Inducible nuclear factors binding the IgM heavy chain pre-mRNA secretory poly(A) site
✍ Scribed by Cathy Phillips; Anneliese Schimpl; Walter Dietrich-Goetz; J. Barklie Clements; Anders Virtanen
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 939 KB
- Volume
- 26
- Category
- Article
- ISSN
- 0014-2980
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✦ Synopsis
Inducible nuclear factors binding the IgM heavy chain pre-mRNA secretory poly(A) site
Two alternative forms of IgM heavy-chain mRNA are produced from a common 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 splicing. The efficiency of cleavage at the secretory poly(A) site is thought 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 site. Purified primary B cells produce the secretory form of p mRNA when stimulated with lipopolysaccharide (LPS) and the membrane form of p mRNA when their antigen receptors are ligated by anti-p antibodies. We compared RNA binding factors in nuclear extracts from cells produced by these different stimulatory conditions and show that induction of the secretory form of p mRNA by LPS correlates with the induction of a 28-32-kDa secretory poly(A) site-specific polypeptide which is also present in the plasmacytoma cell line J558L. Visualization of the 28-32-kDa polypeptide in UV cross-linking assays depends on a GU-rich element downstream of the secretory poly(A) site. We show that this GU-rich region enhances polyadenylation efficiency in vivo by transfection of luciferase reporter constructs into the plasmacytoma J558L. We also examined nuclear extracts from Bcells doubly stimulated with LPS and anti-p antibodies in which expression of the secretory form of p mRNA is selectively inhibited. This inhibition may be due to a down-regulation of polyadenylation at the secretory poly(A) site or an up-regulation of the competitive splicing process. This form of stimulation does 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 secretory and membrane poly(A) site. We report the first detection of changes in RNA binding factors taking place at the secretory poly(A) site which correlate with the expression of different forms of p mRNA produced by primary B cells under different stimulation conditions.