Regulation of Poly(A) Site Use during Mouse B-Cell Development Involves a Change in the Binding of a General Polyadenylation Factor in a B-Cell Stage-Specific Manner

Abstract

Immunoglobulin (Ig) heavy-chain proteins exist in two forms, differing only at their carboxyl termini. The membranebound antigen receptor is found on the surface of early or memory B cells, while the secreted form is produced by plasma cells. The mRNAs for the two protein forms are derived from a single pre-mRNA that is alternatively processed at the 3Ј end (1,8,44,45). The 3Ј end of the secretion-specific (sec) mRNA is encoded by the last constant-region exon; the sec poly(A) site is about 100 nucleotides downstream of the last constantregion exon. The 3Ј end of the membrane-specific (mb) mRNA is encoded by a large portion of the last constant-region exon and by two downstream exons, M1 and M2. mb mRNA is produced when splicing of the last constant-region exon to M1 takes place using a 5Ј splice site within the last constant-region exon and polyadenylation occurs at the poly(A) site at the end of M2.The ratio of sec to mb mRNA is indicative of a B cell's developmental stage (reviewed in reference 18). Early B, pre-B, and memory cells and their tumor analogs, lymphomas, make approximately equal amounts of sec and mb mRNA. Plasma cells and their tumor counterparts, myelomas, make 10-to 100-fold more sec mRNA than mb mRNA. While translational and posttranslational control mechanisms play a small role in contributing to the sec-versus-mb protein ratios of Igproducing cells, the major contribution to the phenotype comes from differential production of sec and mb mRNAs (13,24,25,41). Studies of the mouse Ig ␥2b locus have shown that transcription termination occurs downstream of the mb poly(A) site in both early and late-stage B cells (11). This finding is in contrast to some studies of the Ig transcription unit (13,19), in which differential transcription termination has been suggested as a possible mechanism for the switch in poly(A) site use during B-cell development. The stability of both ␥ sec and ␥ mb mRNAs increases in late-stage or plasma cells, and differential mRNA stability therefore cannot account for the major shift in the ratio of the two mRNAs (33).The key point in understanding posttranscriptional processing of Ig heavy-chain mRNAs is that splicing of the last constant-region exon to M1 and polyadenylation at the sec poly(A) site are two mutually exclusive events. The balance between the two is reflected in the final sec-to-mb mRNA ratio and could potentially be controlled by regulation of either event. However, when the efficiency of splicing of the last constant-region exon to M1 exons was examined in either the or ␥ gene, there was no change in early versus late-stage B cells or in lymphoid versus nonlymphoid cell lines (3,38,40,56), indicating that splicing is a constitutive, nonregulated step. In contrast, experiments using stable transfections have shown that regulated expression of the mouse Ig ␥2b gene is influenced by poly(A) site order and strength (26).The biochemistry of cleavage and polyadenylation and the sequences required in cis in the pre-mRNA for 3Ј-end formation have been well chara...

Chimeric human CstF-77/ Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3′ end processing in Drosophila

“…In human, a shift in poly(A) site choice in the IgM heavy-chain gene occurs during B cell maturation. An increase in CstF-64 protein amount or activity during differentiation has been proposed to be involved in utilization of a weak proximal poly(A) site of the gene, which generates a transcript coding for a secreted protein in mature B cells (24,25). In Drosophila, we previously showed that the Su(f) protein is required for tissue-specific utilization of a regulated poly(A) site in intron 4 of the su(f) gene (19,20).…”

“…In vitro, affinity of CstF for the U͞GU-rich elements, which are highly variable in sequence, defines the efficiency of poly(A) sites by determining the stability of the cleavage complex on the pre-mRNA (22). In mammalian cells, several studies have correlated shifts in the choice of poly(A) sites with quantitative or qualitative variations of CstF-64 (23)(24)(25)(26)(27). In Drosophila, we and others have shown that modulation of su(f) activity in su(f) mutants affects the utilization of alternative poly(A) sites in the f 1 mutation, the Adh͞Adhr locus, and the su(f) gene itself (19,20,28,29).…”

AID−/−μs−/− Mice Are Agammaglobulinemic and Fail to Maintain B220−CD138+ Plasma Cells

“…The decision between the synthesis of membrane vs secreted Igs is made by choice of polyadenylation site (9), but exactly what regulates the levels of these different mRNAs is not known. Clearly, the inability to generate an mRNA that encodes the secreted -chain is not automatically linked to an increase in the synthesis of the membrane bound product, notwithstanding seemingly adequate secretory capacity (7).…”

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

“…Protein concentration was measured by the Bradford assay. UV crosslinking reactions contained 5 to 10 μg of nuclear extract protein, 10 to 20 fmol of substrate RNA (50,000 to 100,000 cpm), 1 mM ATP, 0.7 mM MgCl 2 , and 40 ng of carrier tRNA in a 25 μl final volume of buffer (20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) pH 7.9, 0.2 mM EDTA, 10% glycerol, and 150 mM KCl) [26]. Reaction mixtures were incubated for 5 to 10 min at 30°C and then subjected to UV crosslinking on ice for 10 min in a Stratalinker (1.8 x 10 6 μJ/cm 2 ).…”

“…Samples were treated with (1 mg/ml) at 37°C for 30 min. Proteins were boiled for 5 min in cracking buffer (80 mM Tris-Cl, pH 6.8, 0,1 M dithiothreitol, 2% sodium dodecyl sulfate (SDS), 10% glycerol, and 0.2% bromophenol blue) and separated in 10% SDS-polyacrylamide gels [26]. Binding of proteins to the RNA was visualized using storage phosphor imaging and quantified using ImageQuaNT software.…”