INDUCIBLE NUCLEAR FACTORS BINDING THE IGM HEAVY-CHAIN PRE-MESSENGER-RNA SECRETORY POLY(A) SITE

Two alternative forms of IgM heavy-chain mRNA are produced from a comm on precursor mRNA as a result of competition between cleavage/poly(A) addition at the upstream (secretory) poly(A) site and cleavage/poly(A) addition at the downstream (membrane) poly(A) site coupled with splic ing. The efficiency of cleavage at the secretory poly(A) site is thoug ht to play a crucial role in this alternative processing. We therefore examined RNA binding factors recognizing the secretory poly(A) site, in the absence of the splicing option, to look for transacting factors that may play a role in cleavage/polyadenylation efficiency at this s ite. Purified primary B cells produce the secretory form of mu mRNA wh en stimulated with lipopolysaccharide (LPS) and the membrane form of m u mRNA when their antigen receptors are ligated by anti-mu antibodies. We compared RNA binding factors in nuclear extracts from cells produc ed by these different stimulatory conditions and show that induction o f the secretory form of mu mRNA by LPS correlates with the induction o f a 28-32-kDa secretory poly(A) site-specific polypeptide which is als o present in the plasmacytoma cell line J558L. Visualization of the 28 -32-kDa polypeptide in UV cross-linking assays depends on a GU-rich el ement downstream of the secretory poly(A) site. We show that this GU-r ich region enhances polyadenylation efficiency in vivo by transfection of luciferase reporter constructs into the plasmacytoma J558L. We als o examined nuclear extracts from B cells doubly stimulated with LPS an d anti-mu antibodies in which expression of the secretory form of mu m RNA is selectively inhibited. This inhibition may be due to a down-reg ulation of polyadenylation at the secretory poly(A) site or an up-regu lation of the competitive splicing process. This form of stimulation d oes not lead to the disappearance of the 28-32-kDa polypeptide, but to an enhanced binding of a 50-55-kDa factor which binds both the secret ory and membrane poly(A) site. We report the first detection of change s in RNA binding factors taking place at the secretory poly(A) site wh ich correlate with the expression of different forms of mu mRNA produc ed by primary B cells under different stimulation conditions.