Identification of mRNA targets and functional characterization of tristetraprolin (TTP) family proteins in Drosophila and mouse preadipocytes
Date Issued
2012
Date
2012
Author(s)
Yang, Wen-Hsuan
Abstract
The turnover of AU-rich element (ARE)-containing mRNAs is post-transcriptionally regulated by one or more RNA-binding proteins, such as tristetraprolin (TTP) family proteins. In rodent, there are four proteins belonging to TTP family: TTP (TIS11, ZFP36), ZFP36L1 (TIS11b, BRF-1), ZFP36L2 (TIS11d, BRF-2), and ZFP36L3. In Drosophila, only one TTP family protein has been reported (DTIS11). TTP family proteins have two tandem CCCH zinc finger (TZF) domains that bind to ARE of target mRNAs and then cause the mRNAs to be destabilized by recruitment of 5'' and 3'' mRNA degradation complexes. To identify their mRNA targets and the functional effect of TTP family proteins, the overexpression and knockdown were performed in Drosophila cells and mouse preadipocytes, respectively. The eyes absent (eya) transcript is one of many ARE-containing mRNAs in Drosophila. RNA pull-down and luciferase reporter analyses demonstrated that the DTIS11 RNA-binding domain is required for DTIS11 to bind the eya 3'' UTR and reduce levels of eya mRNA. Moreover, ectopic expression of DTIS11 in Drosophila Schneider 2 (S2) cells decreased levels of eya mRNA and reduced cell viability. Consistent with these results, TTP proteins overexpressed in MCF7 human breast cancer cells were associated with eya homologue 2 (EYA2) mRNA, and caused a decrease in EYA2 mRNA stability and cell viability which may be caused by apoptosis. Our results demonstrated that eya mRNA is a novel target of TTP protein. In the second part of the thesis, the functional analyses of ZFP36L1 and ZFP36L2 in regulating mitogen-activated protein kinase (MAPK) phosphatase-1 (Mkp-1) mRNA and adipogenesis were investigated. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3'' untranslated region (UTR) of Mkp-1 mRNA and downregulated Mkp-1 3''UTR-mediated luciferase activity. Knockdown of ZFP36L1, but not ZFP36L2, increased basal levels of immediate early genes including Mkp-1 mRNA and decreased ERK activation. We also found that knockdown of constitutive expression of ZFP36L1 and ZFP36L2 would inhibit and slightly enhance differentiation of mouse 3T3-L1 preadipocyte, respectively. These results suggested ZFP36L1 and ZFP36L2 may regulate the expression of ARE-containing mRNA and thus adipogenesis differently. Collectively, our findings indicate that TTP family proteins modulate cell viability or cell differentiation through destabilizing some mRNA targets.
Subjects
post-transcriptional regulation
tristetraprolin (TTP)
eyes absent (eya)
MAPK phosphatase-1 (MKP-1)
ZFP36L1
SDGs
Type
thesis
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