FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation.
FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation.
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DOI：
10.1093/nar/gkq1295
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发表时间：
2011-05
影响因子：
14.9
通讯作者：
Sun HS
中科院分区：
文献类型：
--
作者：
Gau BH;Chen TM;Shih YH;Sun HS
FGF 9 mRNA的3′-非翻译区（UTR）中的TG微卫星先前已被证明可调节FGF 9的表达。在本研究中，我们研究了可能的相互作用蛋白，结合FGF 9 3′-UTR UG-重复序列，并研究这种蛋白质-RNA相互作用的机制。我们首先应用RNA下拉分析和LC-MS分析来鉴定与该重复序列相关的蛋白质。在所鉴定的蛋白质中，FUBP 3特异性结合合成的（UG）15寡核糖核苷酸，如RNA-EMSA实验中的超位移所示。在HEK 293细胞中，内源性FGF 9蛋白响应于瞬时过表达而上调，并且在FUBP 3敲低后下调。由于FGF 9 mRNA的相对水平在这两种条件下相似，并且FUBP 3的消耗对FGF 9 mRNA的转换率没有影响，因此这些数据表明FUBP 3在转录后水平调节FGF 9表达。使用核糖体复合物下拉分析的进一步检查显示FUBP 3的过表达促进FGF 9表达。相反，FUBP 3敲低的HEK 293细胞中多核糖体相关的FGF 9 mRNA显著降低。最后，报告基因分析表明，（UG）-基序与FUBP 3的协同效应，微调FGF 9的表达。总之，本研究的结果表明FUBP 3的新的RNA结合特性和FUBP 3与FGF 9 3′-UTR UG-repeat之间的相互作用促进FGF 9 mRNA的翻译。
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.
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