缺氧(Hypoxia)是一種細胞或組織中氧分壓下降的現象。 已知許多心血管疾病，肺部疾病和癌症等人類疾病常常都伴隨著組織和細胞的缺氧。由於氧氣分壓的改變，許多的細胞生理機制都會有所調整，以適應環境的改變。基因的轉譯(Translation)便是其中一個例子，已知在缺氧的情況下一般基因轉譯的機制(Cap-dependent translation) 會被抑制，而一些在缺氧時維持細胞生存必須的基因為了維持其表現量，便會利用其他的轉譯起始機制，如核醣體插入位點(Internal ribosome binding site; IRES)啟動的轉譯調控來達到目的。 IRES序列位於訊息核糖酸(mRNA) 的五端未轉譯區(5’UTR) 內， 並且會形成複雜的三級結構。過去的研究已證實了一群被稱之為IRES作用因子(IRES trans-acting factor ; ITAF)的蛋白會直接的參與在基因轉譯的起始，協助帶有IRES序列的mRNA召集轉譯起始因子(initiation factors)以及核醣體(ribosome)開始進行轉譯。本實驗室先前的研究發現，人類第九號纖維母細胞生長因子(Fibroblast growth factor 9; FGF9) 的mRNA 5’UTR帶有IRES序列，且在缺氧時FGF9 mRNA會藉由IRES轉譯機制增加其蛋白的產量。本研究的目的是為了更進一步了解FGF9 在缺氧情況下是如何啟動IRES轉譯機制以及找出參與在其中的ITAF。 我們合成不同片段FGF9 5’UTR 的RNA探針藉由RNA下拉反應分離出會與不同片段RNA結合的RNA結合蛋白(RNA binding protein ; RBPs)，接著利用質譜儀鑑定技術找出了hnRNP M和 NF90 會與FGF9的IRES結合。我們亦利用西方點墨法(Western blot)證實了此蛋白與FGF9 5’UTR IRES結合的特異性。此外，我們也藉由western blot的實驗發現了HuR與hnRNP C1/C2這幾個常見的ITAF與FGF9 5’UTR mRNA非IRES的區域結合。此外， 我們將細胞經過不同時間的缺氧處理並研究ITAF在細胞核及細胞質之間的分布情形。結果顯示NF90和hnRNP M在細胞缺氧狀態下會自細胞核往細胞質移動，這樣的結果暗示了NF90和hnRNP M極有可能參與在缺氧下FGF9利用IRES進行轉譯的機制中。藉由短小核酸 (shRNA)干擾技術，我們發現到抑制hnRNP M或NF90的表現皆會同時抑制FGF9蛋白在缺氧狀態下的轉譯。這樣的結果顯示了hnRNP M和NF90在缺氧下FGF9利用IRES進行轉譯的機制中扮演著重要的角色。總和以上的結果，我們認為NF90和hnRNP M可能是FGF9 IRES的作用因子，而HuR以及hnRNP C1/C2則可能扮演穩定FGF9 mRNA的角色。到目前為止，我們的實驗結果顯示了NF90和hnRNP M是FGF9 IRES的潛在ITAF ，在缺氧的環境下，這些ITAFs會與其他RNA結合蛋白如 HuR 及hnRNP C1/C2一起作用來啟動IRES轉譯機制，進而達到快速增加FGF9蛋白質量的目的。

Hypoxia is a reduction in the normal level of tissue oxygen tension, and involved in many human diseases including vascular diseases, pulmonary diseases and cancers. Under hypoxia, canonical cap-dependent translation is generally inhibited, many hypoxia-responding genes initiate their translations through an alternative mechanism known as internal ribosome entry site (IRES)-mediated translation. The IRES-mediated translation requires RNA sequence to form a complex tertiary structure in the 5’untranslated region (5’UTR). It has been demonstrated that some proteins, named IRES trans-acting factors or ITAFs, are interacting with IRES-containing mRNA and recruiting ribosomes to the IRES element. Thus, ITAFs directly involve in the initiation of IRES-dependent translation. Our previous study showed that FGF9 5’UTR contains an IRES element and FGF9 protein expression is up-regulated through IRES-mediated transactivation during hypoxia. To identify the ITAFs interact with FGF9 5’UTR, we performed RNA pull-down assay followed by mass spectrometry analysis. The heterogeneous nuclear ribonucleoprotein M (hnRNP M) and Double strand RNA-binding protein 76 (NF90) were pulled down using FGF9 IRES and confirmed the specific binding using western blots. In addition, screening the association of FGF9 mRNA 5’UTR and common ITAFs have identified Hu antigen R (HuR) and heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C1/C2) bound to the non-IRES region of FGF9 5’UTR. Time course experiment was performed and fractionated proteins were collected to show the effect of hypoxia on relative abundance and subcellular distribution of these ITAFs. Our data showed that both NF90 and hnRNP M relocated from nucleus to cytoplasm after hypoxic treatment. The results suggest that NF90 and hnRNP M may participate in the hypoxia-induced IRES-mediated FGF9 protein expression. In addition, by using the shRNA knockdown strategy, we found that both of NF90 and hnRNP M played important role in hypoxia-induced, FGF9 IRES-mediated translation. Based on our findings, we propose that hnRNP M and NF90 are the potential ITAFs of FGF9 IRES, together with other well characterized RNA binding proteins like hnRNP C1/C2 and HuR, these proteins coordinately regulated FGF9 protein expression during hypoxia.

Abdel-Rahman, W.M., Kalinina, J., Shoman, S., Eissa, S., Ollikainen, M., Elomaa, O., Eliseenkova, A.V., Butzow, R., Mohammadi, M., and Peltoma¨ki, P.i. (2008). Somatic FGF9 Mutations in Colorectal and Endometrial Carcinomas Associated With Membranous b-Catenin. Human Mutation 29, 390-397.
Balvay, L., Soto Rifo, R., Ricci, E.P., Decimo, D., and Ohlmann, T. (2009). Structural and functional diversity of viral IRESes. Biochimica et Biophysica Acta 1789, 542-557.
Bergalet, J., Fawal, M., Lopez, C., Desjobert, C., Lamant, L., Delsol, G., Morello, D., and Espinos, E. (2011). HuR-Mediated Control of C/EBP mRNA Stability and Translation in ALK-Positive Anaplastic Large Cell Lymphomas. Molecular Cancer Research 9, 485-496.
Bert Schepens, S.A.T., Yanik Bruynooghe, Rudi Beyaert and Sigrid Cornelis (2005). The polypyrimidine tract-binding protein stimulates HIF-1α IRES-mediated translation during hypoxia.pdf. Nucleic Acids Research 33, 6884–6894.
Bertout, J.A., Patel, S.A., and Simon, M.C. (2008). The impact of O2 availability on human cancer. Nature Reviews Cancer 8, 967-975.
Bonnal, S., Pileur, F., Orsini, C., Parker, F., Pujol, F., Prats, A.C., and Vagner, S. (2005). Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA. The Journal of Biological Chemistry 280, 4144-4153.
Chen, T.M., Hsu, C.H., Tsai, S.J., and Sun, H.S. (2010). AUF1 p42 isoform selectively controls both steady-state and PGE2-induced FGF9 mRNA decay. Nucleic Acids Research 38, 8061-8071.
Chen, T.M., Kuo, P.L., Hsu, C.H., Tsai, S.J., Chen, M.J., Lin, C.W., and Sun, H.S. (2007). Microsatellite in the 3' untranslated region of human fibroblast growth factor 9 (FGF9) gene exhibits pleiotropic effect on modulating FGF9 protein expression. Human Mutation 28, 98.
Chen, T.M., Shih, Y.-H., Tseng, J.T., Lai, M.-C., Wu, C.-H., Li, Y.-H., Tsai, S.-J., and Sun, H.S. (2013). Overexpression of FGF9 in colon cancer cells is mediated by hypoxia-induced translational activation.
Chuang, P.C., Sun, H.S., Chen, T.M., and Tsai, S.J. (2006). Prostaglandin E2 induces fibroblast growth factor 9 via EP3-dependent protein kinase Cdelta and Elk-1 signaling. Molecular and Cellular Biology 26, 8281-8292.
Colvin, J.S., Green, R.P., Schmahl, J., Capel, B., and Ornitz, D.M. (2001). Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 104, 875-889.
Cornelis, S. (2007). A role for hnRNP C1/C2 and Unr in internal
initiation of translation during mitosis. The EMBO journal 26, 158-169.
Dewhirst, M.W., Cao, Y., and Moeller, B. (2008). Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response. Nature Reviews Cancer 8, 425-437.
Dobbyn, H.C., Hill, K., Hamilton, T.L., Spriggs, K.A., Pickering, B.M., Coldwell, M.J., de Moor, C.H., Bushell, M., and Willis, A.E. (2008). Regulation of BAG-1 IRES-mediated translation following chemotoxic stress. Oncogene 27, 1167-1174.
Durie, D., Lewis, S.M., Liwak, U., Kisilewicz, M., Gorospe, M., and Holcik, M. (2011). RNA-binding protein HuR mediates cytoprotection through stimulation of XIAP translation. Oncogene 30, 1460-1469.
Fitzgerald, K.D., and Semler, B.L. (2009). Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. Biochimica et Biophysica Acta 1789, 518-528.
Fuchs, J.-P. (1996). The human hnRNP-M proteins: structure and relation
with early heat shock-induced splicing arrest and chromosome mapping. Nucleic Acids Research, 24, 2535-2542.

Galban, S., Kuwano, Y., Pullmann, R., Jr., Martindale, J.L., Kim, H.H., Lal, A., Abdelmohsen, K., Yang, X., Dang, Y., Liu, J.O., et al. (2008). RNA-binding proteins HuR and PTB promote the translation of hypoxia-inducible factor 1alpha. Molecular and Cellular Biology 28, 93-107.
Gau, B.H., Chen, T.M., Shih, Y.H., and Sun, H.S. (2011). FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation. Nucleic Acids Research 39, 3582-3593.
Gingras, A.C., Raught, B., and Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annual Review of Biochemistry 68, 913-963.
Gorospe, M. (2010). NF90 selectively represses the translation of target mRNAs bearing an AUrich signature motif. Nucleic Acids Research 38, 225-238.
Harris, A.L. (2002). Hypoxia--a key regulatory factor in tumour growth. Nature reviews Cancer 2, 38-47.
Hellen, C.U. (2009). IRES-induced conformational changes in the ribosome and the mechanism of translation initiation by internal ribosomal entry. Biochimica et Biophysica Acta 1789, 558-570.
Hendrix, N.D., Wu, R., Kuick, R., Schwartz, D.R., Fearon, E.R., and Cho, K.R. (2006). Fibroblast growth factor 9 has oncogenic activity and is a downstream target of Wnt signaling in ovarian endometrioid adenocarcinomas. Cancer Research 66, 1354-1362.
Holcik, M., Gordon, B.W., and Korneluk, R.G. (2003). The Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Protein XIAP Is Modulated by the Heterogeneous Nuclear Ribonucleoproteins C1 and C2. Molecular and Cellular Biology 23, 280-288.
Holcik, M., and Sonenberg, N. (2005). Translational control in stress and apoptosis. Nature Reviews Molecular Cell Biology 6, 318-327.
Hovhannisyan, R.H., and Carstens, R.P. (2007). Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons. The Journal of Biological Chemistry 282, 36265-36274.
Hsiao, C.D., Ekker, M., and Tsai, H.J. (2003). Skin-specific expression of ictacalcin, a homolog of the S100 genes, during zebrafish embryogenesis. Developmental Dynamics 228, 745-750.
Jang, S.K., Krausslich, H.G., Nicklin, M.J., Duke, G.M., Palmenberg, A.C., and Wimmer, E. (1988). A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. Journal of Virology 62, 2636-2643.
Kettunen, P., and Thesleff, I. (1998). Expression and function of FGFs-4, -8, and -9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis. Developmental Dynamics 211, 256-268.
Komar, A.A., and Hatzoglou, M. (2005). Internal ribosome entry sites in cellular mRNAs: mystery of their existence. The Journal of Biological Chemistry 280, 23425-23428.
Komar, A.A., and Hatzoglou, M. (2011). Cellular IRES-mediated translation: The war of ITAFs in pathophysiological states. Cell Cycle 10, 229-240.
Krecic, A.M., and Swanson, M.S. (1999). hnRNP complexes: composition, structure, and function. Current Opinion in Cell Biology 11, 363-371.
Kuwano, Y., Kim, H.H., Abdelmohsen, K., Pullmann, R., Jr., Martindale, J.L., Yang, X., and Gorospe, M. (2008). MKP-1 mRNA stabilization and translational control by RNA-binding proteins HuR and NF90. Molecular and Cellular Biology 28, 4562-4575.
Lazaris-Karatzas, A., Montine, K.S., and Sonenberg, N. (1990). Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5' cap. Nature 345, 544-547.
Leppek, K., Schott, J., and Stoecklin, G. (2011). Protein synthesis and translational control: at eye level with the ribosome. EMBO Reports 12, 1214-1216.
Leushacke, M., Sporle, R., Bernemann, C., Brouwer-Lehmitz, A., Fritzmann, J., Theis, M., Buchholz, F., Herrmann, B.G., and Morkel, M. (2011). An RNA interference phenotypic screen identifies a role for FGF signals in colon cancer progression. PloS One 6, e23381.
Lewis, S.M., and Holcik, M. (2008). For IRES trans-acting factors, it is all about location. Oncogene 27, 1033-1035.
Lewis, S.M., Veyrier, A., Hosszu Ungureanu, N., Bonnal, S., Vagner, S., and Holcik, M. (2007). Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation. Molecular Biology of the Cell 18, 1302-1311.
Li, Z.G., Mathew, P., Yang, J., Starbuck, M.W., Zurita, A.J., Liu, J., Sikes, C., Multani, A.S., Efstathiou, E., Lopez, A., et al. (2008). Androgen receptor-negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms. The Journal of Clinical Investigation 118, 2697-2710.
Lin, J.C., Hsu, M., and Tarn, W.Y. (2007). Cell stress modulates the function of splicing regulatory protein RBM4 in translation control. PNAS 104, 2235-2240.
Lovicu, F.J., and Overbeek, P.A. (1998). Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice. Development 125, 3365-3377.
Macejak, D.G., and Sarnow, P. (1991). Internal initiation of translation mediated by the 5' leader of a cellular mRNA. Nature 353, 90-94.
Majumder, M., Yaman, I., Gaccioli, F., Zeenko, V.V., Wang, C., Caprara, M.G., Venema, R.C., Komar, A.A., Snider, M.D., and Hatzoglou, M. (2009). The hnRNA-binding proteins hnRNP L and PTB are required for efficient translation of the Cat-1 arginine lysine transporter mRNA during amino acid starvation. Molecular and Cellular Biology 29, 2899-2912.
Mamane, Y., Petroulakis, E., LeBacquer, O., and Sonenberg, N. (2006). mTOR, translation initiation and cancer. Oncogene 25, 6416-6422.
Mamane, Y., Petroulakis, E., Rong, L., Yoshida, K., Ler, L.W., and Sonenberg, N. (2004). eIF4E--from translation to transformation. Oncogene 23, 3172-3179.
Marcoulatos, P., Koussidis, G., Mamuris, Z., Velissariou, V., and Vamvakopoulos, N.C. (1996). Mapping interleukin enhancer binding factor 2 gene (ILF2) to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates. Journal of Interferon & Cytokine Research 16, 1035-1038.
Mariani, F.V., Ahn, C.P., and Martin, G.R. (2008). Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning. Nature 453, 401-405.
Mattei, M.G., Penault-Llorca, F., Coulier, F., and Birnbaum, D. (1995). The human FGF9 gene maps to chromosomal region 13q11-q12. Genomics 29, 811-812.
Mazan-Mamczarz, K., Galban, S., de Silanes, I.L., Martindale, J.L., Atasoy, U., Keene, J.D., and Gorospe, M. (2003). RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation. PNAS 100, 8354-8359.
Miyamoto, M., Naruo, K., Seko, C., Matsumoto, S., Kondo, T., and Kurokawa, T. (1993). Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property. Molecular and Cellular Biology 13, 4251-4259.
Mokrejs, M., Masek, T., Vopalensky, V., Hlubucek, P., Delbos, P., and Pospisek, M. (2010). IRESite--a tool for the examination of viral and cellular internal ribosome entry sites. Nucleic Acids Research 38, D131-136.
Parrott, A.M., Walsh, M.R., Reichman, T.W., and Mathews, M.B. (2005). RNA binding and phosphorylation determine the intracellular distribution of nuclear factors 90 and 110. Journal of Molecular Biology 348, 281-293.
Pelletier, J., Kaplan, G., Racaniello, V.R., and Sonenberg, N. (1988). Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Molecular and Cellular Biology 8, 1103-1112.
Pelletier, J., and Sonenberg, N. (1988). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320-325.
Petz, M., Them, N., Huber, H., Beug, H., and Mikulits, W. (2012). La enhances IRES-mediated translation of laminin B1 during malignant epithelial to mesenchymal transition. Nucleic Acids Research 40, 290-302.
Pirvola, U., Zhang, X., Mantela, J., Ornitz, D.M., and Ylikoski, J. (2004). Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions. Developmental Biology 273, 350-360.
Pogach, M.S., Cao, Y., Millien, G., Ramirez, M.I., and Williams, M.C. (2007). Key developmental regulators change during hyperoxia-induced injury and recovery in adult mouse lung. Journal of Cellular Biochemistry 100, 1415-1429.
Robinson, D., Hasharoni, A., Oganesian, A., Sandell, L.J., Yayon, A., and Nevo, Z. (2001). Role of FGF9 and FGF receptor 3 in osteochondroma formation. Orthopedics 24, 783-787.
Schepens, B., Tinton, S.A., Bruynooghe, Y., Beyaert, R., and Cornelis, S. (2005). The polypyrimidine tract-binding protein stimulates HIF-1alpha IRES-mediated translation during hypoxia. Nucleic Acids Research 33, 6884-6894.
Schmidt, E.V. (2004). The role of c-myc in regulation of translation initiation. Oncogene 23, 3217-3221.
Seo, M., and Noguchi, K. (1995). Retinoic acid induces gene expression of fibroblast growth factor-9 during induction of neuronal differentiation of mouse embryonal carcinoma P19 cells. FEBS letters 370, 231-235.
Shamanna, R.A., Hoque, M., Lewis-Antes, A., Azzam, E.I., Lagunoff, D., Pe'ery, T., and Mathews, M.B. (2011). The NF90/NF45 complex participates in DNA break repair via nonhomologous end joining. Molecular and Cellular Biology 31, 4832-4843.
Shi, L., Zhao, G., Qiu, D., Godfrey, W.R., Vogel, H., Rando, T.A., Hu, H., and Kao, P.N. (2005). NF90 regulates cell cycle exit and terminal myogenic differentiation by direct binding to the 3'-untranslated region of MyoD and p21WAF1/CIP1 mRNAs. The Journal of Biological Chemistry 280, 18981-18989.
Sonenberg, N., and Hinnebusch, A.G. (2009). Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136, 731-745.
Spriggs, K.A., Bushell, M., Mitchell, S.A., and Willis, A.E. (2005). Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors. Cell Death and Differentiation 12, 585-591.
Spriggs, K.A., Bushell, M., and Willis, A.E. (2010). Translational regulation of gene expression during conditions of cell stress. Molecular Cell 40, 228-237.
Steitz, C.M.B.a.J.A. (2001). HuR and mRNA stability. Cellular And Molecular Life Sciences 58, 266-277.
Stoneley, M., Paulin, F.E., Quesne, J.P.L., Chappell, S.A., and Willis, A.E. (1998). C-Myc 5' untranslated region contains an internal ribosome entry segment. Oncogene 16, 423-428.
Teishima, J., Shoji, K., Hayashi, T., Miyamoto, K., Ohara, S., and Matsubara, A. (2012). Relationship between the localization of fibroblast growth factor 9 in prostate cancer cells and postoperative recurrence. Prostate Cancer and Prostatic Diseases 15, 8-14.
Uniacke, J., Holterman, C.E., Lachance, G., Franovic, A., Jacob, M.D., Fabian, M.R., Payette, J., Holcik, M., Pause, A., and Lee, S. (2012). An oxygen-regulated switch in the protein synthesis machinery. Nature 486, 126-129.
Vumbaca, F., Phoenix, K.N., Rodriguez-Pinto, D., Han, D.K., and Claffey, K.P. (2008). Double-stranded RNA-binding protein regulates vascular endothelial growth factor mRNA stability, translation, and breast cancer angiogenesis. Molecular and Cellular Biology 28, 772-783.
Waskiewicz, A.J., Flynn, A., Proud, C.G., and Cooper, J.A. (1997). Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. The EMBO Journal 16, 1909-1920.
Wing, L.Y., Chuang, P.C., Wu, M.H., Chen, H.M., and Tsai, S.J. (2003). Expression and mitogenic effect of fibroblast growth factor-9 in human endometriotic implant is regulated by aberrant production of estrogen. The Journal of Clinical Endocrinology and Metabolism 88, 5547-5554.
Wouters, B.G., and Koritzinsky, M. (2008). Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nature Reviews Cancer 8, 851-864.
Wu, X.L., Gu, M.M., Huang, L., Liu, X.S., Zhang, H.X., Ding, X.Y., Xu, J.Q., Cui, B., Wang, L., Lu, S.Y., et al. (2009). Multiple synostoses syndrome is due to a missense mutation in exon 2 of FGF9 gene. American Journal of Human Genetics 85, 53-63.
Young, R.M., Wang, S.J., Gordan, J.D., Ji, X., Liebhaber, S.A., and Simon, M.C. (2008). Hypoxia-mediated selective mRNA translation by an internal ribosome entry site-independent mechanism. The Journal of Biological Chemistry 283, 16309-16319.