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.
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.
Shell
Scott A
SA
Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15221, USA.
Hesse
Candice
C
Morris
Sidney M
SM
Milcarek
Christine
C
D020413:3' Untranslated Regions; D000465:Algorithms; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D039241:Cleavage Stimulation Factor / Q000737:chemistry* / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D008957:Models, Genetic; D015233:Models, Statistical; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D011061:Poly A / Q000737:chemistry*; D026723:Polyadenylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; D039241:Cleavage Stimulation Factor; C492658:Cstf2t protein, mouse; D008070:Lipopolysaccharides; D009841:Oligonucleotides; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA