FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation - PubMed (original) (raw)

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

Bing-Huang Gau et al. Nucleic Acids Res. 2011 May.

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.

© The Author(s) 2011. Published by Oxford University Press.

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Figures

Figure 1.

Figure 1.

FGF9 3′-UTR RNA structure prediction. (A) Sequence analysis of human FGF9 3′-UTR. The sequence corresponding to the 3′-end of the gene is depicted. The TGA nucleotide sequence marked in bold type corresponds to the translation stop codon. The (GA) and (TG) MS motifs are marked in bold and underlines, and the AU-rich element (ARE) sequence and poly-A signals are marked with double underlines. (B) Representative RNA structure of full-length FGF9 3′-UTR sequence (NM_002010; 613 nt). (C) Enlargement of boxed area in B. Curved lines mark the locations of indicated UG repeats. MFE of predicted structure under each indicated UG repeat in units of kcal/mol.

Figure 2.

Figure 2.

Riboprotein complex formation by UG repeats of FGF9 3′-UTR. (A) UV-cross-linking was conducted using biotinylated (UG)15 oligoribonucleotide and protein extracts (cytoplasmic extract, CE; nuclear extract, NE) prepared from HEK293 (left) and NT2D1 (right) cells. Excess molar ratio of unlabeled cold probe was added as indicated in the competition assay. (B) The same labeled probe, cold probe and protein extracts as in (A), were used in RNA-EMSA and showed similar results in HEK293 (left) and NT2D1 (right) cells. C1–C6: complexes 1–6. (C) (UG)15 binding proteins identified by LC–MS. Proteins pulled down in the absence (left lane) or the presence of riboprobe (UG)15 (right lane) were assayed by silver stain. Differentially expressed bands (marked by 1–8) were excised for protein identification by LC–MS.

Figure 3.

Figure 3.

Identification of FUBP3 as UG-repeat binding protein. (A) The RNA–protein complex was detected using specific antibodies as indicated in RNA-EMSA in the protein extracts from NT2D1 (left) and HEK293 (right) cells. CE, cytoplasmic extract; NE, nuclear extract. IgG was used as a control. (B) Pull-down assays were performed using biotinylated (UG)15 riboprobe and _in vitro_-transcribed FGF9 3′-UTR containing (UG)15 repeats in the cytoplasmic extract from HEK293 cells. Specific antibodies to FUBP3, GAPDH and AUF1 were used to detect the pulled-down proteins. H2O and _in vitro_-transcribed GAPDH probe were used as negative controls for pull-down assays. (C) Immunoprecipitated RNA–protein complexes using anti-FUBP3 antibody showed the presence of endogenous FGF9, but not GAPDH mRNA in HEK293 cells (lane 2). Goat normal IgG was used as a control for the immunoprecipitation reaction. The enrichment of FGF9 mRNA precipitated by anti-FUBP3 antibody is shown relative to goat normal IgG. Data were normalized to GAPDH.

Figure 4.

Figure 4.

FUBP3 increased the expression of endogenous FGF9 and reporter proteins containing FGF9 3′-UTR. (A) Representative western blots and RT-PCR results from transient FUBP3 overexpression and shRNA knockdown. Endogenous FUBP3 (filled triangle) and overexpressed FUBP3 (open triangle) were shown in the top panel. Relative mRNA level (RT-PCR; middle panel) and protein amount (ELISA; bottom panel) were shown under each treatments. (B) RNA stability assay showed that knockdown of FUBP3 had no effect on FGF9 turn-over rate. (C) FUBP3 overexpression promotes FGF9 protein synthesis in HEK293 cells. The ribosome complexes were pulled down by 40S ribosomal protein S6 antibody from HEK293 cells overexpressing recombinant FUBP3. The mRNAs bound with ribosome complexes were extracted and analyzed by quantitative RT-PCR. FGF9 mRNA expression level was detected and normalized with the mRNA expression level of GAPDH. The total RNA was also extracted from HEK293 cell with or without FUBP3-overexpression (Right), and the FGF9 mRNA level was detected as an experimental comparison to the results showed in (Left). (D) Relative translational efficiency representation of FGF9 and GAPDH in the polysomal fractions. Ribosome-associated transcripts were measured using quantitative RT-PCR and translational efficiency of FGF9 and GAPDH under FUBP3-knockdown were normalized with the translational efficiency in GFP-knockdown cells. **P < 0.01; ***P < 0.001.

Figure 5.

Figure 5.

Synergistic effect of FUBP3 with FGF9 (UG)n repeats. (A) Schematic diagram of reporter constructs containing partial FGF9 3′-UTR with various TG repeats. Reporter activities of various constructs from HEK293 cells overexpressing (B) or knockdown (C) FUBP3. All values were normalized to Renilla luciferase activity produced from a cotransfected control plasmid. Error bars represent standard deviations from three independent transfections. Statistical analyses were conducted by one-way ANOVA. *P < 0.05; **P < 0.01.

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