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Inducible nuclear factors binding the IgM heavy chain pre-mRNA secretory poly(A) site
C Phillips1, A Schimpl, W Dietrich-Goetz
1Institut für Virologie und Immunologie der Universität Würzburg, Germany. cathy.phillips@medgen.uu.se
Abstract:
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 mu mRNA when stimulated with lipopolysaccharide (LPS) and the membrane form of mu mRNA when their antigen receptors are ligated by anti-mu 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 mu 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 B cells doubly stimulated with LPS and anti-mu antibodies in which expression of the secretory form of mu 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 mu mRNA produced by primary B cells under different stimulation conditions.
Insights
Researchers identified specific RNA binding factors that control the production of secretory and membrane forms of IgM heavy-chain mRNA in B cells. This discovery sheds light on the regulation of alternative mRNA processing in immune cells.
Area of Science:
- Molecular Biology
- Immunology
- RNA Processing
Background:
- Alternative polyadenylation of IgM heavy-chain mRNA generates distinct secretory and membrane-bound forms.
- The efficiency of cleavage at the upstream secretory polyadenylation site is critical for this alternative processing.
- Understanding the RNA-binding factors involved is key to elucidating the regulatory mechanisms.
Purpose of the Study:
- To identify and characterize RNA-binding factors that interact with the secretory polyadenylation site.
- To investigate the role of these factors in regulating the cleavage/polyadenylation efficiency at the secretory poly(A) site.
- To correlate changes in RNA-binding factors with the differential expression of IgM heavy-chain mRNA isoforms in primary B cells.
Main Methods:
- Nuclear extracts from primary B cells, stimulated with lipopolysaccharide (LPS) or anti-mu antibodies, were analyzed.
- UV cross-linking assays were employed to visualize RNA-binding polypeptides.
- Luciferase reporter constructs were used to assess polyadenylation efficiency in vivo via transfection.
Main Results:
- LPS stimulation, favoring secretory IgM mRNA, correlated with the induction of a 28-32 kDa secretory poly(A) site-specific polypeptide.
- This 28-32 kDa polypeptide's binding depended on a downstream GU-rich element, which enhanced polyadenylation efficiency.
- Dual stimulation (LPS and anti-mu), inhibiting secretory IgM mRNA, showed enhanced binding of a 50-55 kDa factor to both poly(A) sites.
Conclusions:
- Changes in RNA-binding factors at the secretory poly(A) site directly correlate with the differential expression of IgM heavy-chain mRNA forms.
- A 28-32 kDa polypeptide, associated with a GU-rich element, plays a role in promoting secretory polyadenylation.
- The study provides the first evidence of altered RNA-binding factors at the secretory poly(A) site linked to specific B cell stimulation conditions.