INTRODUCTION

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The immunoglobulin (Ig) genes were among the first discovered to encode more than one gene product; the Ig pre-mRNA is differentially processed at its 3' end into mRNAs that encode secreted and membrane-associated forms of the Ig protein (1, 8, 30). The mRNA encoding the secreted IgM protein (µs mRNA) is produced when the precursor RNA is cleaved and polyadenylated at the promoter-proximal µs poly(A) site. If the precursor RNA is instead spliced between the Cµ4 and M1 exons, which removes the µs poly(A) site, and is cleaved and polyadenylated at the downstream µm poly(A) site, the mRNA encoding the membrane-associated IgM protein (µm mRNA) is produced. During B-lymphocyte maturation from a resting B cell to an activated B cell or plasma cell, there is a shift in the relative amounts of µs and µm mRNAs produced; plasma cells express relatively more µs mRNA than B cells. How this and other Ig genes are regulated by alternative RNA processing during B-cell maturation has been studied mainly by introducing modified Ig genes into cell lines representing different stages of B-cell maturation (reviewed in reference 26).

By analyzing the processing patterns of modified µ genes, it has been established that cleavage-polyadenylation at the µs poly(A) site and splicing between Cµ4 and M1 are mutually exclusive RNA processing reactions whose efficiencies are suboptimal but balanced. The balance between the efficiencies of the reactions, but not their suboptimal nature, is required for regulation; if the efficiencies of both reactions are improved, regulation is maintained. However, if either reaction is made too strong relative to the other, only one RNA is produced from the modified gene in all cell types (24, 29). These observations led us to propose that a µ gene-like structure, with competing cleavage-polyadenylation and splicing reactions, is sufficient for µ processing regulation. This predicts that alternative RNAs from a gene with a similar arrangement of processing signals would be regulated like µ RNA in B cells and plasma cells. We confirmed this prediction by demonstrating that RNAs from two different non-Ig genes modified to have a poly(A) site within an intron are indeed regulated when expression in B-cell and plasma cell lines is compared (25). Thus, changes in RNA processing patterns during lymphocyte maturation must be mediated, at least in part, by general processing factors rather than gene-specific factors.

In the past, studies of Ig gene regulation have been conducted by using tissue culture cell lines derived from mouse lymphomas, myelomas, and plasmacytomas. The stage of lymphocyte development that each cell line most closely resembles is determined based on the presence of specific cell surface molecules and their Ig secretion status (see, e.g., references 15 and 17). Over the years and in a number of different laboratories, more than 16 different cell lines representing B-cell and plasma cell stages have been transfected with Ig genes, both stably and transiently. While the exact µs/µm, s/m, or s/m mRNA expression ratio varied among cell lines and experiments, this ratio is always higher in cells of the plasma cell stage than in cells of the pre-B- and B-cell stages. Therefore, tissue culture cells have been considered a valid system with which to study the mechanism regulating alternative processing of Ig RNA. Nevertheless, tissue culture cell lines, since they are derived from tumors and are maintained in continuous culture in vitro, are not normal lymphocytes. In fact, the experimental results obtained from cell lines and from resting and activated natural mouse B cells differ with respect to increases in the rate of Ig gene transcription initiation during lymphocyte maturation (11, 14, 16, 36). The model we have proposed for µ regulation, i.e., that a µ gene-like structure is required and that changes in general processing factors mediate the alternative processing, suggests a very different future experimental approach than models that propose µ gene-specific sequences. Therefore, we felt that it was essential to validate this model by using a more natural experimental system.

Transgenic-mouse technology is a powerful tool that has been used to gain insight into protein function, immunological responses, and transcriptional regulation of gene expression (12). However, this technology has been used only once to address a question of regulation at the level of alternative RNA processing: a transgenic mouse was produced to identify the non-cell-type-specific mRNA processing pattern of a ubiquitously expressed calcitonin/calcitonin gene-related peptide (CGRP) transgene (6). Most tissues in this mouse expressed calcitonin mRNA; CGRP mRNA expression was primarily restricted to neurons. The authors concluded that the CGRPprocessing pathway requires a tissue-specific cellular environment, while production of calcitonin mRNA is the default or nonregulated pathway. We describe here transgenic mice carrying a non-Ig gene, the modified class I major histocompatibility complex (MHC) gene D^ds (25), that is properly regulated in tissue culture cell lines to test our model that a gene with balanced competing cleavage-polyadenylation and splice reactions will be regulated during normal lymphocyte maturation. Transgene mRNA processing should mimic the endogenous µ gene mRNA processing during normal B-cell differentiation if the balanced competition model holds true in nontransformed cells. In addition, since the transgene is expressed in many tissues, we can assess the contributions that unique cellular environments make to the expression patterns of genes with competing splice and cleavage-polyadenylation reactions.

	MATERIALS AND METHODS

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Transgene construction, preparation, and injection. To construct the transgene D^dsTG (Fig. 1), a 2.7-kb EcoRI-BamHI fragment containing the D^d promoter (10) was first cloned into EcoRI-BamHI-cut pGEM4Z (Promega) to obtain the plasmid pGEM4Z-2.7. Next, a 4.5-kb BamHI-HindIII fragment containing the D^dBsm(H)s structural gene (25) was ligated into BamHI- and HindIII-cut pGEM4Z-2.7 to obtain D^dsTG.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Structure of the endogenous µ gene compared to that of the Dds transgene. The transgene was designed to have processing options similar to those in the µ gene, i.e., a poly(A) site (A) in competition with a splice reaction (angled lines above). Two RNAs are processed from each of these genes. Large solid boxes, common exons; open boxes, "exons" ending with a poly(A) site that are unique to poly(A) or µs mRNA; hatched boxes, exons that are unique to RNA that is spliced to remove the competing poly(A) site; smaller stippled boxes, transcriptional control regions.

Transgenic-animal screening. Two to 3 weeks after birth, pups were screened for transgene integration by Southern blot analysis of DNA obtained from tail biopsies (18). DNAs were digested with PstI and probed with a DNA fragment that hybridizes to both the transgenic and endogenous s poly(A) sites, producing a 2.4-kb endogenous fragment and a 1.7-kb transgenic fragment (data not shown). Individual lines were established from 3 of the 11 originally obtained transgenic founder mice. Imaging analysis of Southern blots allowed us to estimate that the 888 line has 20 copies, the 911 line has 6 copies, and the 916 line has 4 copies of the transgene (data not shown).

Preparation of primary B cells. Splenocytes from 6- to 8-week-old transgenic littermates were isolated and treated with anti-Thy 1.2 antibodies (hybridoma HO13-4.6; ATCC TIB 99), L3T4 antibodies (hybridoma RL-172/4) (5), and baby rabbit complement (Pel Freez, Inc., Brown Deer, Wis.) to remove T cells. High-density resting B cells isolated from the 70-60% interface of Percoll buoyant density gradients (Pharmacia LKB Biotechnology, Piscataway, N.J.) (7) were washed and diluted to 5 × 10⁶ cells per ml for bulk culture and to 2 × 10⁶ cells per ml for plating in RPMI 1640 medium containing 50 µM 2-mercaptoethanol, 24 mM sodium biocarbonate, 100 U of penicillin and streptomycin (Sigma) per ml, 2 mM glutamine, and 50 µg of gentamicin (BioWhittaker, Walkersville, Md.) per ml, with 10% heat-inactivated fetal calf serum (Sigma). Where indicated, lipopolysaccharide (LPS) from Salmonella enteritidis (Sigma) was used at 50 µg per ml. Untreated cells usually were harvested immediately, while treated cells were harvested after 72 h in culture. In two experiments, treated cells were cultured for 48 h; this did not influence the RNA expression data.

Carbon tetrachloride liver treatment. Transgenic littermates, aged 6 to 8 weeks, were injected intraperitoneally with 50 µl of 100% mineral oil (Sigma) or 10% carbon tetrachloride-90% mineral oil (J. T. Baker, Phillipsburg, N.J.). Animals were sacrificed at 48 or 72 h postinjection and their liver tissues were removed for RNA analysis; there was no difference in the RNA expression data for these two time points.

RNA preparation and analysis. Primary B cells were harvested by centrifugation; tissues were collected by dissection. RNA was isolated by using Trizol reagent as directed by the manufacturer (Gibco BRL).

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Expression of endogenous µ and transgene RNAs in response to LPS treatment. (A) Small resting splenic B lymphocytes isolated from three different lines of transgenic mice, 888, 911, and 916, were treated with LPS for 72 h (+) and compared to nontreated resting cells (). RNAs from these cell populations were isolated, and expression of the endogenous µ gene (Cµ) and Dd transgene (Dd tg) was analyzed by S1 nuclease protection with the probes diagrammed in panel B. The positions of the probes and the fragments protected by the cleaved-polyadenylated RNA (pA), the spliced RNA (splice), and the endogenous class I MHC gene (Db/k) are shown. (B) Diagram of the S1 nuclease protection probes that distinguish alternatively processed RNAs. For the Dd tg probe, the triangle indicates the 18-nucleotide insertion into the transgene that distinguishes it from the closely related endogenous MHC Db/k mRNA. P, PstI; H, HindIII; Bn, BanI; Ac, AccI. (C) Quantitation of the regulation index (pA/splice ratios for cells treated with LPS divided by pA/splice ratios for cells not treated with LPS) for individual preparations of B cells from the transgenic mouse lines indicated. TG, transgene.

	RESULTS

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The class I MHC D^d gene was modified previously to contain a poly(A) site within the third intron so that its structure was similar to that of the Ig µ gene; a 184-bp fragment containing the s poly(A) site was inserted into the third intron to compete with the exon 3-to-exon 4 splice reaction (Fig. 1). Although the s site is from the regulated Ig  gene, we previously established that sequences within the secreted poly(A) sites are not specifically required for regulation, because both the µm and simian virus 40 late poly(A) sites could substitute for the µs poly(A) site (29). Two alternative mRNAs were produced from this gene, and their expression was regulated when compared in B-cell and plasma cell lines; like µ mRNA, relatively more spliced RNA was produced in B cells (25). Twelve different poly(A) sites have been placed into this D^d gene; expression from all of these chimeric genes is regulated in cell lines (23). The D^ds gene also has an 18-bp insert in exon 3 to distinguish it from the endogenous D^d gene. In the cell lines, the D^ds gene was expressed from the Ig  promoter linked to the simian virus 40 enhancer. So that it would be more widely expressed in the mouse, the D^ds gene was cloned back under the control of the natural D^d transcriptional control elements. Previous studies have shown that this D^d gene fragment is expressed similarly to the endogenous D^d gene in transgenic mice (3). Genomic DNA obtained from tail biopsies was used to determine transgene integration into the mouse genome and to screen for germ line transmission to progeny.

mRNA processing in LPS-treated transgenic B cells. High-density resting splenic B cells isolated with Percoll buoyant density gradients can differentiate in culture to antibody-secreting cells upon treatment with LPS. LPS stimulates resting B cells both to proliferate and to differentiate along the path towards a plasma cell fate, and this is a standard procedure used to study molecular events that occur during these processes (see, e.g., references 19 and 36). In such bulk cultures of resting cells, approximately 25% of the cells respond to the LPS stimulation as measured by limiting dilution (35). The µs-to-µm mRNA expression pattern gradually changed from predominantly µm RNA at time zero of culture to approximately 90% µs mRNA by 72 h of treatment (19). We measured the µs/µm mRNA ratio from the endogenous µ gene in the resting transgenic B cells and in transgenic B cells stimulated for 72 h with LPS to monitor the resting and activated states of the cells. We then measured the pA/splice expression pattern of the transgene RNA in these B-cell populations to determine whether it was able to respond to the changes that occurred in the RNA processing environment within these cells.

Transgene expression in mouse tissues. When two different cell types process the same pre-mRNA in different ways, it is generally assumed that one cell type contains a trans-acting RNA processing regulator and that the other cell type, lacking the regulator, processes the pre-mRNA along a default pathway that is dependent only on the general processing machinery (22). This is clearly the case with the splice regulators that participate in sex determination of Drosophila (see, e.g., reference 22). One approach that has been taken previously to identify the cell type that contains trans-acting processing regulators is to ectopically express the regulated gene (see, e.g., reference 6). It was assumed that cell types that normally do not encounter the transcripts to be regulated will process them along a default processing pathway. This then identifies the regulated pathway and thus the cellular environment needed to process the transcript in this way. Since the D^ds transgene is widely expressed in our mice, we analyzed transgene mRNA processing in a number of different tissues in an attempt to identify the regulated and default processing pathways for this gene. Tissues were isolated from several mice of each transgenic line, and the RNAs were analyzed by S1 nuclease protection (Fig. 3A; Table 2). The pA/splice expression ratio showed variability between mice, but these deviations were not unlike those observed in other transgene studies (32). The results did not provide a clear answer to the question of whether tissues expressed the transgene like resting B cells or like LPS-activated B cells. The resting B-cell pA/splice expression ratio ranged from 0.19 to 0.43, while LPS-treated B-cell expression ratios ranged from 0.64 to 2.0 (Table 1). Mouse tissues had pA/splice expression ratios that ranged from 0.08 to 1.2, overlapping both resting and LPS-activated B-cell expression ratios, with more values falling into the resting B-cell range (compare Tables 1 and 2). Since there was variability among tissues and individual animals and among different preparations of resting and LPS-activated B cells, it could be argued that a distinction between regulated and default cannot easily be made for this gene. This interpretation was also proposed to explain data showing that nonlymphoid cells expressed variable ratios of µs and µm mRNAs. While the µs/µm mRNA ratios in nonlymphoid cells were more similar to those in a plasma cell than to those in a B cell, because of the variability, we suggested that the B-cell and plasma cell ratios were two along a continuum of possible processing patterns rather than one being regulated and one not being regulated (25). One major difference between our current experiments and those published previously was that this study tested expression in normal tissue which was generally not proliferating, while the others examined expression in transformed cell lines which were continually proliferating. To test whether any RNA processing differences were related simply to cell proliferation, we examined transgene expression during liver regeneration in vivo.

View larger version (40K): [in this window] [in a new window]   FIG. 3.   Transgene expression in mouse tissues. (A) S1 nuclease protection analysis of RNAs from the tissues labeled, from both a line 916 transgenic mouse (+) and a nontransgenic littermate (). (B) S1 nuclease protection analysis of liver RNAs from carbon tetrachloride-treated animals (+) and from mineral oil-treated littermates (). The probe is as diagrammed in Fig. 2B; protected fragments are labeled.

	DISCUSSION

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Previously we have shown that regulated µ mRNA processing depends on both the balance and arrangement of the competing splice and cleavage-polyadenylation reactions; no gene-specific sequences are required for this regulation (25). Thus, it is likely that changes in general cleavage-polyadenylation and/or splicing components mediate µ regulation. Since this model is based on results obtained with established tumor cell lines, we felt it was essential to examine this model in vivo during normal B-lymphocyte maturation. Now, using transgenic mice, we show that a non-Ig gene modified to have balanced competing splice and cleavage-polyadenylation reactions is regulated similarly to the endogenous µ gene during LPS-stimulated B-cell activation. These results establish that the balance and arrangement of the two processing signals are indeed sufficient for RNA processing regulation. Therefore, these studies validate previous and continuing regulatory experiments using tissue culture cell lines; the data generated in vivo is consistent with data generated from in vitro tissue culture.

While the pA/splice RNA ratio for the transgene and the µ gene shift in the same direction in resting and LPS-treated B cells, the regulation index, which quantitates the change in this ratio, varies among individual experiments. This is due to minor differences in the B cells isolated from the mice. However, despite this variability, the transgene regulation index is consistently about half of that of the endogenous µ gene for each experiment (Fig. 2; Table 1). There are at least two possible explanations for this 50% difference. One is that there are specific sequences, either in the µ gene or in the transgene, that contribute to the difference seen. The other possibility is that the precise balance between the efficiencies of the splice and cleavage-polyadenylation reactions affects the sensitivity of a transcript to changes in the cellular environment. The latter idea is supported by past experiments with closely related µ genes that differ in the strengths of their splice and cleavage-polyadenylation reactions (24, 27-29). The regulation indices of these µ gene derivatives in B-cell and plasma cell lines range from 2 to 22, a much greater difference than that seen between the transgene and endogenous µ gene in this study. These results with related µ genes are more difficult to reconcile with the former explanation, i.e., the presence or absence of specific sequences.

That gene-specific sequences are not required for regulation implies that changes in the levels or activities of general processing factors must mediate the change in RNA processing patterns. For the µ gene this could mean changes in cleavage-polyadenylation factors, splicing factors, or both. Indeed, we recently demonstrated that the level of the polyadenylation component CstF-64, the 64-kDa subunit of cleavage stimulation factor, is modulated between resting and LPS-stimulated B cells and that overexpression of this protein affects endogenous µ RNA processing in a chicken B-cell line (33). This experiment, along with others (20, 28), establishes that changes in cleavage-polyadenylation efficiency are an important component of µ processing regulation. However, while natural mouse B cells respond to LPS stimulation with about a 10-fold increase in CstF-64, consistent differences in the levels of this protein are not seen in tissue culture cell lines (9). This implies that there are additional mechanisms that contribute to µ processing regulation; possibilities include changes in the activity of cleavage-polyadenylation components (9), changes in the splice components (4), and changes in factors that affect the communication between the splice and cleavage-polyadenylation machinery (see, e.g., references 21 and 34). It is easy to imagine that changes in the amounts or activities of general trans-acting factors, relative to each other, can have dramatic effects on an RNA that can be alternatively processed by competing reactions.

The idea that cell-specific levels of several different general factors affect alternative RNA processing complicates the definition of the classical regulated and default pathways wherein a specific trans-acting factor affects processing choices, usually in an all-or-none way. Indeed, we have not been able to clearly assign the B cell or plasma cell as having the regulated or default RNA processing environment. In a survey of µ gene expression in non-B-cell lines, the pA/splice (µs/µm) ratio varied widely but was more similar to that seen in a plasma cell line than to that seen in a B-cell line (25). However, more recently we have examined several µ gene variants in a nonlymphoid cell line and have obtained different results; some µ genes were processed more like a plasma cell, while some were processed more like a B cell (23). In the current study, transgene mRNA expression in different mouse tissues is shown to vary over a range that overlaps both resting and LPS-activated B-cell expression patterns. Thus, the paradigm of regulated and default RNA processing patterns is probably not appropriate for the µ gene, where both alternate RNAs are always coexpressed and it is the ratio of the two that is modulated. Changes in the components for splicing, cleavage-polyadenylation, or both, relative to each other, are likely to be the essential features of µ regulation. Thus, each cell type, both lymphoid and nonlymphoid, may be expected to contain a characteristic concentration of processing factors; these concentrations change, relative to each other, during B-lymphocyte maturation.