The cleavage and polyadenylation reaction has been shown to play an important role in modulating Ig heavy chain poly(A) site use in B-cell development. This application focuses on defining the mechanisms of how these changes in the molecular nature, quantity, affinity for RNA, and interactions of CstF-64 (64 kDa subunit of cleavage stimulatory factor of the polyadenylation reaction) with other components of the polyadenylation/cleavage machinery operate and influence poly(A) site choice during the induction of plasma and memory cells. We propose to test the following:
Aim 1 Three proteins have been identified by our lab whose amount or activity varies between plasma cells and earlier B-cell stages. We will test the hypothesis that their interactions with CstF-64, CPSF100 or the downstream region of RNA can influence polyadenylation patterns in B-cell stages.
Aim 2. Experiments are proposed to address our hypothesis that the changes in CstF-64 binding activity we previously observed are brought about in part by mechanisms intrinsic to the CstF-64 protein.
Aim 3. We will to test the hypothesis that CstF-64 up-regulation is mediated by NF-kB in lipopolysaccharide and interferon-gamma stimulation and CD 40 ligand-mediated B-cell growth. The experiments described in this application attempt to find differences in cells that may serve as markers to identify memory, naive, and plasma B-cells as well as to understand the molecular events that determine how memory an/or B-cells may be activated to differentiate into plasma cells. Understanding immunological memory is important for vaccine development to eradicate infectious diseases, mediating allergic responses and autoimmune diseases, and limiting tumor growth. Understanding the control of polyadenylation is important for understanding and possibly controlling many aspects of cell growth, differentiation and malignancy.
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