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Papers In Press, published online ahead of print October 5, 2005
Immunology Dept., University of Pittsburgh, School of Medicine, Pittsburgh, PA 15221
Corresponding Author: milcarek{at}pitt.edu
LPS activation of murine RAW 264.7 macrophages influences the expression of multiple genes through transcriptional and post-transcriptional mechanisms. We observed a 5-fold increase in CstF-64 expression following LPS treatment of RAW macrophages. The increase in CstF-64 protein was specific in that several other factors involved in 3’-end processing were not affected by LPS stimulation. Activation of RAW macrophages with LPS caused an increase in proximal poly(A) site selection within a reporter mini-gene containing two linked poly(A) sites that occurred concomitant with the increase in CstF-64 expression. Furthermore, forced over-expression of CstF-64 protein also induced alternative poly(A) site selection on the reporter mini-gene. Microarray analysis performed on CstF-64 over-expressing RAW macrophages revealed that elevated levels of CstF-64 altered the expression of 51 genes, 14 of which showed similar changes in gene expression with LPS stimulation. Sequence analysis of the 3’-untranslated regions of these 51 genes revealed that over 45% possess multiple putative poly(A) sites. Two of these 51 genes demonstrated alternative polyadenylation under both LPS-stimulating and CstF-64 over-expressing conditions. We conclude that the physiologically increased levels of CstF-64 observed in LPS-stimulated RAW macrophages contribute to the changes in expression and alternative polyadenylation of a number of genes, thus identifying another level of gene regulation that occurs in macrophages activated with LPS.
J. Biol. Chem, 10.1074/jbc.M508848200
Submitted on August 10, 2005
Accepted on October 5, 2005
Elevated levels of the 64-kDa cleavage stimulatory factor (CstF-64) in LPS-stimulated macrophages influence gene expression and induce alternative poly(A) site selection
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