Elevated Levels of the 64-kDa Cleavage Stimulatory Factor (CstF-64) in Lipopolysaccharide-stimulated Macrophages Influence Gene Expression and Induce Alternative Poly(A) Site Selection

Scott A Shell,Candice Hesse,Sidney M Morris,Christine Milcarek

doi:10.1074/jbc.m508848200

Scott A Shell, Candice Hesse + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m508848200

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Abstract

Lipopolysaccharide (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 overexpression of the CstF-64 protein also induced alternative poly(A) site selection on the reporter minigene. Microarray analysis performed on CstF-64 overexpressing 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-overexpressing conditions. We concluded 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.

Highlights

We have shown that LPS stimulation of murine RAW macrophages stimulated with LPS for 18 h exhibited cleavage stimulatory factor (CstF)-64 expression levels ϳ5-fold over basal expression and occurred during the cessation of the cell cycle (Fig. 1)
We further showed that LPS stimulation of both RAW macrophages and murine bone marrow-derived macrophages (BMDMs) increased CstF-64 in that several other proteins associated with pre-mRNA cleavage/polyadenylation did not change expression (Fig. 2)
Microarray analysis revealed that LPS stimulation of RAW macrophages for 18 h significantly increased the expression of 245 known genes and 45 unique expressed sequence tags (ESTs)

Summary

Introduction

CstF-64 Induces Alternative Polyadenylation in Macrophages product [12], we hypothesized that the increased levels of CstF-64 observed in LPS-stimulated macrophages would have a similar effect on poly(A) site choice in many genes. Closer analysis of two of the genes whose expression changed under both LPS-stimulating and CstF-64overexpressing conditions, Id-2 and Mmp-9, revealed that an alternative polyadenylation event does occur on these gene transcripts and that this change in poly(A) site choice may contribute to the increase of mature transcripts by the removal of mRNA instability elements. We conclude that increases in expression of the pre-mRNA cleavage factor CstF-64 observed in LPS-stimulated macrophages significantly contribute to changes in the expression of a multitude of genes through alternative polyadenylation events that occur in the context of infection by Gram-negative bacteria

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