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

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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 γ2b 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 ≤ 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 ≤ 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.