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The Journal of Immunology, 2003, 170: 979-988.
Copyright © 2003 by The American Association of Immunologists

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

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3' untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed {Delta}1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the {Delta}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 nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interaction between CD40 expressed on B cells, macrophages, and other APCs with its ligand CD40 ligand (CD40L3; also designated CD154) expressed on activated CD4+ T cells has been shown to be a critical parameter in eliciting both humoral and cell-mediated immune responses. The absolute requirement for CD40 signaling in a wide range of immune responses has been demonstrated in animal models lacking either functional CD40 (1) or CD154 (2) and in humans suffering from both X-linked and CD40-associated hyper-IgM syndrome (hyper-IgM X-1 and hyper-IgM 3, respectively) (3, 4, 5, 6, 7, 8, 9). In these cases, there is clear absence of isotype switching in B cells, a lack of germinal centers, and deficient primary and secondary responses to thymus-dependent Ags. In addition to its role in the humoral immune response, a number of studies have revealed the importance of CD40 signaling in other immune functions including 1) the rescue from apoptosis of both Ag-induced and naive B cells, 2) the induction of peripheral tolerance, 3) the selection of self-reactive T cells, and 4) the activation of cellular immunity primarily through the interaction of activated CD4+ T cells with CD40-expressing macrophages (reviewed in Refs. 10, 11, 12).

Early studies examining the kinetics of CD154 expression showed rapid and transient surface expression on activated T cells (13, 14). The transient nature of CD154 expression on APC-activated T cells corresponded to the internalization of CD154 by CD40-expressing APCs through receptor-mediated endocytosis (15). However, multiple studies showed that the time course and extent of CD154 expression depended on the type of stimuli as well as on costimulatory interactions provided by B cells or APCs (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Specifically, studies from our laboratory and others revealed that CD154 is expressed on purified T cells for an extended period of time in response to anti-CD3 signaling either with or without costimulatory signals (18, 25, 26, 27) or in the presence of different cytokines (28, 29). Engagement of CD154 with CD40 on Ag-selected B cells initially induces B cells to proliferate and undergo differentiation into Ab-secreting cells (reviewed in Ref. 10). However, it has been shown that continued CD40 signaling in activated B cells prevents terminal differentiation as measured by a decrease in both secretory Ig and the transcriptional regulator B lymphocyte-induced maturation protein 1 (30). Together, these studies suggest that CD154 expression is critical for regulating the immune response at early and late times after activation. Furthermore, it is clear that CD154 expression is induced by the integration of signals from multiple pathways that ultimately regulate the functional response of the CD4+ T cell at a specific time in the ongoing immune response.

Although CD154 is regulated both temporally and with respect to cell type, the underlying mechanisms responsible for this control are just beginning to be defined. CD154 gene transcription is induced by TCR signaling and expression is enhanced in response to costimulatory signals (31, 32, 33). Transcriptional regulation appears to be dependent on NF-AT factor binding sites located in a region 5' of the TATA box and start sites (34, 35, 36) and a T cell-dependent enhancer element has been located 3' of the coding region that is NF-{kappa}B dependent (35). In addition to transcriptional regulation, we and others have shown that posttranscriptional regulation plays a critical role in modulating the steady-state level of CD154 mRNA (25, 37, 38). Specifically, CD154 transcripts are degraded in an activation-dependent manner (25). At early times after CD3-dependent signaling, CD154 mRNA is rapidly degraded (t1/2 < 40 min) which is a feature shared by numerous cytokine, growth factor, and cell cycle protein mRNAs including TNF-{alpha} (reviewed in Ref. 39). However, unlike the TNF-{alpha} (25, 40) or c-myc (40, 41) transcript which remains unstable in activated T cells, the stability of the CD154 mRNA shows an unusual pattern of "regulated instability" that corresponds to a 3- to 4-fold increase in stability after extended CD3 activation (25). The stability of the CD154 transcript is only marginally increased by costimulatory signals but is highly stabilized by activators of protein kinase C (25, 37, 38). In a separate study, it was demonstrated that human endothelial cells enhance the stability of CD154 message in PHA-stimulated human T cells through an LFA3-dependent mechanism (42), supporting the premise that posttranscriptional regulation is an important factor in CD154 expression.

To extend our initial observations, we used the Jurkat D1.1 T cell line, a line that constitutively expresses CD154 mRNA with a relatively stable half-life (t1/2 = 2.3 h), to identify a putative stability complex (termed complex I) (43). This complex bound specifically to a highly pyrimidine-rich region in the 3' untranslated region (UTR) and contained a poly(CU) RNA-binding protein that cross-linked only under conditions where the CD154 mRNA was highly stabilized. This was most clearly seen in comparing the binding patterns of extracts from resting, 2-h, 24-h, and 48-h anti-CD3-activated CD4+ T cells. With these extracts, complex formation and cross-linking were only observed in T cells stimulated for 48 h and, to a much lesser extent, in cells stimulated for 24 h. This work strongly suggested that complex I is directly involved in regulating the variable decay rate of CD154 mRNA during T cell activation.

In our current study, we have extended our analysis of the "stability" phase of CD154 mRNA decay and identified three different binding regions within the pyrimidine-rich region. We have identified one protein, polypyrimidine tract-binding protein (PTB) as a component of complex I. Finally, we have shown both by in vitro and in vivo assays, that complex I binding results in an increase in stability of the CD154 transcript.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Abs

The Jurkat D1.1 T cell line have been previously described (17, 44). Rabbit anti-human PTB antisera was a gift from Dr. J. Patton (Vanderbilt University, Nashville, TN). Anti-Y box-binding protein (YB) antisera was a gift 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 x 107 D1.1 cells were harvested, washed with PBS (pH 7.4), and lysed with 200 µl of lysis buffer (0.2% Nonidet P-40, 40 mM KCl, 10 mM HEPES (pH 7.9), 3 mM MgCl2, 5% glycerol, 1 mM DTT, 8 ng/ml aprotinin, and 0.5 mM PMSF) for 5 min. Lysates were centrifuged at 16,000 x g for 2 min and supernatants were stored at -80°C. The protein concentration of extracts was determined by the Bradford assay (Bio-Rad, Hercules, CA).

Deletion mutagenesis

The {Delta}E1'–E5', {Delta}1506, and the {Delta}1515 mutations were generated using the Quick Change Mutagenesis kit (Stratagene, La Jolla, CA). For {Delta}E1'–E5', the BamHI-BamHI fragment of CD154 containing the entire 3' UTR and downstream sequences was used as a template and amplified with primers 5' mutE1E5 (5'-cctctttcaatctctctctctccatctcctctagtctcttccctcccccagtctctcttctc-3') and 3' mutE1E5 (5'-gagaagagagactagggggagggaaagagactagagagagatgg agagagagagattgaaagag-3'). To generate the {Delta}1506, the above procedure was used except the {Delta}E1'–E5' plasmid and primers {Delta}1377–1506 (5'-cgccaccctctcggacagttattcattctccccctttctaacacacacacacacacacac-3') and {Delta}1506–1377 (5'-gtgtgtgtgtgtgtgtgtgttagaaagggggagaatgaataactgtccgagagggtggcg-3') were used. To generate the {Delta}1515 mutation, the {Delta}1506 template was used with primers mut1348–517 (5'-ggagaaccgaaacccccccccccccccccacacacacacacacacacacacacacacac-3') and mut1517–1348 (5'-gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgggtgggtgggtgggtgtttcggttctcc-3'). All reactions were conducted according to the manufacturer’s protocols.

RNA probes

The XbaI-E1', E1'–E5', E1'-BsrI and HaeIII-DraI templates were generated as previously described (43). The T7-XbaI-E1' template was PCR amplified with the following primers: 5'-cgtaatacgactcactatagggctagaacgtctaacacagtggaga-3' (forward) and 5'-tgaaagagagagatggagagagagagagagatt-3' (reverse). The T7-E5'-HaeIII template was synthesized using the following primers: 5'-cgtaatacgactcactatagggagtctcttccctcccccagtctctctt-3' (forward) and 5'-agagaactgactagcaacggcctg a-3' (reverse). The XbaI-HaeIII, {Delta}1506, and {Delta}1515 templates were synthesized using the corresponding CD154 3' UTR template (see above) with the following primers: XbaI-HaeIII, 5'-cgtaatacgacgcactatagggctagaacgtctaacacagtggaga-3' (forward), and 5'-agagaactgactagcaacggcctga-3' (reverse). PCR were prepared in 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 U Taq polymerase (Promega, Madison, WI). Cycling parameters were as follows: 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 {Delta}1515-poly(T) templates were synthesized as above except the 3' primer contained a 60 nt poly(T) sequence.

RNA probes were synthesized using 0.5 µg of the PCR DNA template; 0.4 mM each of rGTP, rATP, and rCTP; 0.04 mM rUTP; 30 mM DTT; 20 U of RNasin; 1x T7 transcription buffer (40 mM Tris-HCl (pH 7.9); 6 mM MgCl2; 2 mM spermidine; and 10 mM NaCl); 25–40 µCi of [{alpha}-32P]rUTP; and 4.25 U of T7 RNA polymerase (Promega) at 37°C for 1 h. Reactions were treated with DNase RQ1 for 15 min and centrifuged through G25 columns (Amersham Biosciences, Piscataway, NJ) to remove unincorporated nucleotides.

EMSA

Twenty-microliter reactions were prepared in EMSA binding buffer (40 mM KCl, 10 mM HEPES (pH 7.9), 3 mM MgCl2, 1 mM DTT, and 5% glycerol), 4 ng of Escherichia coli tRNA, 5–10 µg of D1.1 extract, and 4 x 104 cpm of in vitro-synthesized RNA probe. In supershift experiments, 1-µl Abs at a concentration of 1 µg/µl was added to the reaction for 1.5 h before addition of the probe. Following a 30-min incubation at room temperature, 2 µl of a RNase mix of either 40 U of RNase T1 and 100 pg of RNase A or 40 U of RNase T1, 100 pg of RNase A, and 0.015 U of RNase V1 was added, and the reactions were incubated for an additional 40 min at 37°C. After addition of 100 µg of heparin, the reactions were incubated for 10 min on ice and separated on a 7% native acrylamide gel in 0.25x Tris-borate-EDTA at 200 V for 2–4 h. In competition experiments, unlabeled RNA synthesized in vitro was diluted 1/25 in binding buffer (referred to as 1x). Increasing amounts of unlabeled transcript was added to the reaction before addition of the probe. In experiments using depleted extracts, 1.3 mg of D1.1 extract in 100 µl was incubated with 1–2 µl of 1 µg/µl Abs overnight at 4°C followed by addition of protein A-Sepharose. The extracts were centrifuged and the supernatant was depleted two additional times.

In vitro decay assays

Capped RNA transcripts were synthesized in vitro as described above with 624 µM Cap analog (Amersham Biosciences). Reactions were incubated at 37°C for 20 min and stopped with 100 µl ULB (7 M urea, 2% SDS, 0.35 M NaCl, 10 mM EDTA, and 10 mM Tris (pH 7.5)). Samples were electrophoresed through a 5% acrylamide (29/1)/7 M urea gel and the appropriate bands were excised from the gel and eluted with 150 µl of elution buffer (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 diethyl pyrocarbonate ddH2O.

Twenty-five-microliter reactions containing 50–100 µg of extract, 1 x 104 cpm in vitro-synthesized capped RNA, 1x IVD-1 buffer (100 mM KOAc, 2 mM MgOAc, 2 mM DTT, 10 mM creatine phosphate, 1 mM ATP, 0.4 mM GTP, 10 mM Tris (pH 7.5), and 0.1 mM spermine) were incubated at 37°C for indicated time points. To monitor degradation of the RNA within the reaction, 150 µl of ULB buffer containing 1 x 102 cpm of kinased oligonucleotide was added to the reaction. The reaction was extracted with phenol/chloroform, ethanol precipitated, and resuspended with 22 µl of 80% formamide/dye. The samples were boiled and 10 µl of each reaction was electrophoresed through a 5% acrylamide (29/1)/7 M urea gel 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 Medicine and Dentistry of New Jersey, Piscataway, NJ) and contains the human elongation factor 1{alpha} promoter. The poly(A)- pcDEF3 was constructed by digesting 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)- pcDEF3 construct. A 2.3-kb BamHI-BamHI fragment containing part of the 3' UTR and 1.5 kb of genomic sequence was subcloned from a phage clone containing the 3' region of the CD154 gene. A similar construct was made that contained the {Delta}1515 deletion. The mutDef3 and mut{Delta}1515Def3 constructs were engineered using the Erase-a-base kit (Promega) which introduced a 388-bp deletion into the coding region of the CD154 cDNA. This coding region replaced the wild-type coding region in both wtDef3 and {Delta}1515Def3.

Stable transfection

Jurkat D1.1 cells (5 x 106) were resuspended in 0.4 ml of RPMI 1640 and 25 µg of linearized construct DNA was introduced by electroporation using 250 mV, 960 µF. The pulsed cells were stored on ice for 10 min and incubated at 37°C in 5% CO2 for 36–48 h. To select for stable transfectants, cells were resuspended in medium containing 1 mg/ml G418.

5,6-dichloro-{alpha}-D-ribofuranosylbenzimidazole (DRB) treatment and RNA isolation

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

RT-PCR to analyze mRNA decay

Reverse transcription was conducted in a 30-µl volume with 3 µg of RNA, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 300 U 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 for 1 h followed by 10 min at 68°C.

PCR was performed using 2.5 µl of cDNA in a 40-µl reaction volume containing 10 mM Tris (pH 8.8), 1.5 mM MgCl2, 25 mM KCl, 0.25 mM dNTPs, 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 cycling conditions were 5 min at 92°C, then 25 cycles of 57°C for 1 min, 72°C for 1.5 min, and 92°C for 1 min, followed by 1 cycle of 72°C for 5 min. Ten microliters of each reaction was electrophoresed through a 1.5% agarose/Tris-borate-EDTA gel and bands were quantitated using the Kodak 1D analysis software.

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 U of RNasin, 20 µg of extract, and 2 x 104 cpm of in vitro-synthesized RNA probe were incubated for 15 min at room temperature. Proteins were UV cross-linked to probes by exposing the reactions with 254 nm UV light for 15 min at 0°C. RNase mix (2 µl; 40 U of RNase T1 and 100 pg of RNase A or 40 U of RNase T1, and 1 µg of RNase A) was added to the reaction and incubated for 30 min at 37°C. Samples were boiled for 5 min and electrophoresed through a 5% stacking gel/12% separating gel for 4.5 h at 30 mA. The gel was fixed in 10% acetic acid, dried, and visualized by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The region defined by {Delta}1515 completely lacks complex binding

Our previous work demonstrated that complex I binding to the CD154 transcript directly corresponds to an increase in stability of the message at extended times of T cell activation. Furthermore, we localized the complex I minimal binding site to a 63-nt stretch within the XbaI-HaeIII region in the 3' UTR (termed E1'–E5') (Fig. 1A). To extend these findings and determine whether complex I binding was required for CD154 mRNA stability, we designed experiments to directly test the effect of deleting the E1'–E5' region on CD154 mRNA decay. To this end, probes were generated that either retained (XbaI-HaeIII) or lacked ({Delta}E1'–E5'), the minimal binding sequence within the context of the XbaI to HaeIII sequence and conducted RNA-binding assays to detect complex I binding. Surprisingly, complex formation was obtained with the {Delta}E1'-E5' probe lacking the minimal binding site (Fig. 1B, lane 13) as well as with the control probes (Fig. 1B, lanes 11 and 12). In an attempt to comprehensively define the region of complex I binding, we synthesized two additional templates that lacked sequences between 1377 and 1506 and 1349 and 1515 (designated {Delta}1506 and {Delta}1515, respectively). These regions were chosen based on the fact that they lacked most ({Delta}1506) and all ({Delta}1515) of the CU-rich sequences in the XbaI-HaeIII region (see Fig. 1). Using the {Delta}1506 probe, we observed a slower migrating complex (Fig. 1B, lane 14), whereas no complex formed with the {Delta}1515 probe (Fig. 1B, lane 15). Because of the absence of complex formation with the {Delta}1515 probe, RNA containing this deletion was used in all subsequent experiments designed to define the relationship of complex I binding to CD154 mRNA stability.



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  		FIGURE 1. Analysis of the complex I cis-acting binding element in the CD154 3' UTR. A, Schematic of the CD154 mRNA with the 5' UTR, coding region, and the 988-nt 3' UTR indicated. The complete sequence of the XbaI to 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 µg of Jurkat D1.1 total cell extract (lanes 11–15) with 4 x 104 cpm of either probe XbaI-HaeIII (lanes 6 and 11), E1'–E5' (lanes 7 and 12), {Delta}E1'–E5' (lanes 8 and 13), {Delta}1506 (lanes 9 and 14), and {Delta}1515 (lanes 10 and 15). The individual probes are shown in lanes 1–5. Below the figure is a schematic of the in vitro synthesized probes used in RNA-binding studies: The E1'–E5' probe contains only the minimal binding region, the XbaI-HaeIII probe contains all sequences between the XbaI and HaeIII sites, the {Delta}E1'–E5' probe lacks the minimal binding region, the {Delta}1506 mutant contains the XbaI-HaeIII sequence with a deletion of the region between nt 1377 and 1506, and the {Delta}1515 probe consists of the XbaI-HaeIII region with a deletion of the sequence between 1349 and 1515.

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

We were intrigued by the finding that complex I binding occurred in the absence of the minimal E1'–E5' binding site. This result suggested that either complex I was able to form on sequences outside this region or that bringing two noncontiguous regions together through deletion created a new, but physiologically irrelevant binding site. To distinguish between these two possibilities, RNA-binding experiments were conducted with probes synthesized from templates that included sequences upstream and downstream of the E1'–E5' region (probes XbaI-E1' and E5'–HaeIII). We found that a complex migrating with the same mobility as complex I formed with the upstream XbaI-E1' probe and a faint, slower migrating complex bound to the downstream E5'-HaeIII probe (Fig. 2, arrows). To determine whether these two complexes were related to each other and/or to complex I, we set up RNA-binding experiments with capped and labeled XbaI-E1', E1'–E5', and E5'-HaeIII probes and competed complex formation with increasing concentrations of unlabeled self- and non-self RNA. As shown in Fig. 3, all three complexes were competed by cold self-RNA and to a large extent by non-self RNA as well (A–C, lanes 4–15). In contrast, there was very little competition for any of the three complexes with the irrelevant HaeIII-DraI RNA that has been previously shown to lack complex I binding (43) (Fig. 3, lanes 16–19). A consistent finding was that complexes were better competed by cold self than by non-self RNA, suggesting that the complexes were similar but not identical. This was particularly true for the slower migrating complex which bound to the E5'-HaeIII probe. Here, the complex was fully competed by 50-fold addition of competitor self-RNA whereas the same complex required the addition of 100-fold cold Xba-E1' and E1'–E5' RNA to be fully competed (Fig. 3C, lanes 4–15). These data strongly suggest that the three complexes binding within the XbaI-HaeIII region are related to each other and support the idea that at least three independent complex binding sites are adjacently located in the CD154 3' UTR.



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  		FIGURE 2. Complex formation occurs on at least three distinct sites within the XbaI-HaeIII region. RNA-binding assays were conducted with the 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 were incubated with RNase in the presence (lanes 3, 6, and 9 (E)) or absence (lanes 2, 5, and 8 (R)) of 5 µg of D1.1 total extract. Lanes 1, 4, and 7 show the probes alone (P) in the absence of RNase and extract. The sequence of the individual probes is shown below.

 


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  		FIGURE 3. Complexes formed within the XbaI-HaeIII region are related to each other. Competition assays were established with in vitro-synthesized, -labeled, and -capped RNA probes in the presence of increasing amounts of unlabeled and capped competitor RNA. Lanes 1-3 show the different labeled probes alone, with RNase in the absence of extract, and with RNase plus extract, respectively. Unlabeled transcript was either undiluted or diluted 1/25 in binding buffer and added to the different fractions such that the amount of cold probe increased from 1x to 25x, 50x, and 100x. Competition of the different labeled probes was conducted in the presence of increasing concentrations of cold XbaI-E1' (lanes 4–7), cold E1'–E5' (lanes 8–11), cold E5'-HaeIII (lanes 12–15), and cold irrelevant HaeIII-DraI RNA (lanes 16–19).

 
The complex I element confers stability on the CD154 transcript in vitro

To begin to decipher the role complex I plays in CD154 mRNA decay, we conducted in vitro decay assays using transcripts containing the XbaI-HaeIII region of the 3' UTR with or without the 1349–1515 sequences. In vitro-transcribed, -capped, and -polyadenylated XbaI-HaeIII (309 nt) and {Delta}1515 (135 nt) transcripts were incubated with D1.1 total extract over a 1-h time course. As shown in Fig. 4, the {Delta}1515 transcript was less stable than the XbaI-HaeIII wild-type transcript during the first 15 min of incubation, with ~25% loss during this time interval. In contrast, between 15 and 60 min, the {Delta}1515 transcript was significantly more stable than the XbaI-HaeIII transcript. To ensure that the smaller size of the {Delta}1515 transcript was not a factor in its instability during the first 15 min, we tested the E1'-BsrI transcript, a 79-nt RNA containing the E1'–E5' minimal binding site, and found it significantly more stable than either the XbaI-HaeIII or the {Delta}1515 transcript. One interpretation of these results is that complex I is acting as a stability factor by protecting a site that is a "hot spot" for nuclease attack.



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  		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), {Delta}1515 (lanes 5–8), and E1'-BsrI (lanes 9–12) probes and 50 µg of extract from Jurkat D1.1 cells. Percentage of the remaining RNA was determined after 60 min by comparing the band intensity to the intensity of the input RNA signal (designated as 100%). Internal control bands refer to a labeled oligonucleotide that was included in the stop buffer to normalize for RNA extractions and gel loading. B, The semilog graph depicts the averaged values and SEM for nine ({Delta}1515 and E1'-BsrI) and three (XbaI-HaeIII) independent experiments.

 
The complex I element confers stability on the CD154 transcript in vivo

Our in vitro results suggested that complex I binding was involved in regulating the decay pathway of CD154 mRNA. However, to address questions about the in vivo significance of complex I binding within the context of the complete CD154 mRNA, we introduced constructs containing the full-length CD154 cDNA, or the same region with the {Delta}1515 deletion, into Jurkat T cells. The CD154-specific sequences were subcloned into the pcDEF3 vector (designated wtDEF3 and {Delta}1515DEF3) in which transcription is under the control of the human elongation factor 1{alpha} promoter and is active in Jurkat T cells independent of cell activation (B. Barnhart and L. R. Covey, unpublished observations). To ensure that regulatory elements within the vector were not influencing the decay rate of the transcribed RNA, the bovine papilloma virus poly(A) signals were removed from the pcDEF3 construct and replaced with the CD154 poly(A) site plus 1000 bp of downstream sequence (Fig. 5A). Additionally, since one specific objective was to introduce the constructs into the CD154+ D1.1 Jurkat T cell line, the constructs were engineered with a 388-bp deletion in the coding region that allows discrimination between transcripts originating from the construct and endogenously expressed CD154 transcripts. The new constructs were termed mutDef3 and mut{Delta}1515Def3 and were introduced by stable transfection into the Jurkat D1.1 cell line. Individual mutDef3 and mut{Delta}1515Def3 subclones were screened by RNase protection assays to identify those expressing the construct wild-type and {Delta}1515 RNAs at similar levels (data not shown). Furthermore, preliminary PCR were conducted with varying amounts of cDNA to ensure that amplification was taking place within a linear range of the assay (data not shown). RNA decay was measured by incubating the cells with the RNA polymerase II inhibitor DRB over a 6-h time course.



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  		FIGURE 5. The complex I binding region increases the stability of the CD154 mRNA in vivo. A, Schematic of CD154 full-length and {Delta}1515 regions, including the 388-bp coding region deletion, subcloned into the pcDEF3 vector to give the mutDef3 and mut{Delta}1515Def3 constructs. The insert contains the 1800-bp CD154 cDNA sequence plus a 1000-bp genomic sequence which includes the CD154 poly(A) addition site and 950 bp of additional downstream sequence. The {Delta}1515 construct lacks the 166 bp denoted by an inverted triangle. The arrows indicate the relative location of the primers used to amplify the construct-specific transcripts. B, Shown is a representative PCR experiment which analyzed the decay pattern of CD154 mRNA in DRB-treated Jurkat D1.1 subclones containing either the mutDef3 or mut{Delta}1515Def3 constructs. RT-PCR were conducted with RNA from cells incubated with DRB for 0 h (lanes 1 and 6), 1 h (lanes 2 and 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 range of amplification. Primers specific to CD154 were used to amplify the fragment spanning the coding region deletion shown in A and GAPDH-specific primers were also included in the reaction to control for differences in loading. C, The semilog graph represents the results of in vivo DRB experiments using the Jurkat D1.1 cells expressing either the mutDef3 ({blacksquare}) or mut{Delta}1515Def3 (•) constructs. The fraction of mRNA remaining at each time point was quantitated and was normalized relative to the GAPDH band. The values represent the average of three independent experiments ± SEM.

 
RNA was isolated and RT-PCR was conducted using primers that hybridized to either side of the coding region deletion and gave an 839-bp and 451-bp fragment for the endogenous and construct-specific transcripts, respectively. To control for equal RNA amounts and loading errors, a 400-bp GAPDH fragment was amplified in all samples. As shown in Fig. 5, B and C, the mutDef3 transcript was more stable than the mut{Delta}1515Def3 transcript over the time course of DRB inhibition. This difference in stability was directly related to the {Delta}1515 deletion since the two transcripts were identical except for this deletion. The difference in decay rate between the two transcripts was greatest at the early time points after transcription inhibition, which corresponds to what was observed in vitro. Also, since the level of construct-specific expression was very similar between the two cell lines, specific kinetics of transcript decay in each line was not caused by measurable differences in the substrate to nuclease ratio. These data identify complex I binding as being a factor in regulating CD154 transcript stability in vivo.

A 55-kDa protein binds to three independent sites within the CD154 3' UTR

To begin to identify proteins that were part of the stability complex, we initiated UV cross-linking studies using our different RNA probes and Jurkat D1.1 extract. We previously demonstrated that a 90-kDa protein cross-linked to the minimal binding site in both Jurkat and late-activated CD4+ T cell extracts (43). However, we consistently observed nonspecific bands in these binding reactions, indicating that the RNase treatment after UV exposure was too mild. To view the cross-linking in the absence of the nonspecific bands, we instituted a more stringent RNase A treatment which included a 10,000-fold increase after UV exposure. Using these conditions, we no longer observed a 90-kDa protein but found that a protein of ~55 kDa bound to the XbaI-HaeIII and E1'–E5' probes but not to the {Delta}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 {Delta}1515 probe. All three bands were competed with an excess of poly(dCT) and cold E1'–E5' RNA but were not competed with the nonspecific competitor (data not shown).



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  		FIGURE 6. Identification of a 55-kDa protein that cross-links to the complex 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 µg of Jurkat D1.1 total cell extract and 1 x 104 cpm of uniformly labeled XbaI-HaeIII (lanes 1 and 4), E1'–E5' (lanes 2 and 5), or {Delta}1515 (lanes 3 and 6) transcripts with 40 U of RNase T1 and 1 µg of RNase A. Position and molecular mass (kDa) of protein standards are indicated. The arrow notes the 55-kDa protein. B, UV cross-linking was conducted as in A with uniformly 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 that is present with all three probes.

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

PTB (heterogeneous nuclear ribonucleoprotein I (hnRNPI) binds to the CD154 stability region in the 3' UTR

Before initiating protein purification experiments to identify the 55-kDa protein, we wanted to establish whether it was a known poly(CU)-binding protein. Two poly(CU)-binding proteins that were approximately the correct size were the human YB-1 (45) and the PTB (also called hnRNPI) (46, 47, 48). It has previously been reported that YB-1 binds to a pyrimidine-rich element that is involved in c-Jun N-terminal-mediated stability of the IL-2 mRNA in activated Jurkat T cells (41). Furthermore, PTB binds to pyrimidine-rich stretches in RNA and appears to have multiple functions in splicing as well as in other posttranscriptional processes (reviewed in Ref. 49). Thus, both proteins appeared to be candidates 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 three rounds of depletion with either Abs against PTB, YB-1, or NF90 (a dsRNA-binding protein initially identified in T cells (50)). Our results demonstrated that compared with the undepleted extract, there was no effect on the intensity of binding with extract depleted of NF90 and a modest decrease in binding with the YB-1-depleted extract (Fig. 7A). In contrast, we observed a marked decrease in complex binding with extract that was depleted of PTB. We checked the depleted extract for the presence of the different proteins and were unable to detect either PTB or NF90 by Western blot analysis, demonstrating that the depletion had been complete. Unfortunately, we could not conclusively determine whether YB-1 was completely removed from the depleted extract since its molecular 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.



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  		FIGURE 7. Identification of PTB as the 55-kDa protein that binds to the complex I binding site. A,. RNA EMSA was conducted with the uniformly labeled E1'–E5' probe and 5 µg of either Jurkat D1.1 total extract (lane 3) or extract 3x depleted with either anti-PTB antisera (lane 4), anti-YB-1 antisera (lane 5), or anti-NF90 antisera (lane 6). Lane 1 shows probe alone and lane 2 shows probe with RNase in the absence of extract. B, Western blot analysis of 50 µg of D1.1 extract before and after 3x depletion with specific Abs. The large dark band in the YB-1- and PTB-depleted extracts is 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 µg of Jurkat D1.1 total extract (lanes 3–5). One microliter of 1 µg/µl anti-YB-1 (lane 4) and anti-PTB (lane 5) was added to the reaction before incubation with the probe. Lane 1 is probe in the absence of extract and RNase and lane 2 is probe with RNase in the absence of extract.

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


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have extended our previous studies of CD154 mRNA stability by presenting data on the nature of both the binding site and protein composition of complex I. Previously, we defined the E1' to E5' minimal binding site for complex I as a 63-nt region located in the 3' UTR at nt 1411–1473. In our present study, we were surprised to find that the E1'–E5' sequence could be deleted from the XbaI-HaeIII region and complex I still formed on the remaining sequences. This observation led to the identification of two additional CU-rich binding regions within the XbaI-HaeIII region. All three regions were found by competition experiments to bind related complexes although the relationship between the different complexes has not been fully determined. The similarity of the complexes is reinforced by the fact that all three have as an RNA-binding component a 55-kDa protein whose identification is consistent with PTB, or hnRNP-I. It is possible that the larger E5'-HaeIII complex is comprised of complex I plus additional proteins as suggested from the faint band that migrates with the same mobility as complex I. Additional experiments are in progress to address this possibility.

Results from our in vitro decay assays suggest that complex I binding plays a role in regulating the turnover of CD154 mRNA. An interesting observation from these experiments is that both the XbaI-HaeIII and {Delta}1515 transcripts decay with unique second order kinetics throughout the 60-min time course. The rapid decay of the {Delta}1515 transcript in the first 15 min, vs relative stability of the XbaI-HaeIII during this interval, suggests that complex I facilitates the formation of a nuclease-resistant structure. However, once the complex is removed there is rapid degradation of the XbaI-HaeIII but not the {Delta}1515 RNA. This observation supports the idea that a major factor driving the rapid kinetics of the XbaI-HaeIII RNA are the sequences bound by complex I. That the E1'-BsrI transcript is highly stable under the same conditions would implicate sequences outside E1'-BsrI, but within the adjacent CU-rich sequences, for targeting nuclease activity. This type of model is reminiscent of recent examples of endonuclease-catalyzed decay which is regulated by RNA-binding proteins interacting with cis-acting mRNA stability elements. For example, the vigilin protein binds to the vitellogenin mRNA 3' UTR and inhibits transcript cleavage by the polysomal ribonuclease 1 endonuclease (51). Also, it has been recently shown that the {alpha} complex, which forms on a specific site in the 3' UTR of {alpha}-globin and confers transcript stability, masks a unique cleavage site that is recognized by an erythroid-enriched endonuclease (termed ErEN) (52, 53). Although we did not observe specific cleavage intermediates under our in vitro conditions, it is possible that slowing the reaction rate would allow for detection of these species. Alternatively, once complex I is removed, exonuclease may act more aggressively on the XbaI-HaeIII transcript vs the {Delta}1515 transcript. Further experimentation will allow us to test these different models.

In accordance with our in vitro data, our in vivo results support a role for complex I binding notably in the early hours of the time course. The delay in the overall decay of both the mutDef3 or mut{Delta}1515Def3 RNA, compared with the smaller transcripts in vitro, could be explained by the significantly different sizes of the transcripts as well as a cell environment that may limit exposure to nuclease. Based on our in vitro findings, it is quite possible that by removing complex I from the transfected cell instead of removing its binding site from the transcript would measurably affect the decay rate of the CD154 transcript in vivo.

Our finding that PTB is a major component of complex I is in agreement with others showing that PTB carries out diverse functions in posttranscriptional processes (reviewed in Ref. 49). PTB is known to be a homodimer that belongs to a family of RNA-binding proteins characterized by possession of at least one RNA recognition motif (54) and was originally identified as a nuclear-localized splicing factor that binds to pyrimidine-rich tracts within pre-mRNAs of a large number of genes (Refs. 46, 47, 55 ; reviewed in Ref. 49). PTB is also implicated in the control of polyadenylation (56, 57), mRNA localization (58), and activates internal ribosome entry site-driven translation in picornaviruses (59, 60). That PTB may also be involved in RNA stability is supported by results demonstrating that PTB binds to an element in the 3' UTR of GTPase-activating protein 43 mRNA identified as an instability element (61). The fact that PTB may be just one component of a larger complex is indicated by our finding that complex I is at least 150-kDa in size (K. Singh and L. R. Covey, unpublished data) and the presence of a different, but related complex that binds the E5'-HaeIII transcript. PTB is expressed ubiquitously and as three alternatively spliced isoforms in many different cells and tissue types (46, 48). We have previously shown that complex I only forms in T cells after extended activation. This suggests that either PTB is differentially expressed in activated T cells or that other proteins in the complex may regulate the specificity of complex I function in T cell activation.

Our studies showing a differential stability program for CD154 mRNA at early and extended time of T cell activation suggests a functional role for CD40 signaling at different stages of the immune response. It has been recently reported that the two phases of CD154 expression are differentially regulated; the first stage being responsive to signaling through the TCR and the second phase being modulated by IL-4 and IL-12 cytokines (62). We have yet to analyze CD154 mRNA stability under these different conditions of stimulation. However, further characterization of the CD154-specific complexes and their role in message stability will allow us to identify how these proteins function to regulate CD154 expression under different physiological conditions.


   Acknowledgments
 
We thank Drs. Mike Kiledjian and Carol Wilusz for insightful discussions and critical reading of this manuscript. We are grateful to Drs. Wanda Reynolds, Peter Kao, and James Patton for the generous gift of Abs and Dr. Jerry Langer for the pcDef3 vector. Also, we thank Dr. Zouran Wang and other members of the Kiledjian laboratory for their help and advice with the in vitro decay assays. Finally, we acknowledge past and present members of the Covey laboratory for continual advice and helpful discussions.


   Footnotes
 
1 This work was supported by grants from the Charles and Joanna Busch Biomedical Foundation (Rutgers University) and the American Heart Association (Grant 0051487T) to L.R.C. Back

2 Address correspondence and reprint requests to Dr. Lori R. Covey, Department of Cell Biology and Neuroscience, Rutgers, State University of New Jersey, 604 Allison Road, Piscataway, NJ 08854. E-mail address: covey{at}biology.rutgers.edu Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; UTR, untranslated region; PTB, polypyrimidine tract-binding protein; NF90, nuclear factor 90; DRB, 5,6-dichloro-{alpha}-D-ribofuranosylbenzimidazole; hnRNPI, heterogeneous nuclear ribonucleoprotein; YB-1, Y box-binding protein 1. Back

Received for publication September 16, 2002. Accepted for publication November 13, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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