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FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation.

ABSTRACT: A TG microsatellite in the 3'-untranslated region (UTR) of FGF9 mRNA has previously been shown to modulate FGF9 expression. In the present study, we investigate the possible interacting protein that binds to FGF9 3'-UTR UG-repeat and study the mechanism underlying this protein-RNA interaction. We first applied RNA pull-down assays and LC-MS analysis to identify proteins associated with this repetitive sequence. Among the identified proteins, FUBP3 specifically bound to the synthetic (UG)(15) oligoribonucleotide as shown by supershift in RNA-EMSA experiments. The endogenous FGF9 protein was upregulated in response to transient overexpression and downregulated after knockdown of FUBP3 in HEK293 cells. As the relative levels of FGF9 mRNA were similar in these two conditions, and the depletion of FUBP3 had no effect on the turn-over rate of FGF9 mRNA, these data suggested that FUBP3 regulates FGF9 expression at the post-transcriptional level. Further examination using ribosome complex pull-down assay showed overexpression of FUBP3 promotes FGF9 expression. In contrast, polyribosome-associated FGF9 mRNA decreased significantly in FUBP3-knockdown HEK293 cells. Finally, reporter assay suggested a synergistic effect of the (UG)-motif with FUBP3 to fine-tune the expression of FGF9. Altogether, results from this study showed the novel RNA-binding property of FUBP3 and the interaction between FUBP3 and FGF9 3'-UTR UG-repeat promoting FGF9 mRNA translation.

SUBMITTER: Gau BH

PROVIDER: S-EPMC3089454 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation.

Gau Bing-Huang BH Chen Tsung-Ming TM Shih Yu-Heng J YH Sun H Sunny HS

Nucleic acids research 20110119 9

A TG microsatellite in the 3'-untranslated region (UTR) of FGF9 mRNA has previously been shown to modulate FGF9 expression. In the present study, we investigate the possible interacting protein that binds to FGF9 3'-UTR UG-repeat and study the mechanism underlying this protein-RNA interaction. We first applied RNA pull-down assays and LC-MS analysis to identify proteins associated with this repetitive sequence. Among the identified proteins, FUBP3 specifically bound to the synthetic (UG)(15) oli ...[more]

PMID: 21252297

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Project description:Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation.The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.

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FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation.

Publications

FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation.

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