B-lineage regulated polyadenylation occurs on weak poly(A) sites regardless of sequence composition at the cleavage and downstream regions

Abstract

Early/memory and plasma B-cell lines and fibroblasts were analyzed for their ability to use a 5' proximal (variant) versus a 3' distal (constant) poly(A) site, in the absence of a competing splice, from a set of related constructs. The proximal:distal poly(A) site use (P:D ratio) of the resulting cytoplasmic poly(A)+ mRNA is a measure of poly(A) site strength. In this context the immunoglobulin gamma2b secretory-specific poly(A) site showed a P:D ratio of 1:1 in plasma cells, 0.43:1 in early/memory B-cells and an intermediate value in fibroblasts. Meanwhile, a construct with a proximal SV40 early-like poly(A) site produced mRNA with a P:D ratio of >>50:1 in all cell types. Alterations in the region downstream of the proximal poly(A) addition site and at the site itself resulted in changes in the P:D ratio. However, these poly(A) sites, all with a P:D ratio of < or = 5:1, were used most efficiently in plasma cells. Constructs totally devoid of immunoglobulin sequences, but containing heterologous poly(A) sites producing mRNA with P:D ratios of < or = 5:1, were also used more efficiently in plasma cells. We therefore conclude that weak poly(A) sites, regardless of sequence composition, are used more efficiently in plasma cells than in the other cell types.

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

“…We recently quantitated this data and found that, in two independent experiments, the E1A 12S:13S ratio was 1.7-to 2.1-fold higher in the 3-1 pre-B cells than in the S194 plasma cells. This is similar to the change in use of tandem poly(A) sites that was measured in these same cells (Peterson et al 1991) and in a similar independent experiment (Matis et al 1996). This, then, is a third example of an alternative splice reaction that is differentially modulated between B cells and plasma cells.…”

“…Thus, µ processing is likely controlled by general RNA-processing factors; theoretically, this could be due to changes in general cleavage-polyadenylation activity, general splice activity, or a combination of both. Evidence has accumulated to suggest that cleavagepolyadenylation activity is higher in plasma cells, which preferentially use the µs poly(A) site, than in B cells (Peterson et al 1991;Edwalds-Gilbert and Milcarek 1995;Matis et al 1996;Takagaki et al 1996;Veraldi et al 2001). By using two different RNA substrates that could be alternatively spliced, with no competing cleavage-polyadenylation reaction, we have demonstrated here that the splice environment is also clearly different between B cells and plasma cells.…”

“…Tandem poly(A) sites are differentially used in the two cell types (Peterson et al 1991;Matis et al 1996), the amount of Cleavage-stimulatory Factor (CstF) 64K subunit crosslinked to poly(A) sites differed between extracts from B-cell and plasma-cell lines (Edwalds-Gilbert and Milcarek 1995), the levels of CstF 64K increased when resting mouse B cells were stimulated with LPS (Takagaki et al 1996), and overexpression of CstF 64K in a chicken B-cell line altered the endogenous µs/µm mRNA ratio (Takagaki et al 1996). Thus, CstF 64K is likely to be a component of µ-processing regulation.…”

Inefficient processing of mRNA for the membraneform of IgE is a genetic mechanism to limit recruitment of IgE‐secreting cells

“…The transcription of the membrane forms of Ig is in general favored by "weak" polyA sites for the secreted forms, regardless of the sequence composition at the cleavage and 3 0 regions [19]. Further, the differentiation state of the cell and therewith the composition of the polyadenylation/cleavage complex definitively plays an important role.…”

Hide and Go Seek: Activation of the Secretory-Specific Poly (A) Site of Igh by Transcription Elongation Factors

“…Weak poly(A) sites are used more efficiently in plasma cells than in B/ memory cells even in constructs in which there is no splicing between the sites. Polyadenylation therefore tips the balance towards secretory poly(A) site use in plasma cells (Milcarek and Hall 1985;Kobrin, Milcarek et al 1986;Lassman, Matis et al 1992;Lassman and Milcarek 1992;Matis, Martincic et al 1996). Differences between plasma and memory B-cells are found in the polyadenylation machinery.…”