Multiple RNA Surveillance Mechanisms Cooperate to Reduce the Amount of Nonfunctional Igκ Transcripts

During B cell development, V(D)J recombination begins at the pro-B stage, by the joining of D-J, followed by V-DJ segments at the IgH locus. Then, VJ recombination of IgL chain genes occurs (Igκ then Igλ loci) in pre-B cells (1–3). Provided BCR signaling is optimal, B lymphocytes are positively selected and develop into mature B cells, then able to differentiate into plasma cells and to secrete high amounts of Abs.

In two-thirds of cases, random V(D)J rearrangements induce a frameshift in the open reading frame and the appearance of a premature termination codon (PTC). When an aberrantly rearranged V(D)J junction appears on one allele, the recombination takes place on the second allele and, only B cells expressing functional Ig H and L chains are positively selected (2). Several reports have previously demonstrated that ∼40–50% of B lymphocytes carry biallelic recombination of Ig alleles [i.e., V(D)J⁺ and V(D)J⁻ for functional (F) and nonfunctional (NF) alleles respectively] (4–10). Although monoallelic V(D)J rearrangement may be initially favored by asynchronous replication timing and asymmetric chromatin modifications (2, 6, 11–15), biallelic recombination is finally frequent and is accompanied by primary transcription of rearranged IgH and IgL genes (4, 16–19). In the absence of a transcriptional silencing of V(D)J⁻ genes, the low level of mature V(D)J⁻ transcripts containing a PTC is likely explained by their high sensitivity to RNA surveillance mechanisms (20).

The molecular machinery of RNA surveillance is now known to include various pathways all controlling the quality of pre-mRNA and mRNA and limiting the translation of truncated proteins. Indeed, in eukaryotic cells, a PTC detected within a primary transcript may result in splicing inhibition and preclude mRNA maturation (19, 21, 22). Nonsense-altered splicing (NAS) could also induce the skipping of the PTC⁺ exon and prevent the maturation of the complete mRNA, while generating mRNA and proteins with internal deletions, sometimes preserving part of the function of that protein (23–26). Full-length PTC⁺ processed transcripts escaping both of these mechanisms could finally be subjected to nonsense-mediated decay (NMD) (24, 27–30). Although actors of RNA surveillance, such as up frameshift 1 protein, could contribute to both NMD and NAS, these mechanisms are distinct and the knock-down of up frameshift 2 reduces NAS but not NMD (26, 31).

NF Ig genes are inherently susceptible to PTC and several studies have shown that they could be subjected to a very stringent RNA surveillance (20, 32, 33). Because they are composed of only three exons (L, leader; VJ, variable; C, constant), nonproductive Igκ transcripts constitute a good model to analyze the various pathways mediating their posttranscriptional regulation. Indeed, splicing inhibition of PTC⁺ Igκ transcripts has been reported in hybridoma cell lines (22) and in a cell-free system (21). Although frameshifted VJκ rearrangements should not expose Igκ mature transcripts to classical NMD (because the resulting PTC is close to or within the last exon, Cκ), we have previously demonstrated that an NMD-related mechanism operates to reduce the amount of aberrant Igκ mRNA (17). Yet, due to the positive selection of cells with inframe VJ junctions, this process could not be analyzed in mature B cells and was only amenable to analysis by using mice in which B cell maturation was blocked at the pro-B cell stage (10).

By studying RNA surveillance of PTC⁺ Igκ transcripts arising from “mini-locus” constructs in B cell lines or in the mouse endogenous locus, we now demonstrate that NMD, splicing inhibition and NAS are all recruited for the downregulation of NF Igκ transcripts. Each mechanism individually has a partial effect but their combined actions altogether permit an efficient decrease of PTC⁺ Igκ mRNA. Moreover, the overall RNA surveillance machinery appears increasingly active from pre-B to plasma-cell lines, with a downregulation of 60–90% of NF Igκ mRNA, respectively. Strengthening of RNA surveillance throughout B cell development was also confirmed in primary B cells. Altogether, these data clearly indicate that NF VJ alleles are not subjected to allelic exclusion at the transcriptional level but that, multiple RNA surveillance mechanisms collaborate to decrease the level of nonproductive Igκ mRNA.

Mice were maintained in our animal facilities under specific pathogen-free conditions. Mice aged 1–6 mo old were used in all experiments. All the protocols used have been approved by our institutional review board for animal experimentation.

IgH locus “VH-LMP2A” (LMP2A) knock-in mice have been described previously and harbor a replacement of JH segments by the EBV LMP2A gene controlled by VH-promoter (34). The signaling pathway induced by LMP2A protein can mimic the BCR tonic signal and allow B lymphocytes to differentiate in the absence of a normal BCR.

VJκ rearrangements were built by joining artificially the Vκ1-135 and Jκ5 segments using a Sal I restriction site encoded by PCR primers (Supplemental Fig. 1A). The Vκ1-135 reverse primers were also designed to create either an inframe junction (VJ5^F) or an out-of-frame junction (VJ5^NF) by inserting five additional nucleotides. The frameshift introduced in VJ5^NF created a PTC in the VJ exon, 3 nt upstream from the donor splice site (Fig. 1A). Both VJ5^F and VJ5^NF constructs were subsequently cloned into the previously described “pSPIg8 plasmid” spanning 12.7 kb of murine Igκ locus and containing all the elements required for expression of Igκ L chains (enhancer Eiκ; Cκ exon; polyA) (35).

Murine cell lines included pre-B cells (18.81 and 70Z/3), B cells (Wehi, CH12, and A20) and plasma cells (S194, NS1, and X63Ag8). Erythrocyte depleted spleen cells from LMP2A and wild-type (wt) mice were cultured (10⁶ cells/ml) in RPMI 1640 supplemented with 10% FCS (Invitrogen, San Diego, CA), sodium pyruvate, nonessential amino acids, β-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Rockville, MD).

For analysis of precursor B cells, B220⁺ CD43⁺ bone marrow cells were sorted from LMP2A mice on a FACS-Vantage (BD Biosciences, San Jose, CA), with a purity >90%. Staining was performed with anti-B220 (RA3-6B2, eBioscience, San Diego, CA) and anti-CD43 (S7, BD Biosciences) Abs.

Cell lines (2 × 10⁶) were stably transfected by electroporation according to the manufacturer instructions (Lonza, Basel, Switzerland). Each transfection was performed with 2 μg linear plasmid DNA from VJ5 constructs (F or NF alone or, with an equimolar ratio of F and NF) and, with 200 ng linear DNA encoding a neomycin resistance gene. Twenty-four hours after transfection, G418 (800–1200 μg/ml) was added to cultures to select stably transfected cells. Cells were expanded for 10–12 d and then, G418 was removed. Two to 3 d later, dead cells were eliminated by Ficoll centrifugation (Lympholyte-Mammal, Cedarlane Laboratories, Burlington, Ontario, Canada), and living cells (10⁶ cells/ml) were treated or not with 100 μg/ml cycloheximide (CHX) (CHX in DMSO; Sigma-Aldrich, St. Louis, MO) for 4 h. Control cells were treated with DMSO alone (dilution factor 1/1000).

Genomic DNA and total RNA from cell lines or spleen cells were prepared using standard proteinase K (Eurogentec, Liège, Belgium) and Trireagent (Invitrogen) procedures, respectively. Nuclear and cytoplasmic RNA was extracted from transfected cells by using the PARIS kit (Ambion, Austin, TX), according to the manufacturer's protocol. RT-PCR was carried out on DNase I (Invitrogen)-treated RNA and was checked to be negative in the absence of reverse transcription, ruling out contamination by genomic DNA. Reverse transcription was performed using Superscript II (Invitrogen) on 1–5 μg total RNA. Priming for reverse transcription was done with random hexamers.

For analysis of mature transcripts, cDNA was amplified for 25 cycles of 50 s at 94°C, 50 s at 56°C, and 50 s at 72°C, using Taq DNA polymerase (Qbiogen, MP Biomedicals, Illkirch, France). Amplification was performed using a forward primer located in the Vκ1-135 segment (V-for: 5′-TCGGTTACCATTGGACAAC-3′), and a backward primer complementary to the 5′ end of the murine Cκ exon (C-rev: 5′-GCACCTCCAGATGGTTAACTGC-3′). The size of PCR products was 370 and 375 bp for VJ5^F and VJ5^NF constructs, respectively. To identify whether these constructs could be alternatively spliced, PCR (30 cycles otherwise indicated) were performed using a forward primer located in the leader exon of the Vκ1-135 segment (L-for: 5′-ATGATGAGTCCTGCCCAGTT-3′) and the “C-rev” backward primer. The size of PCR products was 463 and 468 bp for VJ5^F and VJ5^NF full length transcripts respectively, and 115 bp for transcripts lacking the VJ exon (leader-Cκ: alt-mRNA).

For primary transcript analysis, amplification was carried out for 30 cycles using the “L-for” forward primer and a backward primer located within the intron following Jκ5 (Int-rev: 5′-CATTTTCTCAAGATTTTCTGAACTGAC-3′). The size of PCR products was 886 and 891 bp for VJ5^F and VJ5^NF primary transcripts, respectively. This RT-PCR allowed amplification of two additional products corresponding to intermediate splicing transcripts having lost the 400 bp intron between the leader and VJ exons (IVS1) but retaining the VJ to Cκ intervening sequence (IVS2).

DNA was amplified in the same conditions (30 cycles) using “V-for” and “Int-rev” primers, the size of PCR products was 392 and 397 bp for VJ5^F and VJ5^NF constructs, respectively.

The quality of subcellular fractions was assessed by real-time PCR using Taqman Universal or SYBR Green Mastermix (Applied Biosystem, Foster City, CA) and primers specific for primary (Mb1-For: 5′-CAGGAGCAGAAAACCCAAGT-3′/Mb1-Rev: 5′-AGGAGGGTGAGGCCCTATAA-3′; V-for/Int-rev) or mature transcripts (Actin-for: 5′-CGATGCCCTGAG-GCTCTT-3′/Actin-rev: 5′-TGGATGCCACAGGATTCCA-3′; GAPDH: Mm99999915_g1, Applied Biosystem). Quantitative PCR (Q-PCR) were performed on cDNA samples equivalent to 10 ng RNA per reaction. Data were analyzed by comparing Ct values from nuclear and cytoplasmic fractions (relative RNA level = 2^−(ΔCt) with ΔCt = Ct_nucleus − Ct_cytoplasm).

In cell lines cotransfected with VJ5^F and VJ5^NF plasmids, we analyzed the PCR products using a high resolution capillary electrophoresis to identify specifically both F and NF constructs harboring a 5 nt difference. For this analysis, a fluorescent forward primer (6-FAM) was used in each PCR. Samples were prepared by mixing 1 μl PCR products with 14 μl deionized formamide (Sigma-Aldrich) and 0.3 μl Genescan-ROX 500 internal standard (Applied Biosystems). This mixture was denatured at 95°C for 3 min and then quickly cooled on ice. Samples were analyzed on a capillary electrophoresis apparatus and the Genemapper software (Abi9000, Applied Biosystems). Computing of the electrophoresis data provided the area of peaks corresponding to F and NF constructs and allowed to calculate for each sample the NF/F ratio. Validation of this method was performed by mixing different ratios of VJ5^F and VJ5^NF constructs (either from plasmid DNA or cDNA) and, by analyzing the experimental NF/F ratios (Supplemental Fig. 1C, 1D).

Northern blotting was conducted by migrating 10 μg total RNA on a 1% agarose denaturing gel, followed by transfer on nylon sheet membranes (MP Biomedicals). The blots were hybridized using [³²P]-labeled probes specific for VJ5 (a 262 bp PCR fragment located inside the Vκ1-135 exon) and actin transcripts (a 252 bp PCR fragment located inside the exon 4 and 5 of actin gene). Blots were revealed on a Cyclone Imager (Perkin Elmer, Waltham, MA) and quantification of bands was performed using OptiQuant Software (Perkin Elmer)

For sequence analysis of VJκ junctions in B cells from LMP2A mice, PCR was performed on cDNA using a previously described “Vκ consensus” forward primer (Vκ-cons: 5′-CAG(G/C)TTCAGTGGCAGTGG(A/G)TC(A/T)GG(A/G)AC-3′) (36), and the backward primer “C-rev”. PCR products were gel-purified (Macherey-Nagel, Düren, Germany) and cloned into pCR2.1-TOPO vector (Invitrogen). DNA sequences were obtained using the M13 Reverse primer (5′-CAGGAAACAGCTATGGAC-3′) and a Cycle Sequencing kit together with an automated sequencer (Applied Biosystems). Sequences from VJ junctions were analyzed using V-QUEST software (IMGT, the international ImMunoGeneTics information system; www.imgt.org).

Results are expressed as mean ± SEM and overall differences between variables were evaluated by a two-tailed unpaired Student t test using Prism GraphPad software (San Diego, CA).

To study RNA surveillance of Igκ transcripts and to evaluate its efficiency throughout B cell development, we created vectors for the expression of F and NF VJκ rearrangements in transfected cells. VJ sequences were assembled by joining Vκ1-135 and Jκ5 segments either inframe (VJ5^F) or out-of-frame (VJ5^NF) by inserting 5 nt at the V-J junction (Fig. 1A, Supplemental Fig. 1A). Equimolar ratios of these VJ5^F and VJ5^NF “mini-locus” constructs were stably transfected in several murine cell lines (representing various developmental stages from pre-B to plasma cells). To determine simultaneously the amount of both NF and F constructs at the DNA or RNA level, we performed PCR and/or RT-PCR, followed by capillary electrophoresis of fluorescent amplification products (5′ primer-FAM). We first analyzed the ratio of NF and F peak areas at the DNA level (NF/F_DNA) and checked that similar copy numbers of both constructs were integrated in genomic DNA of transfected cells, the NF/F_DNA ratio being close to 1 in most experiments (Supplemental Fig. 1). To further homogenize data from independent transfection experiments, only normalized NF/F_RNA ratios were considered (normalized NF/F_RNA = observed NF/F_RNA / observed NF/F_DNA).

As shown in Fig. 1B, the peak corresponding to non-functional mature transcripts (VJ5^NF) was markedly diminished compared with functional mRNA (VJ5^F). Indeed, in all the transfected cell lines, NF/F ratios were between 0.4 and 0.1 corresponding to a downregulation of 60–90% of NF mRNA (i.e., 2.5- to 10-fold decrease) (Fig. 1C). To analyze mRNA surveillance according to B cell developmental stage, we compared the data obtained from pre-B, B, and plasma cell lines and found the NF/F ratio significantly lower in terminally differentiated plasma cells than in B cells and pre-B cells (Fig. 1D).

Of note, RT-PCR and Northern blot quantification have revealed that the level of mature VJ5 transcripts was extremely low in pre-B cells and was increased by 130.9- and 293.3-fold in B and plasma cell lines, respectively (Fig. 1E, 1F). Likewise, a poor transcription of the Igκ locus has been documented in pre-B cell lines (37). Thus, the increase of RNA surveillance observed during B cell maturation seems to be correlated to the level of Igκ mature transcripts.

To investigate the mechanisms responsible for RNA surveillance of NF Igκ transcripts, we analyzed whether NF/F ratios were affected by CHX treatment; this protein synthesis inhibitor is classically used for NMD inhibition (38). Northern blot analysis of A20 transfected with either F (VJ5^F) or NF (VJ5^NF) constructs demonstrated that CHX treatment increased by ∼2-fold the mRNA level of VJ5^NF transcripts without affecting VJ5^F transcripts (Fig. 2A). Because VJ5^NF carries a PTC located at only 3 nt from the last exon-exon junction and mimics nonproductive VJ, the resulting transcripts should not represent good NMD substrates. Indeed, compared with IgH or TCRβ transcripts that are highly sensitive to NMD (18, 20, 32, 39), CHX treatment induced only a modest increase of PTC⁺ transcripts in cell lines cotransfected with both F and NF constructs (Fig. 2B). Although modest, NMD was detectable throughout B cell development and NF/F ratios were increased by CHX in all cell lines (with a 1.27-, 1.84- and 1.90-fold increase for pre-B, B, and plasma cell lines, respectively) (Fig. 2C, 2D).

Interestingly, the overall mRNA surveillance was correlated to the NMD efficiency and cell lines having the lowest NF/F ratio showed the highest increase after CHX treatment (Fig. 2E).

Thus, compared with pre-B cells, the increased surveillance of NF Igκ transcripts in differentiated plasma cells and mature B cells relied in part on a more efficient NMD.

Although NMD modestly controls the amount of NF Igκ mRNA, other mechanisms likely contribute to RNA surveillance of out-of-frame Igκ transcripts. To investigate whether inhibition of splicing occurred, we next analyzed the relative distribution of F and NF primary Igκ transcripts by RT-PCR with a forward primer located in the “leader” exon and a backward primer located in the “Jκ5-Cκ” intron (Fig. 3A). For both VJ5^F and VJ5^NF constructs, we observed two different transcripts: the primary (pre-mRNA) and a splicing intermediate (int-RNA) (Fig. 3B). Sequencing revealed that the int-RNA arose from the splicing of the leader onto the VJ exon but retained the intervening sequence between Jκ5 and Cκ. In cell lines cotransfected with F and NF constructs, we have quantified the NF/F ratio for both pre-mRNA and int-RNA transcripts using capillary electrophoresis (Fig. 3C, 3D). Contrary to the pattern obtained with mature mRNA, VJ5^NF transcripts were more abundant than VJ5^F for both primary (NF/F ratios from 1.2–2.3 with an average of 1.7 ± 0.1) and splicing int-RNA (NF/F ratios from 0.8–5.4 with an average of 2.5 ± 0.4) (Fig. 3C, 3D without CHX). Although variations were observed in some cell lines (with high NF/F int-RNA ratios in NS1 and no accumulation of NF int-RNA in 70Z/3 and S194 cells), other data suggested that the efficiency of splicing inhibition was almost the same throughout B cell development.

In agreement with the translation-independent splicing inhibition previously described for primary Igκ transcripts (21), we found that CHX treatment changed neither pre-mRNA nor int-RNA NF/F ratios, with the sole exception of S194 cell line in which CHX induced an unexpected increase in NF/F int-RNA ratios (Fig. 3C, 3D).

Next, we performed Q-PCR on nuclear and cytoplasmic fractions to analyze the cellular distribution of these RNA species (Fig. 3E). Purity of subcellular fractions was assessed by monitoring β-actin mRNA, GAPDH mRNA, and Mb-1 pre-mRNA levels. Compared with cytoplasm, nuclear fractions were enriched in pre-mRNA and poor in mature mRNA (Fig. 3E). We also observed that amplification of unspliced VJ5 transcripts (i.e., pre-mRNA and int-RNA) were 16.6-fold higher in the nucleus than in the cytoplasm (Fig. 3E). Thus, unspliced VJ5 transcripts were mostly confined in the nucleus. In agreement with their predominant nuclear location, we confirmed that NF/F ratios of nuclear and total pre-mRNA and int-RNA species were similar (NF/F[pre-mRNA] = 1.6 ± 0.2 and 1.5 ± 0.2 and NF/F[int-RNA] = 2.3 ± 0.2 and 2.1 ± 0.2 in total and nuclear fraction, respectively).

Therefore, a modest accumulation of unspliced or partially spliced NF Igκ transcripts occurred in the nucleus. Unlike the NMD efficiency that increases with B cell maturation, inhibition of splicing and/or intron retention remains constant in most cell lines.

We also examined whether the mechanism of NAS could participate in the RNA surveillance of nonsense Igκ transcripts. Using forward and backward primers located in the leader and Cκ exons, we identified alternatively spliced transcripts that had skipped the VJ exon (“L-Cκ” transcripts: alt-mRNA) (Fig. 4A). Although full-length mRNA was visualized in cells transfected with both VJ5^F and VJ5^NF constructs, alternative splicing yielding “L-Cκ” transcripts only showed up clearly in VJ5^NF transfected cells (Fig. 4B).

As shown in Fig. 4C, NF Igκ transcripts could be alternatively spliced in all the cotransfected cells used in this study and this process was not altered by CHX treatment.

After capillary electrophoresis, we could distinguish three differ-ent mRNA species: the alt-mRNA and both the F and NF mRNA (Fig. 4D). To more precisely quantify NAS during B cell development, we analyzed the amount of “L-Cκ” transcripts after normalization to F mRNA from a cotransfected construct not exposed to NMD (alt-mRNA/F ratios) (Fig. 4D–F). In cotransfections with VJ5^F and VJ5^NF constructs, NAS showed few variations between cell lines except that it was significantly higher (by 1.75-fold, p < 0.001) in plasma cells than B cells (Fig. 4F). Increased NAS might thus contribute to the globally more efficient mRNA surveillance of NF Igκ transcripts observed in plasma cell lines.

Although NAS was strongly induced for out-of-frame transcripts, it might involve modifications of exonic-splicing-enhancer (ESE) motifs (class I NAS) rather than the disruption of the reading frame (class II NAS) (23, 24, 40, 41). To distinguish between these two classes of NAS, both VJ5^F and VJ5^NF sequences were analyzed using RESCUE-ESE (http://genes.mit.edu/burgelab/rescue-ese/). This program can predict the fixation of serine/arginine-rich proteins to putative ESE sequences in different vertebrate genes, including the mouse (42). The analysis performed using RESCUE-ESE did not reveal any modification of ESE and suggested that the NAS observed for Igκ transcripts strictly belonged to class II NAS (data not shown). However, discrepancies between available software have been previously reported (40), making it difficult to definitely exclude the occurrence of some class I NAS.

We next analyzed the RNA surveillance of Igκ transcripts in LMP2A mice that freely accumulate random Igκ rearrangements in B cells without any selection for a functional BCR.

These mice exhibit an insertion of the EBV LMP2A gene within the IgH locus and, the LMP2A protein mimics BCR signaling (34). In these animals, we asked whether the mRNA surveillance efficiency was increased along B cell development. For this purpose, we sorted bone marrow B220⁺ CD43⁺ cells (precursor B cells) and compared their NF/F ratios with resting or LPS-stimulated splenic B cells (Table I). Reminiscent to cell line data (Fig.1D), NF/F ratio was highest in precursor B cells and decreased in resting and activated mature B cells (Table I, control lanes), indicating that the overall surveillance appeared to gradually increase along B cell maturation. Moreover, the CHX-mediated increase of NF/F ratios was more pronounced in mature B cells (1.89- and 2.5-fold in resting and activated cells, respectively) than in precursors (1.64-fold). These data demonstrated that the NMD efficiency increased during B cell differentiation. Because NF/F ratios were almost identical in CHX treated cells (Table I, CHX lanes), strengthening of the NMD activity mainly contributed to the increased mRNA surveillance of NF Igκ transcripts throughout B cell development.

Next, we analyzed whether skipping of the VJ exon (alt-mRNA), most likely from out-of-frame junctions, occurred in primary B cells (Fig. 5). We observed that NAS occurred in B cells from LMP2A and wt mice and was not altered by CHX treatment (Fig. 5A). In LMP2A mice, NAS was readily observed in all B cell compartments (precursor, resting and activated B cells) (Fig. 5B). However, the alt-mRNA amount was very low making hazardous to quantify fine variations of NAS efficiency along B cell development.

Altogether transcripts arising from nonproductive Igκ alleles are subjected to efficient posttranscriptional regulation mediated by NMD and NAS mechanisms (Fig. 6). This analysis in primary B cells further demonstrates that the overall RNA surveillance and the NMD efficiency were increased during B cell differentiation.

In B cells harboring V(D)J rearrangement on both Ig alleles, it is now clear that there is no transcriptional silencing of the nonproductive allele (4, 16–18, 43, 44). In this study, we examined the posttranscriptional regulation of NF Igκ transcripts from such alleles in B lymphocytes from mice and cell lines. The analysis of RNA surveillance pathways along B cell development demonstrated the coexistence of splicing inhibition, NMD and NAS, cooperating to control the amount of NF Igκ transcripts (Fig. 6). Downregulation of PTC⁺ Igκ mRNA also appeared stronger in B- and plasma-cells than in pre-B cell lines, with an elimination of 60–90% of NF mRNA (i.e., 2.5- to 10-fold decrease) in pre-B and plasma cells respectively. Moreover, the increase of RNA surveillance observed during B cell maturation seems to correlate to the Igκ mRNA level supporting the idea that RNA surveillance could be linked to splicing efficiency and translation (30, 45), although other molecular changes associated with B cell maturation cannot be excluded.

Previous analysis have demonstrated that PTC⁺ Igκ genes could be actively transcribed (17, 21, 22). The data reported in this study provide a quantitative analysis of the mechanisms implicated in RNA surveillance of PTC⁺ Igκ transcripts. In B cell lines spanning the main developmental stages, we observed that inhibition of splicing induces a modest increase (<2-fold) in NF pre-mRNA. Interestingly, out-of-frame rearrangement also leads to an accumulation of splicing intermediates that have eliminated the first intron (IVS1) but retained the intervening sequence between VJ and Cκ exons (IVS2). As previously reported for PTC⁺ Igκ primary transcripts (21, 22), the herein described accumulation of splicing intermediates is mainly CHX-independent. Our data also suggest that both unspliced and partially spliced Igκ RNA are mainly confined in the nucleus and that, accumulation of these NF primary transcripts remains constant in most cell lines. These results are reminiscent of the recently observed nonsense-mediated upregulation of TCRβ pre-mRNA that occurs in the nucleus in a translation-independent fashion (46).

We have previously demonstrated in mice having a blockade of B cell maturation at the pro-B stage that NF Igκ transcripts could be degraded by NMD (17). Using LMP2A mice, we extend this observation to mature B cells and we provide evidence for increased RNA surveillance and NMD efficiency along B cell differentiation. In agreement with the results from cell lines and primary cells, nonfunctionally rearranged Igκ genes should constitute poor NMD substrates and indeed, CHX treatment only induced increase of ∼2-fold in PTC⁺ mRNA in mature B cells. Because the PTC induced by an out-of-frame VJ rearrangement are located to close to the last exon-exon junction (or even in the penultimate exon), such aberrant Igκ transcripts are not predisposed to the classical EJC-dependent NMD (29). Therefore, PTC⁺ Igκ mRNA arising from NF VJ junctions should be degraded by an EJC-independent NMD as previously described for Igμ “minigenes” harboring a PTC in the last constant exon (47). However, additional experiments are needed to decipher which alternative branch of the NMD pathway downregulates NF Igκ mRNA (27).

In cell lines, the NMD efficiency was correlated to the overall mRNA surveillance of PTC⁺ Igκ mRNA (Fig. 2E). However, B and plasma cell lines exhibit a significant difference in mRNA surveillance (as assayed by the NF/F ratio) despite displaying a similar NMD efficiency. Because we found that skipping of the PTC⁺ VJ exon was more frequent in plasma cells than in B cell lines, a difference in NAS could explain the higher susceptibility of plasma cells to mRNA surveillance. Thus, alternative splicing contributes to mRNA surveillance of NF Igκ transcripts and, fluctuation of NAS during B cell development lead to a more efficient downregulation of PTC⁺ mRNA in plasma cell lines. Moreover, we also demonstrated that NAS of Igκ transcripts occurred in B cells from either wt or LMP2A mice.

Because computational analysis gives inconsistent results (40), it might be hazardous to conclude that alt-mRNA is rather induced by the disruption of the reading frame than by a modification of an ESE sequence. Moreover, the evidence for frame-dependent NAS previously published in (41) has been challenged by Mohn and colleagues (48). Whatever the class of NAS involved for Igκ genes, this mechanism seems “purposeless” because the alternatively spliced transcripts encode a Cκ domain devoid of V region that has never been detected at the cell membrane or as a secreted product. Our data reinforce the assumption that NAS is rather an opportunistic phenomenon (29, 49). Such a truncated Cκ protein might even be deleterious for B cells by interfering with the assembly of productive Ig chains. Indeed, alternatively spliced mRNAs encoding structurally abnormal Ig are frequently observed in various lymphoproliferative disorders including myeloma and Burkitt lymphoma (50, 51).

In the current work, we demonstrated for the first time that the combined action of splicing inhibition, NMD and NAS, each being individually weak, contributes to an efficient downregulation of NF Igκ transcripts arising from nonproductive rearrangement. Moreover, these data suggest that differences in NMD and NAS are responsible for the higher mRNA surveillance observed in plasma cells. Because terminally differentiated plasma cells are dedicated to produce high amount of Ab, the strong downregulation of NF Igκ transcripts observed in these cells highlight the link between translation, NMD and RNA surveillance.

We thank the staff of the animal facility at the Institut Fédératif de Recherche 145 (Institut Génomique Environnement Immunité Santé et Thérapeutiques, Limoges, France), M.C. Baclet (Sequencing platform), and C. Ouk-Martin (Flow cytometric platform) for technical assistance. LMP2A mice were kindly provided by Dr. S. Casola (Institute of Molecular Oncology Foundation, Milano, Italy).

Disclosures The authors have no financial conflicts of interest.