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

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.As a critical component of the innate immune system, macrophages respond to LPS 2 from Gram-negative bacteria by activating multiple signaling pathways that result in the rapid expression of pro-inflammatory cytokines and arachidonic acid metabolites. The induced expression of many of these genes is attributed to the activation of transcription factors linked to Toll-like receptor 4-related signal transduction pathways (reviewed in Refs. 1 and 2). Aside from transcriptional contributions to gene expression in stimulated macrophages, post-transcriptional mechanisms have also been shown to influence gene expression. For example, the half-lives of TNF-␣ (3) and lysozyme (4) mRNAs are increased following LPS stimulation by a lengthening of the poly(A) tail. Additionally, translational regulatory and mRNA stability elements have been identified in the 3Ј-untranslated region (3Ј-UTR) of the LPSinducible mRNAs encoding COX-2 (5-7) and TNF-␣ (8, 9).Post-transcriptional pre-mRNA cleavage and polyadenylation, although essential in creating translationally competent mRNA, is a greatly under-appreciated contributor to the regulation of gene expression. Because it has been estimated that only 30% of primary transcripts are polyadenylated (10), changes in 3Ј-end processing can have a profound effect on the amount of mature transcript produced. The frequency with which a poly(A) site is selected for the cleavage/polyadenylation reaction is greatly impacted by the strength of that poly(A) site. Relevant to t...

CstF64: Cell Cycle Regulation and Functional Role in 3′ End Processing of Replication-Dependent Histone mRNAs

“…Based on previous indications that CstF64 might be regulated in the cell cycle or in response to the proliferation state of certain cells (29)(30)(31) and also based on the presence of CstF64 in the histone processing factor HLF (12), we decided to analyze the levels of CstF64 and CstF64 as well as those of other proven or potential HLF members during the cell cycle of HeLa cells. For this purpose, HeLa cells were arrested in G 2 /M by incubation with nocodazole and then released from this block.…”

MARCKS as a Negative Regulator of Lipopolysaccharide Signaling

“…Transcriptomic data in the NCBI Gene Expression Omnibus database (40), including more than 40 human and 60 murine data sets, overwhelmingly show that both MARCKS and MRP transcripts are strongly increased upon LPS stimulation (41,42). Upregulation was observed in monocytes, dendritic cells, epithelial cells, brain, spleen, and lung tissue cells.…”

The nuclear experience of CPEB: Implications for RNA processing and translational control

“…Antibodies used in this study include: rabbit anti-CPEB (Hake and Richter 1994), rabbit anti-HA (Covance), mouse antia-tubulin (Sigma-Aldrich), rabbit anti-histone H4 (Upstate), mouse anti-symplekin (BD Transduction Laboratories), mouse anti-RNA polymerase II 8WG16 (Upstate), rabbit anti-CPSF73 (a gift from D. Bentley, University of Colorado), rabbit anti-CPSF100 (a gift from J. Manley, Columbia University) (Takagaki and Manley 2000), rabbit anti-CstF64 (a gift from C. Milcarek, University of Pittsburgh) (Shell et al 2005), rabbit anti-CBP80 (a gift from E. Izaurralde, Max Planck Institute) (Izaurralde et al 1994), rabbit anti-PARN (a gift from M. Wormington, University of Virginia), rabbit anti-PAB2 (a gift from E. Wahle, University of Halle) (Krause et al 1994), mouse anti-actin, rabbit anti-Maskin (Stebbins-Boaz et al 1999), mouse anti-eIF4A3 (a gift from A. Krainer, Cold Spring Harbor Laboratory), and mouse anti-myc 9E10 (Hake and Richter 1994).…”