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The Journal of Immunology, 2000, 164: 3659-3665.
Copyright © 2000 by The American Association of Immunologists

Correct Immunoglobulin {alpha} mRNA Processing Depends on Specific Sequence in the C{alpha}3-{alpha}M Intron1

John H. Coyle and Deborah A. Lebman2

Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The maturation of IgM-expressing B cells to IgM-secreting plasma cells is associated with both an increase in µ mRNA and the ratio of secreted to membrane forms of µ mRNA which differ at the 3' termini. In contrast, both in vitro and in vivo the secreted form of {alpha} mRNA is predominant at all stages in the development of a secretory IgA response. Previous studies demonstrated that preferential usage of the {alpha}s poly(A) site does not result from transcription termination and is independent of either the poly(A) sites or the 3' splice site associated with the exon encoding the membrane exon of IgA ({alpha}M). The present study demonstrates that a 349-bp region located 774 bp 3' to the {alpha}s poly(A) site is required for the preferential usage of the {alpha}s terminus. This region, which is the first isotype-specific cis-acting regulatory sequence not immediately adjacent to a secretory poly(A) site to be identified, contains regulatory elements that increase the efficiency of polyadenylation/cleavage. A ubiquitous, ~58-kDa RNA-binding protein interacts specifically with this regulatory region. These studies support the premise that cis-acting elements unique to each CH gene can impinge upon a common mechanism regulating Ig mRNA processing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The maturation of B cells to Ig-secreting plasma cells is associated with an increase in both the steady-state level of Ig mRNA and the ratio of secreted (s)3 to membrane (m) form of Ig mRNA which differ in 3' termini (1). All CH genes are structurally similar and form mIg mRNAs by splicing the exon encoding the transmembrane region and the associated poly(A) site to the terminal Ig domain. The sIg mRNAs employ a proximal, intronic poly(A) signal immediately 3' to the terminal Ig domain exon (2, 3). Although some studies have argued that the increased ratio of s:m mRNA accompanying B cell maturation results from transcription termination (4, 5, 6), the majority of studies favor a model in which choice of 3' terminus is regulated at the level of mRNA processing (7, 8, 9, 10). Attempts to establish whether the competing RNA processing reactions are polyadenylation/cleavage at the membrane vs secreted poly(A) sites or polyadenylation/cleavage at the secreted poly(A) site vs splicing of the membrane exon have yielded conflicting results (7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19). Regardless, the observation that the maturation of B cells to plasma cells is associated with an increase in the efficiency of polyadenylation/cleavage reactions but not splicing suggests that polyadenylation/cleavage at the proximal site is the regulated process (8).

Although the overall mechanism of regulation is the same for all CH genes, elements within each CH gene appear to contribute to determining the ratio of s:m mRNA at different stages in B cell development (10, 18, 19, 20, 21). For example, in cultures of B cells stimulated with LPS and TGF-ß, {alpha}s mRNA predominates regardless of the ratio of µs:µm mRNA (22). Similarly, {epsilon}s mRNA predominates at all time points in cultures of B cells stimulated with LPS and IL-4 (23). The observation that the ratios of µs:µm and {alpha}s:{alpha}m are 1:2 and >20:1, respectively, in an IgM-expressing B cell line transfected with C{alpha} argues that the isotype-specific differences in ratio of s:m mRNAs reflect differences in mRNA processing and not differences in the populations induced to isotype switch (10). One possible explanation for isotype-specific regulation is the diversity in secreted poly(A) signals. All C{gamma}s have a 13-bp GU-rich sequence located 33 bp 3' to the AAUAAA signal for the secreted terminus. The same consensus sequence is located 16 bp 3' to the AAUAAA for the µs poly(A) site (24). In addition, unlike {gamma}s poly(A) sites, the µs AAUAAA signal is embedded in an AU-rich region and is associated with a second GU-rich region (20). All of these features of the µs poly(A) site have been shown to be involved in proper polyadenylation/cleavage (20), and the 13-bp consensus sequence has also been shown to be involved in increased usage of {gamma}s poly(A) sites in plasma cells (18, 19). Interestingly, the {alpha}s poly(A) site lacks the GU-rich consensus sequence (20, 24). The observation that preferential usage of the proximal terminus in the context of C{alpha} is independent of the termini indicates that cis-acting elements that are not associated with the poly(A) signal can be involved in regulation of Ig mRNA processing (21).

The role of cis-acting elements in the intron between the exons encoding the terminal CH domain and the membrane region is controversial. It has been shown that regulation of 3' terminus usage by µ mRNAs is affected by the length of the Cµ4-µM intron such that decreasing the length of the intron favors usage of the µm terminus (7, 25). Although these studies eliminated the role of one specific region of the intron in regulation, they did not completely rule out a regulatory role for specific sequences in that region. In the same vein, it has been argued that the preferential usage of the {alpha}s poly(A) site reflects the fact that the C{alpha}3-{alpha}M intron is longer than the Cµ4-µM intron (26). The present study demonstrates that specific sequence in the C{alpha}3-{alpha}M intron is required for the predominant usage of the {alpha}s poly(A) site. This region specifically binds an ~58-kDa protein and increases the efficiency of polyadenylation/cleavage at the {alpha}s poly(A) site. These findings demonstrate that isotype-specific cis-acting elements play a role in regulating 3' terminus usage in Ig mRNAs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction

The expression vector SR{alpha} 296pA-, which contains the SR{alpha} promoter (SV40 early promoter plus R-U5 segment of HTLV-1 long terminal repeat), was prepared from pcDL-SR{alpha}296 as described (10). The preparation of SR{alpha}C{alpha}, which contains a 4.5-kb EcoRI fragment of C{alpha} including one-half of C{alpha}2, C{alpha}3, {alpha}M, and the first poly(A) site was also described previously (10). SR{alpha}C{alpha} I-3, which lacks the C{alpha}3-{alpha}M intron sequence from the SmaI to the XbaI restriction enzyme sites was prepared as described previously (21). SR{alpha}C{alpha} I-3 sub was created as follows. The EcoRI-BanI fragment from p{alpha}4.5, which includes one-half of C{alpha}2, C{alpha}3, and part of the C{alpha}3-{alpha}M intron, was isolated with a blunt end created at the XbaI site by treatment with the Klenow fragment of DNA polymerase (Promega, Madison, WI). This fragment was ligated into pBSK+ restriction enzyme digested with EcoRI and SmaI to create pBSK+ I-3. Next, the EcoRI-HaeII fragment from pBSK+ I-3 containing C{alpha} and 317 bp of pBSK+ was isolated with a blunt end created at the HaeII site by the Klenow fragment of DNA polymerase (Promega) and ligated to pBS- restriction enzyme digested with EcoRI and SmaI to generate pBS I-3 sub. An EcoRI-HincII fragment containing the C{alpha} fragment and 334 additional bases, 17 of which were from pBS-, was isolated from pBS I-3 sub. The {alpha}M region was isolated from pBC{alpha}M as a 1713-bp XbaI-EcoRI fragment with a blunt end created at the XbaI restriction enzyme site. Finally, SR{alpha}C{alpha} I-3 sub was generated by ligating the pBS I-3 sub fragment and the {alpha}M fragment to SR{alpha} 296pA- restriction enzyme digested with EcoRI. SR{alpha}C{alpha} H3/ScaI sub was generated by substitution of the HindIII-ScaI region in the C{alpha}3-{alpha}M intron with 430 bp of pBS-. The C{alpha} fragment between the restriction enzyme sites EcoRI and HindIII, which contains one-half of C{alpha}2, C{alpha}3, and the {alpha}s poly(A) signal along with 430 bases from the pBS- plasmid, was amplified from pBC{alpha}s by PCR. The 5' primer (gatctattgtaatacg actcactatagggc) contained an EcoRI site and the 3' primer (gatctatcgatttac ggttcctggccttt) contained a ClaI site. The C{alpha}M sequence was prepared by PCR amplification of pBC{alpha}M. The 5' (gatctatcgatctg agtggtgggtggtgtg) primer contained a ClaI restriction enzyme site and the 3' primer (gatcttgaagggga agatactgttgacggg) contained an EcoRI restriction enzyme site. Following digestion with EcoRI and ClaI, the PCR amplicons were isolated and ligated to SR{alpha}296 pA- digested with EcoRI to generate SR{alpha}C{alpha} H3/ScaI sub. The luciferase expression plasmids were generated as follows. The minimal {alpha}s poly(A) site was isolated from pBSK 2.75 as a 181-bp DpnI-HindIII fragment which contains 61 bp 5' to the {alpha}s AATAAA hexamer and 120 bp 3' to the {alpha}s AATAAA hexamer. The {alpha}s DpnI-HindIII fragment was ligated into pBSK+ digested with the restriction enzymes SmaI and HindIII to generate pBSK{alpha}s. The XbaI-SalI fragment from pBSK{alpha}s was isolated and ligated into the pGL3 promoter plasmid (Promega) digested with XbaI and SalI. pGL3{alpha}sSca/Ban and pGL3{alpha}sBan/Sma were prepared as follows. Both the ScaI-BanI fragment and the BanI-SmaI fragment from the C{alpha}3-{alpha}M intron were PCR amplified from p{alpha}4.5. The 5' primer for both fragments contained a HindIII restriction enzyme site and the 3' primers for both fragments contained a SalI restriction enzyme site. Amplicons were digested with HindIII and SalI and isolated by electrophoresis on agarose gels. To generate pGL3{alpha}sSca/Ban and pGL3{alpha}sBan/Sma, a trimolecular ligation was performed with the HindIII-SalI PCR fragment, the XbaI-SalI fragment from pBSK{alpha}s, and the pGL3 promoter plasmid (Promega) digested with XbaI and SalI. To create pBS I-3, the 349-bp BanI-SmaI fragment was isolated from p{alpha}4.5 with a blunt end at the BanI restriction enzyme site and ligated into pBS- digested with SmaI. pBS I-3 140–349 was generated by PCR amplification of bp 140–349 of the I-3 region. The 5' primer (gatctgaattcggtg ccaagacatataaca) contained an EcoRI site and the 3' primer (gatcttctagaccggg ggcttggagaagccc) contained an XbaI site. Following digestion with EcoRI and XbaI restriction enzymes, the PCR-amplified fragments were ligated to pBS- digested with EcoRI and XbaI. To generate pBS{alpha}M34, an XbaI-EcoRI fragment from 3AE9.3 (provided by Dr. Barbara Birshtein, Albert Einstein College of Medicine, Bronx, NY) which includes the third and fourth {alpha}M poly(A) sites was isolated and ligated to pBS- digested with XbaI and EcoRI.

Analysis of RNA-binding proteins

Linearized templates for in vitro transcription of the 349-bp, I-3 sequence, or {alpha}M34 sequence were prepared by digestion of pBS I-3 or pBS{alpha}M34 with XbaI. The template for bp 1–128 in the 349-bp region was prepared by digestion of pBS I-3 with BsteII. The template for bp 140–349 was prepared by digestion of pBS I-3 140–349 with XbaI. Radiolabeled cRNA probes were prepared from linearized templates using T7 polymerase, 50 µCi [{alpha}-32P]UTP (800 Ci/mmol; ICN Pharmaceuticals, Costa Mesa, CA), and 0.4 mM unlabeled nucleotides. Radiolabeled cRNAs were purified on 8% polyacrylamide gels as described (27). Nuclear extracts of BCL1 cells were prepared essentially as described (28). Cells were harvested, washed once in HBSS, and resuspended at 0.5–1 x 108 cells/ml in lysis buffer (10 mM Tris-HCl (pH 7.4), 3 mM CaCl2, 5 mM MgCl2, 0.1% Nonidet P-40). Cells were lysed in a Dounce homogenizer by five strokes with a type B pestle and centrifuged for 10 min at 4°C at 15,000 rpm. Pellets were resuspended in 1 ml lysis buffer, centrifuged for 30 min at 4°C at 15,000 rpm, resuspended in 30 µl of buffer (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT, and 0.5 mM PMSF), and lysed in a Dounce homogenizer by 10 strokes. The solution was mixed with magnetic triangular fleas at 4°C for 30 min, centrifuged for 30 min at 15,000 rpm, and dialyzed for 5–8 h in buffer (20 mM HEPES (pH 7.9), 25% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). Protein extract concentrations were determined by a colorimetric assay (Bio-Rad, Hercules, CA). Splicing competent HeLa cell nuclear extract was kindly provided by Dr. James Roesser (Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University). Binding reactions consisted of 300,000 cpm of 32P-labeled cRNA, 20 µg of nuclear extract, 40 U of RNasin, 5% glycerol, 10 mM HEPES (pH 7.6), 3 mM MgCl2, and 1 mM EDTA in a final volume of 10 µl. Samples were incubated on ice or at 30°C for a total of 30 min. After a 20-min incubation, heparin was added at 5 mg/ml. UV-cross-linking/label transfer analysis was performed as described elsewhere (29). Finally, samples were treated with 10 U RNase T1 and 10 U RNase One (Promega) for 30 min at 37°C, denatured at 100°C in loading buffer, and analyzed by SDS-PAGE as described previously (29). Gels were analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Cell transfection

Before transfection, cell lines were washed twice and resuspended at 2 x 107 cells/ml in complete medium (RPMI 1640, 10% newborn calf serum, L-glutamine, penicillin/streptomycin, and 50 µM 2-ME) containing 10 mM HEPES. A total of 4 x 106 cells was placed in each cuvette with plasmid DNA and electroporated using a Gene-Pulser with a capacitance extender (Bio-Rad). The optimal electroporation conditions for different cell lines were predetermined. Clones of stable transfectants of the BCL1 lymphoma were established as described previously (21). Because <10% of the wells were positive, there was a >90% probability that individual positive wells were clonal in origin as determined by Poisson analysis. Pooled stables were generated by expansion of the total transfected cell populations in complete medium with 1 mg/ml geneticin (Sigma, St. Louis, MO).

Luciferase expression analysis

Cells were transfected with a total of 30 µg of plasmid DNA. Firefly luciferase (test) plasmid and Renilla luciferase (control) plasmid (Promega) were used at molar ratios between 30:1 and 40:1. Transfected cells were harvested after a 36-h incubation and lysed in 250 µl of passive lysis buffer (Dual Luciferase System; Promega) at room temperature for 15 min. Whole-cell lysates were cleared by centrifugation for 5 min at room temperature. Luminescence was detected in the Chrono-Log Lumi-Vette Luminometer (Chrono-Log, Havertown, PA). Both firefly and Renilla luciferase activity were analyzed in a single cuvette. Firefly luciferase luminescence was detected after the addition of 100 µl of Luciferase Reagent II (Promega) to 20 µl of cleared lysate. Renilla luciferase luminescence was then detected after the addition of 100 µl of Stop-and-Glo substrate (Dual Luciferase System; Promega). Each reading was taken after substrate addition, a 2-s delay, and a 10-s integration. Two separate readings were taken for each sample. The reported data represent readings from five separate transfections. Student’s t test was used to determine the significance of the effect of the I-3 region.

RNA isolation and analysis

Total cellular RNA was isolated using Ultraspec (Biotecx Laboratories, Houston, TX). RNA was quantitated spectrophotometrically, and S1 nuclease analysis was performed as described previously (22). Briefly, the 900-bp BstEII fragment of the plasmid p{alpha}4.5 was end labeled and hybridized with the RNA sample. After hybridization, the samples were digested with S1 nuclease and precipitated with ethanol. The protected fragments were analyzed on 8% polyacrylamide/7 M urea gels. The S1 gels were analyzed using ImageQuant software and a PhosphorImager (Molecular Dynamics).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A 349-bp region in the C{alpha}3-{alpha}M intron is required for preferential usage of the {alpha}s poly(A) site

Previous studies demonstrated that the preferential usage of the proximal poly(A) site in {alpha} mRNAs is independent of the 3' termini. However, analysis of the effect of sequential deletions of regions of the C{alpha}3-{alpha}M intron on the ratio of {alpha}s:{alpha}m indicated that it does depend on either a 349-bp BanI-SmaI region (I-3) or a minimal distance between the competing reactions (21). To resolve this issue, the I-3 region was replaced with 349 bp from pBS- and pBSK+ (I-3 sub, Fig. 1GoA). The ratio of {alpha}s:{alpha}m in both clones of the BCL1 lymphoma transfected with I-3 sub and pools of stable transfectants was significantly lower than in BCL1 transfected with either I-3 or C{alpha} (Fig. 1Go, A and B). To determine whether the effect of this region depended on additional elements in the C{alpha}3-{alpha}M intron, the majority of the intron 5' to I-3 was replaced with an equivalent length of sequence from pBS- (H-S sub, Fig. 1GoC). Similar to BCL1 transfected with C{alpha}, the ratio of {alpha}s:{alpha}m in BCL1 transfected with H-S sub was >20:1. These findings demonstrate that the I-3 region contains the necessary elements for predominant usage of the proximal poly(A) site in {alpha} mRNAs.



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  		FIGURE 1. A 349-bp region in the C{alpha}3-{alpha}M intron is required for the preferential usage of the {alpha}s terminus. A, Map of the C{alpha} 3' terminus. The indicated EcoRI fragment of C{alpha} including one-half of C{alpha}2, C{alpha}3, the C{alpha}3-{alpha}M intron, and {alpha}M was ligated to the pcDLSR{alpha}296 expression vector (cf Materials and Methods). Bars indicate preserved C{alpha} sequence and the gray box indicates substituted sequence. The ratio of {alpha}s:{alpha}m from clones of permanently transfected BCL1 ± SEM for each construct are indicated. Numbers in parentheses are the number of cell lines analyzed. B, S1 nuclease protection analysis of C{alpha}, I-3, and I-3 sub in the BCL1 lymphoma and X63-Ag8.653 (653) plasmacytoma cell line. RNA was isolated from pools of stable transfectants. The {alpha}s:{alpha}m mRNA ratios for each plasmid are shown. C, S1 nuclease protection analysis of C{alpha} H/S sub in BCL1 cells. RNA was isolated from pools of stable transfectants. The {alpha}s:{alpha}m mRNA ratios for each plasmid are shown. H7 is an IgA-expressing subclone of CH12.LX.

 
A 58-kDa protein interacts with the I-3 region of {alpha} pre-mRNA

The I-3 region is capable of forming a stem loop which in and of itself could affect RNA processing (21). However, it is equally possible that this region acts by binding a protein which alters the stability of either the polyadenylation/cleavage complex or spliceosome. To address this, protein binding to cRNAs prepared from the I-3 region and a 399-bp fragment containing the two terminal poly(A) sites associated with {alpha}M ({alpha}M34) was evaluated. Nuclear extracts from BCL1 cells were incubated with the radiolabeled cRNAs and subjected to UV radiation which generates covalent cross-links between RNA bases and associated protein(s) (30). Following digestion with RNases, the protein(s) contacting the cRNA was analyzed by SDS-PAGE. Two proteins of ~58 kDa and 41 kDa interacted with I-3 (Fig. 2GoB). The RNA-protein interaction appears to be relatively temperature independent and occurs in the presence of 500- to 750-fold M excess of tRNA. The larger 58-kDa protein was present in all extracts tested, but the smaller 41-kDa protein was inconsistently detected. In addition to BCL1, the 58-kDa protein was detected in extracts from several clones of the CH12.LX lymphoma (data not shown). Neither protein interacted with a cRNA of similar length prepared from the 5' end of C{alpha} ({alpha}-M34). In addition, unlabeled I-3, but not unlabeled {alpha}-M34, inhibited detection of the 58-kDa protein, suggesting that the 58-kDa protein interacts specifically with the I-3 region (Fig. 2GoC). The 58-kDa protein is also present in a nuclear extract from HeLa cells, suggesting that it is a general processing factor (Fig. 2GoD).



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  		FIGURE 2. A 58-kDa RNA-binding protein interacts specifically with the I-3 region. A, Map of the C{alpha} 3' terminus. Double bars indicate sequence ligated into pBS- for generation of radiolabeled cRNAs. B, Protein binding to cRNAs. cRNAs were incubated with 7.5 µg of nuclear extract from BCL1 at the indicated temperatures, UV-irradiated, treated with RNases, and analyzed by SDS-PAGE. A total of 5 ng of tRNA was used as nonspecific competitor RNA. C, Specificity of binding. Binding reactions contained radiolabeled I-3 and the indicated molar excess of either unlabeled I-3 or {alpha}-M34 (cf Materials and Methods). D, Protein binding in the non-B, HeLa cell line nuclear extract. cRNAs from I-3 were incubated with ~15 µg of either BCL1 or HeLa nuclear extracts.

 
To further map the binding site for the 58-kDa protein, cRNAs were prepared from different regions of I-3 (Fig. 3GoA). The 58-kDa protein did not interact with a cRNA containing nt 1–140 which was prepared from the original I-3 template restriction digested with BstEII (Fig. 3GoB). However, it did interact with a cRNA containing nt 140–349 (Fig. 3GoC). To determine whether this region is sufficient for preferential usage of the {alpha}s poly(A) site, an expression plasmid containing nt 140–349 in lieu of the entire I-3 region was created (I-3140–349, see Fig. 4GoA). The ratio of {alpha}s:{alpha}m in BCL1 transfected with I-3140–349 was >20:1 (Fig. 4GoB). Taken together, these findings indicate that an RNA-protein interaction is involved in regulating 3' terminus usage in {alpha} mRNAs.



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  		FIGURE 3. The 58-kDa protein binds to the 3' end of the I-3 region. A, Map of the I-3 region. cRNAs from bp 1 to BsteII (B) or bp 140–349 (C) in the I-3 region were incubated with 15 µg of nuclear extract from BCL1 at 30°C, UV-irradiated, treated with RNases, and analyzed by SDS-PAGE.

 


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  		FIGURE 4. The 3' end of the I-3 region is sufficient for the preferential usage of the {alpha}s terminus. A, Map of the C{alpha} 3' terminus and SR{alpha}C{alpha} I-3140–349. Bars indicate preserved C{alpha} sequence. The ratio of {alpha}s:{alpha}m from clones of permanently transfected BCL1 ± SEM for each construct are indicated. Numbers in parenthesis are the number of cell lines analyzed. B, S1 nuclease protection analysis. {alpha}s:{alpha}m mRNA ratios for pooled stables are indicated at the bottom.

 
Effect of I-3 on the efficiency of the {alpha}s poly(A) signal

Several lines of evidence indicate that polyadenylation/cleavage at the secreted terminus is regulated during B cell maturation to Ig-secreting cells (8, 17, 18, 19, 20, 31). This suggests that a possible explanation for the effect of the I-3 region is that it increases the efficiency of the {alpha}s poly(A) site in B cells. To address this, the SV40 poly(A) site in a firefly luciferase reporter plasmid was replaced with either the minimal {alpha}s poly(A) site alone or with the I-3 region juxtaposed (Fig. 5GoA). Since the relative level of protein correlates with the efficiency of polyadenylation/cleavage (32), the level of luciferase is an indication of the strength of the poly(A) site. Expression plasmids containing firefly luciferase cDNA with the different poly(A) signals and a control expression plasmid containing a Renilla luciferase cDNA under the control of the thymidine kinase promoter were cotransfected into BCL1. The level of luciferase activity is expressed as the ratio of firefly to Renilla luciferase. The I-3 region caused approximately a 2.5-fold increase in luciferase activity (Fig. 5GoB). This increase, which is statistically significant, suggests that the predominant usage of the {alpha}s poly(A) site in mature B cells results from an RNA-protein interaction that increases the efficiency of the {alpha}s poly(A) site.



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  		FIGURE 5. Effect of the I-3 region on the efficiency of the minimal {alpha}s poly(A) signal. A, Maps of the luciferase constructs. The SV40 poly(A) site in the pGL3 luciferase expression was replaced with the indicated regions: the minimal {alpha}s poly(A) site or the minimal {alpha}s poly(A) site juxtaposed to the Ban-Sma fragment, pGL3{alpha}s I-3. The poly(A) signal was deleted in pGL3pA-. Absolute firefly luciferase expressed as the ratio of firefly luciferase (test) to Renilla luciferase (control) for a representative experiment is indicated in the column on the right. B, I-3 increases the efficiency of the {alpha}s poly(A) site. Luciferase expression was measured from BCL1 cells transfected with each plasmid and reported as fold enhancement over pGL3{alpha}s. Transfection efficiency was standardized by cotransfection of the Renilla luciferase plasmid (cf Materials and Methods). The average fold enhancement ± SEM from five separate experiments is shown, p = 0.007. *, There was no expression from pGL3pA-.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several features of regulation of choice of 3' terminus appear to be common to all isotypes. Although some studies indicated that the increase in µs mRNA accompanying maturation to plasma cells is due in part to transcription termination (4, 5, 6, 33), other studies indicated that the change in relative usage of µs and µm termini resulted from differential µ pre-mRNA processing (7, 8). The latter results are consistent with studies in both C{gamma} and C{alpha} (9, 10). In addition, alterations in CH gene structure affect different isotypes similarly, suggesting that the competing processing reactions are the same. Specifically, decreasing the distance between the secreted poly(A) site and the membrane exon(s) increases the use of the membrane terminus in both µ and {alpha} mRNAs (7, 25, 26). Together with the observation that decreasing the size of either Cµ4 or C{alpha}3 increases usage of the membrane terminus (13, 26), those findings indicate that the competing processing reactions for both genes are polyadenylation/cleavage at the secreted terminus and splicing of the membrane exon(s). Although several models explaining how this competition is regulated have been proposed, none of them conform to all of the data. The observation that usage of 3' termini by {alpha} and µ mRNAs is regulated differently (10) raised the possibility that determining the basis for the difference between these two isotypes could clarify the controversy.

One area of dispute concerns the role of cis-acting sequences in regulating 3' terminus usage. Specific sequence associated with secretory poly(A) sites influences the efficiency of polyadenylation/cleavage (18, 19, 20, 24). For example, a GU-rich region that is conserved among C{gamma} isotypes plays an important role in polyadenylation/cleavage at the {gamma}2bs poly(A) site in plasma cells (18, 19, 24). The µs poly(A) site is associated with two GU-rich regions, both of which are required for appropriate usage of this site (20). However, the role of more distal sequences in regulation has been discounted. Early studies argued that the length of the µs-µM intron was important for regulation, but specific sequence was not (7, 25). The strongest evidence for this contention comes from studies in which competing polyadenylation/cleavage and splicing reactions were introduced into ß-globin and MHC Dd (34, 35). Compared to a lymphoma transiently transfected with the individual chimeric genes, there was an increase in the ratio of mRNAs using the proximal terminus to those using the distal terminus in a plasmacytoma transiently transfected with the same gene. The fact that the increase in ratio was similar to that observed with µ mRNAs was used to argue that regulation is B cell stage specific and does not require any specific sequence in the gene (34, 35). However, it should be noted that although there was an increase in ratio of the two forms of mRNA of the chimeric genes, the actual ratios of proximal to distal termini are not the same for the endogenous µ mRNAs and the chimeric transfectants. These observations are reminiscent of those made with {alpha} mRNAs (10). Although there is an increase in ratio of {alpha}s:{alpha}m mRNA during maturation to plasma cells (36), {alpha}s is used preferentially at all stages in IgA B cell development (22). Furthermore, in B cell lymphomas transfected with C{alpha} expression plasmids, the ratio of {alpha}s:{alpha}m is >20:1 compared to 1:1 for the endogenous Cµ gene (10). The data presented in this study demonstrate that specific sequence in the C{alpha}3-{alpha}M intron is required for the preferential usage of the {alpha}s poly(A) site. Thus, developmental regulation of Ig mRNA processing appears to depend on both B cell stage-specific factors and CH-specific sequence.

The majority of studies support a model in which factors that regulate 3' terminus usage are present in B cells and decrease during maturation to plasma cells. The observation that the secreted form of Ig mRNA is predominant in non-B cells transfected with CH genes (10, 34, 37) suggests that these factors act either to inhibit polyadenylation/cleavage at the sIg termini or enhance splicing. The first evidence that B cell-specific trans-acting factors are involved in regulation was the demonstration that nuclear extracts from a lymphoma, but not a myeloma, caused a decrease in the ratio of µs:µm mRNA in Xenopus oocytes injected with a Cµ expression plasmid (37). More recent studies identified a factor in extracts from a B cell lymphoma that inhibits formation of the polyadenylation/cleavage complex at the µs poly(A) site (38). In contrast to these findings, Takagaki et al. (31) presented data arguing that a relatively low level of the 64-kDa subunit of CstF1 in resting B cells causes the weak µs poly(A) site to be used inefficiently. Interestingly, a second group of investigators observed increased binding of the 64-kDa subunit of CstF1 to several poly(A) sites incubated in extracts from myelomas and hybridomas compared to lymphomas (17). However, they did not observe a change in the overall level of CstF1 and suggested that the presence of an inhibitory factor at the B cell stage was responsible for the decreased binding. Subsequent studies demonstrated that the level of the 64-kDa subunit of CstF1 increases when human B cells enter S phase, but an increase in this factor is neither sufficient nor necessary for the production of secreted Ig mRNA (39). The discrepancy concerning changes in the level of the 64-kDa subunit could reflect differences between resting B cells and lymphomas.

Current opinion favors a paradigm in which immunoglobulin mRNA processing is regulated by changes in polyadenylation/cleavage efficiency during B cell maturation. The present study demonstrates that the preferential usage of the {alpha}s poly(A) site at all stages in the development of a secretory IgA response depends on specific sequence in the {alpha}s-{alpha}M intron. The regulatory I-3 region significantly increases the efficiency of polyadenylation/cleavage at the {alpha}s poly(A) site. This region interacts with a protein with an apparent molecular mass of 58 kDa. Two lines of evidence support the specificity of this RNA-protein interaction. The 58-kDa protein interacts with the regulatory region, but not other sequences in C{alpha}. In addition, binding could be blocked by specific, but not by nonspecific competitor RNA. An apparent increase in binding in the presence of a large molar excess of tRNA could result from the potential ability of the nonspecific competitor to inhibit formation of protein-protein complexes (40). Both the 58-kDa protein and an inconsistently detected 41-kDa protein could result from proteolytic degradation of a larger protein. However, the fact that the protein is also detected in a splicing competent nuclear extract from HeLa cells suggests that this is not the case. This observation also demonstrates that the 58-kDa protein is not B cell specific. Taken together, these findings suggest that the preferential usage of the {alpha}s poly(A) site results from an interaction of a region of the {alpha}s-{alpha}M intron with a ubiquitous RNA-binding protein that increases the efficiency of polyadenylation/cleavage. In a competition between polyadenylation/cleavage at the proximal termini and splicing of membrane exons, the exon definition model (41) predicts that decreasing the distance between the reactions should increase the efficiency of splicing. The effect of manipulations of the length of both the Cµ4-µM and C{alpha}3-{alpha}M introns on 3' terminus usage are consistent with that model (7, 25, 26). Thus, although the regulatory sequence in the I-3 region appears to act independently of other intron sequences, it is likely that the effectiveness of the element depends on the distance between the competing processing reactions.

IgA-expressing B cells develop in mucosal microenvironments that contain a high level of antigen. Controlled humoral immunity at these sites may require the development of mechanisms that alter the sensitivity of IgA-expressing B cells to antigen. Evidence for the evolution of such mechanisms is the observation that IgA B cells respond poorly to BCR cross-linking (42). One explanation for this finding is that IgA B cells express low levels of mIgA. Although posttranslational mechanisms are partly responsible for the inability of B cells and plasma cells to express secreted and membrane forms of IgM, respectively, the mechanisms controlling the level of mIg expression in B cells are not clear (43, 44, 45). However, a relatively low level of {alpha}m mRNA in memory populations could result in a low level of mIgA expression which alters the susceptibility of these cells to Ig cross-linking. Recent studies suggest that the level of BCR expression has profound effects on B cell development (46). Thus, isotype-specific mechanisms of Ig mRNA processing may contribute to the development of effective humoral immunity.


   Acknowledgments
 
We thank Drs. James Roesser and Geoffrey Krystal for helpful discussions and Juan LaCayo for technical assistance.


   Footnotes
 
1 This study was supported by National Institutes of Health Grants AI33451 and AI01344. Back

2 Address correspondence and reprint requests to Dr. Deborah A. Lebman, Department of Microbiology and Immunology, Virginia Commonwealth University, P.O. Box 980678, Richmond, VA 23298-0678. E-mail address: Back

3 Abbreviations used in this paper: m, membrane; s, secreted. Back

Received for publication October 14, 1999. Accepted for publication January 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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