| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 106 === Regulation of mRNA stability is a critical step to control gene expression. It is usually mediated by the RNA-binding proteins (RNA-BP) that bind to the 3’-untranslated region of mRNA, such as AU-rich elements (AREs). Tristetraprolin (TTP) protein family containing conserved CCCH tandem zinc-finger and C-terminal NOT1-binding domains contributes to the destabilization of ARE-containing mRNA. There are four members, TTP (ZFP36), ZFP36L1, ZFP36L2 and ZFP36L3 in TTP protein family. The function of TTP protein family is affected by its protein phosphorylated states and protein-protein interaction.
Our previous study showed that both TTP and Zfp36l1 proteins were phosphorylated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In this report, we demonstrated that TTP was induced and phosphorylated at Ser-316 by ERK-signaling pathway in LPS-stimulated RAW264.7 cells, which disrupted its interaction with CCR4-NOT deadenylase complex and dephosphorylated by protein phosphatase 2A (PP2A). We also generate homozygous and heterozygous TTP knock-out cell lines by CRISPR/Cas9 system in RAW264.7 cell. Moreover, we provide evidence to prove that Ser-334 of Zfp36l1, the conserved residue of Ser-316, was phosphorylated by Rsk1, PKA and MK2. We found that MK2 may phosphorylate C-terminal domain of Zfp36l1 not only at Ser-334 but also at an additional site. In the biochemical function, phosphorylation of Zfp36l1 at Ser-334 would decline the ability to recruit the CCR4-NOT1 complex leading to decrease of MKP-1 ARE-mediated mRNA decay activity.
Additionally, we identified the transiently up-regulated genes after LPS treatment in published microarray data. Through bioinformatics and statistics, we analyze these genes in microRNA database and suggest some potential microRNAs involved in LPS-stimulated gene regulation. One of them, miR-27, was demonstrated to be up-regulated by LPS and correlated in down-regulation of Zfp36L1 through the 3’UTR of Zfp36l1 mRNA.
In our study, we revealed a consensus phosphorylated site on C-terminal domain of TTP protein family, which may regulate the functional activity and protein-protein interaction. Furthermore, miR27 is a potential microRNA that regulates the expression of Zfp36L1 in LPS-stimulated RAW264.7 cell.
|
|---|
