornaviruses (59, 60). That PTB may also be involved in RNAstability is supported by results demonstrating that PTB binds toan element in the 3� UTR of GTPase-activating protein 43mRNA identified as an instability element (61). The fact thatPTB may be just one component of a larger complex isindicated by our finding that complex I is at least 150-kDa insize (K. Singh and L. R. Covey, unpublished data) and thepresence of a different, but related complex that binds theE5�-HaeIII transcript. PTB is expressed ubiquitously and asthree alternatively spliced isoforms in many different cells andtissue types (46, 48). We have previously shown that complexI only forms in T cells after extended activation. This suggeststhat either PTB is differentially expressed in activated T cells orthat other proteins in the complex may regulate the specificityof complex I function in T cell activation.

Our studies showing a differential stability program forCD154 mRNA at early and extended time of T cell activationsuggests a functional role for CD40 signaling at different stagesof the immune response. It has been recently reported that thetwo phases of CD154 expression are differentially regulated;the first stage being responsive to signaling through the TCRand the second phase being modulated by IL-4 and IL-12cytokines (62). We have yet to analyze CD154 mRNA stabilityunder these different conditions of stimulation. However, fur-ther characterization of the CD154-specific complexes and theirrole in message stability will allow us to identify how theseproteins function to regulate CD154 expression under differentphysiological conditions.

AcknowledgmentsWe thank Drs. Mike Kiledjian and Carol Wilusz for insightful discussionsand critical reading of this manuscript. We are grateful to Drs. WandaReynolds, Peter Kao, and James Patton for the generous gift of Abs and Dr.

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Jerry Langer for the pcDef3 vector. Also, we thank Dr. Zouran Wang andother members of the Kiledjian laboratory for their help and advice withthe in vitro decay assays. Finally, we acknowledge past and present mem-bers of the Covey laboratory for continual advice and helpful discussions.

References1. Kawabe, T., T. Naka, K. Yishida, T. Tanaka, H. Fujiwara, S. Suematsu,

N. Yoshida, T. Kishimoto, and H. Kikutani. 1994. The immune response inCD40-deficient mice: impaired immunoglobulin class switching and germinalcenter formation. Immunity 1:167.

2. Xu, J., T. M. Foy, J. D. Laman, E. A. Elliott, J. J. Dunn, T. J. Waldschmidt,J. Elsemore, R. J. Noelle, and R. A. Flavell. 1994. Mice deficient for the CD40ligand. Immunity 1:423.

3. Aruffo, A., M. Farrington, D. Hollenbaugh, X. Li, A. Milatovich, S. Nonoyama,J. Bajorath, L. S. Grosmaire, R. Stenkamp, M. Neubauer, et al. 1993. The CD40ligand, gp39, is defective in activated T cells from patients with X-linked im-munodeficiency with hyper-IgM. Cell 72:291.

4. Korthauer, U., D. Graf, H. W. Mages, F. Briere, M. Padayachee, S. Malcolm,A. G. Ugazio, L. D. Notarangelo, R. J. Levinsky, and R. A. Kroczek. 1993.Defective expression of T-cell CD40 ligand causes X-linked immunodeficiencywith hyper-IgM. Nature 361:539.

5. DiSanto, J. P., J. Y. Bonnefoy, J. F. Gauchat, A. Fischer, and G. de SaintBasile.1993. CD40 ligand mutations in X-linked immunodeficiency with hyper-IgM.Nature 361:541.

6. Allen, R. C., R. J. Armitage, M. E. Conley, H. Rosenblatt, N. A. Jenkins,N. G. Copeland, M. A. Bedell, S. Edelhoff, C. M. Disteche, D. K. Simoneaux, etal. 1993. CD40 ligand gene defects responsible for X-linked hyper-IgM syn-drome. Science 259:990.

7. Fuleihan, R., N. Ramesh, R. Loh, H. Jabara, R. S. Rosen, T. Chatila, S. M. Fu,I. Stamenkovic, and R. S. Geha. 1993. Defective expression of the CD40 ligandin X-chromosome-linked immunoglobulin deficiency with normal or elevatedIgM. Proc. Natl. Acad. Sci. USA 90:2170.

8. Durandy, A., C. Schiff, J. Y. Bonnefoy, M. Forveille, F. Rousset, G. Mazzei,M. Milili, and A. Fischer. 1993. Induction by anti-CD40 antibody or solubleCD40 ligand and cytokines of IgG, IgA, and IgE production by B cells frompatients with X-linked hyper IgM syndrome. Eur. J. Immunol. 23:2294.

9. Ferrari, S., S. Giliani, A. Insalaco, A. Al-Ghonaium, A. R. Soresina, M. Loubser,M. A. Avanzini, M. Marconi, R. Badolato, A. G. Ugazio, et al. 2001. Mutationsof CD40 gene cause an autosomal recessive form of immunodeficiency withhyper IgM. Proc. Natl. Acad. Sci. USA 98:12614.

10. Banchereau, J., F. Bazan, D. Blanchard, F. Briere, J. P. Galizzi, C. v. Kooten,Y. J. Liu, F. Rousset, and S. Saeland. 1994. The CD40 antigen and its ligand.Annu. Rev. Immunol. 12:881.

11. Foy, T. M., A. Aruffo, J. Bajorath, J. E. Buhlmann, and R. J. Noelle. 1996.Immune regulation by CD40 and its ligand GP39. Annu. Rev. Immunol. 14:591.

12. Noelle, R. J. 1996. CD40 and its ligand in host defense. Immunity 4:415.13. Armitage, R. J., W. C. Fanslow, L. Strockbine, T. A. Sato, K. N. Clifford,

B. M. Macduff, D. M. Anderson, S. D. Gimpel, T. Davis-Smith,C. R. Maliszewski, et al. 1992. Molecular and biological characterization of amurine ligand for CD40. Nature 357:80.

14. Castle, B. E., K. Kishimoto, C. Stearns, M. L. Brown, and M. R. Kehry. 1993.Regulation of expression of the ligand for CD40 on T helper lymphocytes. J. Im-munol. 151:1777.

15. Yellin, M. J., K. Sippel, G. Inghirami, L. R. Covey, J. J. Lee, J. Sinning,E. A. Clark, L. Chess, and S. Lederman. 1994. CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. J. Immunol. 152:598.

16. Lane, P., A. Traunecker, S. Inui, A. Lanzavecchia, and D. Gray. 1992. Activatedhuman T cells express a ligand for the human B cell-associated antigen CD40which participates in T cell-dependent activation of B lymphocytes. Eur. J. Im-munol. 22:2573.

17. Lederman, S., M. J. Yellin, A. Krichevsky, J. Belko, J. J. Lee, and L. Chess. 1992.Identification of a novel surface protein on activated CD4� T cells that inducescontact-dependent B cell differentiation (Help). J. Exp. Med. 175:1092.

18. de Boer, M., A. Dasran, J. Kwekkeboom, H. Walter, P. Vandenberghe, andJ. L. Ceupens. 1993. Ligation of B7 with CD28/CTLA-4 on T cells results inCD40 ligand expression, interkeukin-4 secretion and efficient help for antibodyproduction by B cells. Eur. J. Immunol. 23:3120.

19. Hermann, P., D. Blanchard, B. de Saint-Vis, F. Fossiez, C. Gaillard,B. Vanbervliet, F. Briere, J. Banchereau, and J.-P. Galizzi. 1993. Expression ofa 32-kDa ligand for the CD40 antigen on activated human T lymphocytes. Eur.J. Immunol. 23:961.

20. Klaus, S. J., L. M. Pinchuk, H. D. Ochs, C.-L. Law, W. C. Fanslow,R. J. Armitage, and E. A. Clark. 1994. Costimulation through CD28 enhances Tcell-dependent B cell activation via CD40-CD40L interaction. J. Immunol. 152:5643.

21. van Kooten, C., C. Gaillard, J.-P. Galizzi, P. Hermann, F. Fossiez, J. Banchereau,and D. Blanchard. 1994. B cells regulate expression of CD40 ligand on activatedT cells by lowering the mRNA level and through the release of soluble CD40.Eur. J. Immunol. 24:787.

22. Nusslein, H. G., K.-H. Frosch, W. Woith, P. Lane, J. R. Kalden, and B. Manger.1996. Increase of intracellular calcium is the essential signal for the expression ofCD40 ligand. Eur. J. Immunol. 26:846.

23. Ludewig, B., V. Henn, J. M. Schroder, D. Graf, and R. A. Kroczek. 1996. In-duction, regulation, and function of soluble TRAP (CD40 ligand) during inter-

action of primary CD4�CD45RA� T cells with dendritic cells. Eur. J. Immunol.26:3137.

24. Jaiswal, A. I., C. Dubey, S. L. Swain, and M. Croft. 1996. Regulation of CD40ligand expression on naive CD4 T cells: a role for TCR but not co-stimulatorysignals. Int. Immunol. 8:275.

25. Ford, G. S., B. Barnhart, S. Shone, and L. R. Covey. 1999. Regulation of CD154(CD40 ligand) mRNA stability during T cell activation. J. Immunol. 162:4037.

26. Ding, L., J. M. Green, C. B. Thompson, and E. M. Shevach. 1995. B7/CD28-dependent and -independent induction of CD40 ligand expression. J. Immunol.155:5124.

27. Johnson-Leger, C., J. Christensen, and G. G. Klaus. 1998. CD28 co-stimulationstabilizes the expression of the CD40 ligand on T cells. Int. Immunol. 10:1083.

28. Peng, X., J. E. Remacle, A. Kasran, D. Huylebroeck, and J. L. Ceuppens. 1998.IL-12 up-regulates CD40 ligand (CD154) expression on human T cells. J. Im-munol. 160:1166.

29. Skov, S., M. Bonyhadi, N. Odum, and J. A. Ledbetter. 2000. IL-2 and IL-15regulate CD154 expression on activated CD4 T cells. J. Immunol. 164:3500.

30. Randall, T. D., A. W. Heath, L. Satosargumedo, M. C. Howard, I. L. Weissman,and F. E. Lund. 1998. Arrest of B lymphocyte terminal differentiation by CD40signaling-mechanism for lack of antibody-secreting cells in germinal centers.Immunity 8:733.

31. Kwekkeboom, J., D. de Rijk, A. Kasran, S. Barcy, C. de Groot, and M. de boer.1994. Helper effector function of human T cells stimulated by anti-CD3 mAb canbe enhanced by co-stimulatory signals and is partially dependent on CD40-CD40ligand interaction. Eur. J. Immunol. 24:508.

32. Lindgren, H., K. Axcrona, and T. Leanderson. 2001. Regulation of transcriptionalactivity of the murine CD40 ligand promoter in response to signals through TCRand the costimulatory molecules CD28 and CD2. J. Immunol. 166:4578.

33. Parra, E., T. Mustelin, M. Dohlsten, and D. Mercola. 2001. Identification of aCD28 response element in the CD40 ligand promoter. J. Immunol. 166:2437.

34. Tsytsykova, A. V., E. N. Tsitsikov, and R. S. Geha. 1996. The CD40L promotercontains nuclear factor of activated T cells-binding motifs which require AP-1binding for activation of transcription. J. Biol. Chem. 271:3763.

35. Schubert, L. A., R. Q. Cron, A. M. Cleary, M. Brunner, A. Song, L. S. Lu,P. Jullien, A. M. Krensky, and D. B. Lewis. 2002. A T cell-specific enhancer ofthe human CD40 ligand gene. J. Biol. Chem. 277:7386.

36. Srahna, M., J. E. Remacle, K. Annamalai, S. Pype, D. Huylebroeck,M. A. Boogaerts, and P. Vandenberghe. 2001. NF-�B is involved in the regula-tion of CD154 (CD40 ligand) expression in primary human T cells. Clin. Exp.Immunol. 125:229.

37. Suarez, A., L. Mozo, A. Gayo, J. Zamorano, and C. Gutierrez. 1997. Requirementof a second signal via protein kinase C or protein kinase A for maximal expres-sion of CD40 ligand: involvement of transcriptional and posttranscriptionalmechanisms. Eur. J. Immunol. 27:2822.

38. Rigby, W. F., M. G. Waugh, and B. J. Hamilton. 1999. Characterization of RNAbinding proteins associated with CD40 ligand (CD154) mRNA turnover in hu-man T lymphocytes. J. Immunol. 163:4199.

39. Wilusz, C. J., M. Wormington, and S. W. Peltz. 2001. The cap-to-tail guide tomRNA turnover. Nat. Rev. Mol. Cell. Biol. 2:237.

40. Lindsten, T., C. H. June, J. A. Ledbetter, G. Stella, and C. B. Thompson. 1989.Regulation of lymphokine messenger RNA stability by a surface-mediated T cellactivation pathway. Science 244:339.

41. Chen, C.-Y., F. D. Gatto-Konczak, Z. Wu, and M. Karin. 1998. Stabilization ofinterleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science 280:1945.

42. Murakami, K., W. Ma, R. Fuleihan, and J. S. Pober. 1999. Human endothelialcells augment early CD40 ligand expression in activated CD4� T cells throughLFA-3-mediated stabilization of mRNA. J. Immunol. 163:2667.

43. Barnhart, B., P. A. Kosinski, Z. Wang, G. S. Ford, M. Kiledjian, and L. R. Covey.2000. Identification of a complex that binds to the CD154 3� untranslated region:implications for a role in message stability during T cell activation. J. Immunol.165:4478.

44. Yellin, M. J., J. J. Lee, L. Chess, and S. Lederman. 1991. A human CD4-leukemicsubclone with contact-dependent helper function. J. Immunol. 147:3389.

45. Didier, D. K., J. Schiffenbauer, S. L. Woulfe, M. Zacheis, and B. D. Schwartz.1988. Characterization of the cDNA encoding a protein binding to the majorhistocompatibility complex class II Y box. Proc. Natl. Acad. Sci. USA 85:7322.

46. Gil, A., P. A. Sharp, S. F. Jamison, and M. A. Garcia-Blanco. 1991. Character-ization of cDNAs encoding the polypyrimidine tract-binding protein. Genes Dev.5:1224.

47. Patton, J. G., S. A. Mayer, P. Tempst, and B. Nadal-Ginard. 1991. Characteriza-tion and molecular cloning of polypyrimidine tract-binding protein: a componentof a complex necessary for pre-mRNA splicing. Genes Dev. 5:1237.

48. Ghetti, A., S. Pinol-Roma, W. M. Michael, C. Morandi, and G. Dreyfuss. 1992.hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localizationand association with hnRNAs. Nucleic Acids Res. 20:3671.

49. Valcarcel, J., and F. Gebauer. 1997. Post-transcriptional regulation: the dawn ofPTB. Curr. Biol. 7:R705.

50. Langland, J. O., P. N. Kao, and B. L. Jacobs. 1999. Nuclear factor-90 of activatedT-cells: a double-stranded RNA-binding protein and substrate for the double-stranded RNA-dependent protein kinase, PKR. Biochemistry 38:6361.

51. Cunningham, K. S., R. E. Dodson, M. A. Nagel, D. J. Shapiro, andD. R. Schoenberg. 2000. Vigilin binding selectively inhibits cleavage of the vitel-logenin mRNA 3�-untranslated region by the mRNA endonuclease polysomalribonuclease 1. Proc. Natl. Acad. Sci. USA 97:12498.

987The Journal of Immunology

by guest on January 1, 2019http://w

ww

.jimm

unol.org/D

ownloaded from

52. Wang, Z., and M. Kiledjian. 2000. The poly(A)-binding protein and an mRNAstability protein jointly regulate an endoribonuclease activity. Mol. Cell. Biol.20:6334.

53. Wang, Z., and M. Kiledjian. 2000. Identification of an erythroid-enriched en-doribonuclease activity involved in specific mRNA cleavage. EMBO J. 19:295.

54. Perez, I., J. G. McAfee, and J. G. Patton. 1997. Multiple RRMs contribute toRNA binding specificity and affinity for polypyrimidine tract binding protein.Biochemistry 36:11881.

55. Garcia-Blanco, M. A., S. F. Jamison, and P. A. Sharp. 1989. Identification andpurification of a 62,000-dalton protein that binds specifically to the polypyrimi-dine tract of introns. Genes Dev. 3:1874.

56. Moreira, A., Y. Takagaki, S. Brackenridge, M. Wollerton, J. L. Manley, andN. J. Proudfoot. 1998. The upstream sequence element of the C2 complementpoly(A) signal activates mRNA 3� end formation by two distinct mechanisms.Genes Dev. 12:2522.

57. Lou, H., D. M. Helfman, R. F. Gagel, and S. M. Berget. 1999. Polypyrimidinetract-binding protein positively regulates inclusion of an alternative 3�-terminalexon. Mol. Cell. Biol. 19:78.

58. Cote, C. A., D. Gautreau, J. M. Denegre, T. L. Kress, N. A. Terry, andK. L. Mowry. 1999. A Xenopus protein related to hnRNP I has a role in cyto-plasmic RNA localization. Mol. Cell 4:431.

59. Kaminski, A., S. L. Hunt, J. G. Patton, and R. J. Jackson. 1995. Direct evidencethat polypyrimidine tract binding protein (PTB) is essential for internal initiationof translation of encephalomyocarditis virus RNA. RNA 1:924.

60. Hunt, S. L., and R. J. Jackson. 1999. Polypyrimidine-tract binding protein (PTB)is necessary, but not sufficient, for efficient internal initiation of translation ofhuman rhinovirus-2 RNA. RNA 5:344.

61. Irwin, N., V. Baekelandt, L. Goritchenko, and L. I. Benowitz. 1997. Identificationof two proteins that bind to a pyrimidine-rich sequence in the 3�-untranslatedregion of GAP-43 mRNA. Nucleic Acids Res. 25:1281.

62. Lee, B. O., L. Haynes, S. M. Eaton, S. L. Swain, and T. D. Randall. 2002. Thebiological outcome of CD40 signaling is dependent on the duration of CD40ligand expression: reciprocal regulation by interleukin (IL)-4 and IL-12. J. Exp.Med. 196:693.

988 PTB REGULATES CD154 mRNA STABILITY

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