| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 101 === Post-transcriptional control comprises regulation of the stability or translational efficiency of mRNA. Microarray analyses have revealed that 40-50% of changes in gene expression in response to cellular signals occur at the level of mRNA stability. Adenine and uridine -rich element mediated decay is one of several mechanisms the degrade mRNAs in the post-transcriptional regulations. AU-rich elements are always found in 3’UTR of a variety of transcripts that encode immediate response genes such as cytokines and inflammatory mediators, like proto-oncogenes and transcription factors. TTP family proteins bind to ARE of target mRNA by zinc finger domain and promote degradation. There are four members belong to TTP family: TTP (ZFP36), ZFP36L1, ZFP36L2 and ZFP36L3 which is specifically expressed in the placenta and extraembryonic tissues. Currently, the gene regulation and functional characterization of Zfp36l2 are unclear. We observed that Zfp36l2 mRNA expression was decreased in LPS-stimulated RAW264.7 cells. RNA pull down assay have improved that the downregulation of Zfp36l2 mRNA was caused by TTP binding to its ARE, which was induced by NFкB signaling pathway in LPS-stimulated RAW264.7 cells. On the other hand, we have identified two mRNA targets of Zfp36l2, Cox2 and Mkp-1 mRNA, in resting RAW264.7 cells. To investigate the molecular mechanism of Zfp36l2-specific Cox2 mRNA downregulation, we characterized the functional domains of Zfp36l2 by using some deletion constructs. In Immunofluorescence staining, WT- Zfp36l2 dispersed in the cytoplasm evenly, whereas Zfp36l2-N-TZF formed distinct foci and located at processing body, and Zfp36l2 –TZF-C tended to gather at the nuclear membranes. The luciferase reporter analysis showed that N-terminus of Zfp36l2 is required for its mRNA downregulation activity. In addition, we observed that the LPS treatment would lead to a lasting decrease of Zfp36l2 expression for more than 16 h, which might cause a higher induction of anti-inflammatory Mkp-1and IL-10 mRNA and a lower induction of pro-inflammatory cytokine TNFα in response to the second LPS treatment. Our results indicate that the expression levels of Zfp36l2 are tightly controlled to regulate intracellular inflammatory response in LPS-stimulated macrophages.
|
|---|
