B-cell and plasma-cell splicing differences: a potential role in regulated immunoglobulin RNA processing

Abstract

The immunoglobulin micro pre-mRNA is alternatively processed at its 3' end by competing splice and cleavage-polyadenylation reactions to generate mRNAs encoding the membrane-associated or secreted forms of the IgM protein, respectively. The relative use of the competing processing pathways varies during B-lymphocyte development, and it has been established previously that cleavage-polyadenylation activity is higher in plasma cells, which secrete IgM, than in B cells, which produce membrane-associated IgM. To determine whether RNA-splicing activity varies during B-lymphocyte development to contribute to micro RNA-processing regulation, we first demonstrate that micro pre-mRNA processing is sensitive to artificial changes in the splice environment by coexpressing SR proteins with the micro gene. To explore differences between the splice environments of B cells and plasma cells, we analyzed the splicing patterns from two different chimeric non-Ig genes that can be alternatively spliced but have no competing cleavage-polyadenylation reaction. The ratio of intact exon splicing to cryptic splice site use from one chimeric gene differs between several B-cell and several plasma-cell lines. Also, the amount of spliced RNA is higher in B-cell than plasma-cell lines from a set of genes whose splicing is dependent on a functional exonic splice enhancer. Thus, there is clear difference between the B-cell and plasma-cell splicing environments. We propose that both general cleavage-polyadenylation and general splice activities are modulated during B-lymphocyte development to ensure proper regulation of the alternative micro RNA processing pathways.

FIGURE 1.

μ gene–SR protein cotransfections. (A) Diagram of the alternative RNA-processing reactions at the 3′ end of the Cμ gene, and the S1 probe used to distinguish cleaved-polyadenylated μs mRNA from spliced μm mRNA; the sizes of the protected fragments are shown. (B) Equal amounts of pSV5Cμ and the SR protein constructs shown above each lane (pCDM8 is the empty vector) were cotransfected into the M12 B-cell line, and RNA was analyzed by S1 nuclease protection assay. The probe and protected fragments are identified on the right. The S1 protection assays were quantitated on a PhosphorImager and expressed as a ratio of μs to μm mRNA. Each bar represents at least two independent transfections analyzed two or more times. (C) Cotransfection analysis in the S194 plasma-cell line as described in B.

FIGURE 2.

Chimeric D^d-pIgR construct. (A) The 654-bp pIgR exon 4, with 262 bp and 420 bp of surrounding intron sequence, was placed into the KpnI (K) site in intron 3 of the D^d gene. The open box represents the pIgR exon, the black boxes are D^d exons, and thin lines are introns. The 5′ splice-site sequence at the end of pIgR exon 4 is shown; the A at the +4 position was mutated to C in the construct 5′SS (Bruce and Peterson 2001). (B) Diagram of the splicing patterns of the D^d-pIgR RNA and the 5′SS mutant in a nonlymphoid cell line. The full exon is spliced in D^d-pIgR (splice pattern shown above) and a cryptic 5′ splice site 158 nucleotides into exon 4 is activated in 5′SS (splice pattern shown below) (Bruce et al. 1999; Bruce and Peterson 2001).

FIGURE 3.

D^d-pIgR constructs are differentially spliced in B-cell and plasma-cell lines. (A) RNA from S194 plasma cells and M12 B cells mock transfected (−) or transiently transfected with the constructs shown above each lane was analyzed by S1 nuclease protection. The probe and protected fragments are labeled. The S1 reactions were quantitated by PhosphorImager analysis and expressed as a ratio of full-length to cryptically spliced RNA. Multiple protected bands are observed with the cryptic 5′ splice-site RNA due to fortuitous homology between the probe and the sequences in D^d exon 4; the fragments were combined for quantitation. The values shown below each lane are the mean of least two independent transfections, analyzed two or more times; the standard deviation of each mean was <10%. Because of variable lane background, we cannot reliably measure ratios above 30 (Bruce and Peterson 2001); other bands in the lanes are considered background because they do not correspond to bands seen by RT–PCR analysis of this RNA. (B) D^d-pIgR and D^d-5′SS were transiently expressed in the J558L and MPC11 plasma-cell lines and the A20 and 70Z/3 B-cell lines, and the RNA was analyzed by S1 nuclease protection. The probe and protected fragments are labeled and the reactions were quantitated as described above. (C) Diagram of the D^d-pIgR S1 nuclease protection analysis probe that distinguishes full-length from cryptically spliced RNA. RNA that has spliced the full-length pIgR exon into D^d protects the probe to the PpuMI site, and cryptically spliced RNA protects the probe to the cryptic 5′ splice site; the expected sizes of each product are indicated.

FIGURE 4.

μAV-derived RNAs are differentially spliced between B cells and plasma cells. (A) The μAVWT.Bsc vector (μAV) that requires an exonic splice enhancer to be spliced is diagrammed. The arrows below are the PCR primers used to analyze the spliced vs. unspliced RNA; the downstream-most primer was used to monitor the size of the two RNAs, whereas the upstream two primers were used in quantitation. The diagram below identifies the overlapping fragments from the Cμ4 exon that were inserted between the BglII (Bg) and SpeI (S) sites of μAV to test for ESE activity; (L) ApaLI; (P) PstI; (A) ApaI; (M) MspI; (B) BstEII; (D) DpnI; (H) HaeII. (B) Representative RT–PCR reactions of RNA from M12 B cells (B) and S194 plasma cells (PC) that were stably transfected with the construct shown below each pair of lanes. The bands representing the unspliced (308 nucleotides) and spliced (82 nucleotides) RNAs are shown.