Tumor necrosis factor alpha (TNF), a proinflammatory cytokine produced primarily by macrophages and crucial in immune responses, is regulated both transcriptionally and posttranscriptionally upon exposure to various pathogens. Posttranscriptional regulation of TNF expression depends on interaction of RNA-binding proteins with an AU-rich element (ARE) within the 3' untranslated region of TNF mRNA that modulates translational efficacy and mRNA stability. We screened a macrophage protein expression library with a TNF-ARE cRNA probe and identified the RNA-binding protein FXR1P, closely related to fragile X mental retardation syndrome protein FMRP. Macrophage cell lines were generated from FXR1 knockout mice and their littermate controls. The FXR1 knockout macrophages had enhanced TNF protein production following activation compared to control macrophages. Expression of several other proteins encoded by ARE-containing transcripts was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3'-UTR of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. The ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes, demonstrating the important role of FXR1P in the posttranscriptional regulation of TNF expression. Our data suggests that release of this repression by FXR1P occurs during LPS-induced macrophage activation. Complementation of the knockout macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation. Using 2D electrophoresis gel coupled with mass spectrometry to explore the functional role of FXR1P in macrophages, we showed that FXR1P deficiency had an impact on several proteins, including G3BP1 and CBF-A that have been linked to posttranscriptional regulation. Finally, phosphorylation analysis of FXR1P indi
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.85910 |
| Date | January 2005 |
| Creators | Garnon, James |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002260899, proquestno: AAINR21647, Theses scanned by UMI/ProQuest. |
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