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研究生(外文):Ya-Hui Lin
論文名稱(外文):Functional characterization of Rbp1p interacting protein, Nrp1p, in Saccharomyces cerevisiae
外文關鍵詞:mitochondriaporinPUB1NRP1RBP1RNA-binding protein
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真核細胞中,訊息核糖核酸的完全成熟必須經過一連串的修飾過程,包括在五端加上覆蓋、疊接、以及在三端加上多個腺口票呤。在這些轉錄後調控的基因表現過程中,核糖核酸結合蛋白扮演非常重要的角色。我們之前的研究發現酵母菌中的核糖核酸結合蛋白Rbp1p可經由與粒腺體外膜孔蛋白﹙porin﹚訊息核糖核酸﹙mRNA﹚的三端非轉錄區域結合,藉以調控該核糖核酸之降解。在這裡我們將研究另一個與Rbp1p有相互作用的蛋白質:Nrp1p。Nrp1p轉譯出一個七百二十個胺基酸大小的蛋白質,結構上包括有一個核糖核酸結合區域,兩個鋅手指去氧核糖核酸結合區域,以及一個ASN富含區域。酵母菌雙雜合實驗指出Nrp1p與Rbp1p有直接的相互作用,進一步的免疫沉澱實驗更指出內源性的Nrp1p與Rbp1p形成一個蛋白質複合體。北方墨點分析指出,在無法表現Nrp1p的突變株中,porin mRNA的量比野生株要低;而免疫螢光染色及西方墨點分析也顯示porin蛋白質的表現量在過量表現Nrp1p的植株中要比野生株來得高,顯示Nrp1p很可能與porin的代謝有關。此外,Nrp1p與Rbp1p在酵母菌細胞裡的分布有部分相合。這些結果顯示Nrp1p是一個Rbp1p交互作用蛋白,並且可能一起調控porin mRNA的代謝與降解。
In eukaryotic cells, mRNAs undergo sequential processing reactions, including capping, splicing, and polyadenylation. RNA-binding proteins play key roles in these post-transcriptional regulations of gene expression. Our lab has recently demonstrated that Rbp1p can regulate porin mRNA stability through binding to 3’-UTR of porin mRNA. Here, we show that Nrp1p interacts with Rbp1p in two-hybrid interaction analysis. NRP1 encodes a 720 amino-acid protein, Nrp1p, which contains a RRM, two Zn finger motifs, and an ASN-rich region. By co-immunoprecipitation analysis, we showed that Nrp1p could pull down Rbp1p, and vice versa. Tandem affinity purification also showed that endogenous Nrp1p and Rbp1p appeared in the same protein complex. RNA analysis demonstrated that level of porin mRNA in nrp1 mutant strain was lower than that of wild-type strain, and immuno-fluorescence staining shows that porin expression level was enhanced when overexpressed Nrp1p, suggesting that Nrp1p was involved in porin mRNA metabolism. Moreover, subcellular localization analysis showed that Nrp1p, like Rbp1p, is localized to perinuclear region and partially to mitochondria. In conclusion, our results demonstrate that Nrp1p is an Rbp1p interacting protein and is involved in porin mRNA metabolism cooperatively.
中文摘要 4
Abstract 5
Introduction 6
Results 12
I. Characterization of NRP1 12
II. Interactions of Rbp1p, Nrp1p and Pub1p 14
III. Spatial relations among Nrp1p, Rbp1p and Pub1p 17
IV. To study whether Nrp1p is functional related to Rbp1p 19
Discussion 21
Materials and Methods 25
Tables and Figures 36
Table 1. Yeast strains used in this thesis 36
Table 2. Primers used for gene disruption 37
Table3. Primers used to construct NRP1 deletion mutants into pVP16 38
Figure 1. The predicted domains of Nrp1p. 39
Figure 2. Specific immunoactivity and sensitivity of anti-Nrp1p antibodies. 40
Figure 3. NRP1 gene was disrupted by PCR-based technique. 41
Figure 4. NRP1 disruption does not affect the growth rate on SC-media containing different carbon source. 42
Figure 5. NRP1 disruption does not affect the growth rate at different temperature. 43
Figure 6. NRP1 and RBP1 constructs used in the two-hybrid assay. 44
Figure 7. The glutamine stretch 2 of Rbp1p interacts with Nrp1p, and the interaction region of Nrp1p is not clear. 45
Figure 8. Nrp1p interacts with Rbp1p in vivo 47
Figure 9. Pub1p interacts with Nrp1p and Rbp1p. 48
Figure 10. Interaction between Nrp1p and Rbp1p is RNA-independent. 48
Figure 11. Endogenous Rbp1p interacts with Nrp1p in a RNA-independent manner. 49
Figure 12. Pub1p interacts with Nrp1p and Rbp1p in a RNA-independent manner. 50
Figure 13. Rbp1p, Nrp1p and Pub1p are associated with heavy sedimentary structures. 52
Figure 14. Nrp1p is co-localized with Rbp1p. 52
Figure 15. Endogenous Nrp1p is partially co-localized with endogenous Rbp1p. 54
Figure 16. Pub1p is partially co-localized with Nrp1p. 55
Figure 17. RBP1-disruption does not affect Nrp1p distribution. 56
Figure 18. NRP1-disruption does not affect Rbp1p localization. 57
Figure 19. Steady-state level of POR1 mRNA from wild type, rbp1, nrp1 and rbp1nrp1 mutants is detected by Northern blot analysis. 58
Figure 20. Overexpressed Nrp1p enhances porin expression. 59
Figure 21. Overexpressed Nrp1p enhanced porin expression. 60
Figure 22. Rbp1p, but not Nrp1p, may be involved in yeast cell wall maintenance. 61
References 62
Anderson, J.T., Paddy, M.R., Swanson, M.S. (1993) PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae. Mol Cell Biol. 1993 13(10):6102-13.

Beelman, C. A., A. Stevens, G. Caponigro, T. E. LaGrandeur, L. Hatfield, D. M. Fortner, and R. Parker. (1996) An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382:642-646.

Bianchi, M.M., Sartori, G., Vandenbol, M., et al. (1999) How to bring orphan genes into functional families. Yeast 15: 513-526.

Boeck, R., B. Lapeyre, C. E. Brown, and A. B. Sachs. (1998) Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant. Mol. Cell. Biol. 18:5062-5072.

Brown, C.E., Sachs, A.B. (1998) Poly(A) tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation. Mol Cell Biol. 18(11):6548-59.

Butler, J. S. (2002). The yin and yang of the exosome. Trends Cell Biol. 12:90-96.

Buu, L.M., Jang, L.T., Lee, F.J. (2004) The yeast RNA-binding protein Rbp1p modifies the stability of mitochondrial porin mRNA. J Biol Chem. 279(1):453-62.

Chen, C.Y., Gherzi, R., Andersen, J.S., Gaietta, G., Jurchott, K., Royer, H.D., Mann, M., Karin, M. (2000) Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation. Genes Dev. 14(10):1236-48.

Das, B., Butler, J.S., Sherman, F. (2003) Degradation of normal mRNA in the nucleus of Saccharomyces cerevisiae. Mol Cell Biol. 23(16):5502-15.

Daugeron, M. C., F. Mauxion, and B. Seraphin. (2001) The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation. Nucleic Acids Res. 29:2448-2455.

Gavin, A.C. et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature. 10;415(6868):141-7.

Gerster, T., Balmaceda, C.G., Roeder, R.G. (1990) The cell type-specific octamer transcription factor OTF-2 has two domains required for the activation of transcription. EMBO J. 9(5):1635-43.

Goldstein, A.L., McCusker, J.H. (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast. 15(14):1541-53.

Gonzalez, C. I., Ruiz-Echevarria, M. J., Vasudevan, S., Henry, M. F., Peltz, S. W. (2000) The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay. Mol. Cell 5, 489-499

Gorner, W., Durchschlag, E., Martinez-Pastor, M.T., et al. (1998) Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 12: 586-597.

Hentze, M. W., and A. E. Kulozik. (1999) A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96:307-310.

Hsieh, M., Tintut, Y., Gralla, J.D. (1994) Functional roles for the glutamines within the glutamine-rich region of the transcription factor sigma 54. J Biol Chem. 269(1):373-8.

Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., Sakaki, Y. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A. 98(8):4569-74.

Jelinsky, S.A., Samson, L.D. (1999) Global response of Saccharomyces cerevisiae to an alkylating agent. Proc Natl Acad Sci USA 96: 1486-1491.

Kopecka, M., Gabriel, M. (1992) The influence of congo red on the cell wall and (1----3)-beta-D-glucan microfibril biogenesis in Saccharomyces cerevisiae. Arch Microbiol. 158(2):115-26.

Lee F.J., Moss J. (1993) An RNA-binding protein gene (RBP1) of Saccharomyces cerevisiae encodes a putative glucose-repressible protein containing two RNA recognition motifs. J Biol Chem. 268(20):15080-7.

Longtine, M.S., McKenzie, A. 3rd, Demarini, D.J., Shah, N.G., Wach, A., Brachat, A., Philippsen, P., Pringle, J.R. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 14(10):953-61.

Mannella, C.A. The ''ins'' and ''outs'' of mitochondrial membrane channels. Trends Biochem Sci. 17(8):315-20. Review.

Marcotte, E.M., Pellegrini, M., Thompson, M.J., Yeates, T.O., Eisenberg, D. (1999) A combined algorithm for genome-wide prediction of protein function. Nature. 1999 402(6757):83-6.

Mitchell, P., and D. Tollervey. (2001) mRNA turnover. Curr. Opin. Cell Biol. 13:320-325.

Montijn, R.C., Vink, E., Muller, W.H., Verkleij, A.J., Van Den Ende, H., Henrissat, B., Klis, F.M. (1999) Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae. J Bacteriol. 181(24):7414-20.

Proudfoot, N. (2000). Connecting transcription to messenger RNA processing. Trends Biochem Sci. 25(6):290-3. Review.

Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., Seraphin, B. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. 24(3):218-29. Review.

Reynaud, A., Facca, C., Sor, F., Faye, G. (2001) Disruption and functional analysis of six ORFs of chromosome IV: YDL103c (QRI1), YDL105w (QRI2), YDL112w (TRM3), YDL113c, YDL116w (NUP84) and YDL167c (NRP1). Yeast. 18(3):273-82.

Roemer, T., Paravicini, G., Payton, M.A., Bussey, H. (1994) Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly. J Cell Biol. 127(2):567-79.

Ruiz-Echevarria, M.J., Peltz, S.W. (2000) The RNA binding protein Pub1 modulates the stability of transcripts containing upstream open reading frames. Cell. 23;101(7):741-51.

Siomi, H., Dreyfuss, G. (1997) RNA-binding proteins as regulators of gene expression. Curr Opin Genet Dev. 7(3):345-53. Review.

Sweetman, D., Smith, T., Farrell, E.R., Chantry, A., Munsterberg, A. (2003) The conserved glutamine-rich region of chick csal1 and csal3 mediates protein interactions with other spalt family members. Implications for Townes-Brocks syndrome. J Biol Chem. 278(8):6560-6. Epub 2002 Dec 13.

Tucker, M., M. A. Valencia-Sanchez, R. R. Staples, J. Chen, C. L. Denis, and R. Parker. (2001) The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104:377-386.

Vasudevan, S., Peltz, S.W. (2001) Regulated ARE-mediated mRNA decay in Saccharomyces cerevisiae. Mol Cell. 7(6):1191-200.

Veyrune, J.L., Hesketh, J., Blanchard, J.M. (1997) 3'' untranslated regions of c-myc and c-fos mRNAs: multifunctional elements regulating mRNA translation, degradation and subcellular localization. Prog Mol Subcell Biol. 18:35-63. Review.

Wang, W., Czaplinski, K., Rao, Y., Peltz, S. W. (2001) The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts. EMBO J. 20, 880-890

Weng, Y., Czaplinski, K., Peltz, S. W. (1996) Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover. Mol. Cell. Biol. 16, 5491-5506

Wilusz, C.J., Wormington, M., Peltz, S.W. (2001) The cap-to-tail guide to mRNA turnover. Nat Rev Mol Cell Biol. 2(4):237-46. Review.

Wreden, C., Verrotti, A.C., Schisa, J.A., Lieberfarb, M.E., Strickland, S. (1997) Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA. Development. 124(15):3015-23.
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1. 仇德哉:〈清代的雲林縣〉,《雲林文獻》第三十一輯,1987年6月。
2. 松永正義著、何世雄譯:〈台灣的日語文學及台語文學〉《中外文學》第31卷,第10期,2003年3月。
3. 吳文星:〈日據時代書房之研究〉《思與言》第16卷第3期,1978年9月。
4. 吳文星:〈日據時代台灣書房之研究〉,《思與言》16卷3期,1978年9月。
5. 王文顏:〈光復前台灣詩詩社的時代價值〉,《文訊》18期,1985年6月。
6. 呂雲騰:〈史志記載的北港─從五代的北港到魍港、笨港、北港〉,《雲林文獻》第四十三輯,1999年6月。
7. 呂雲騰:〈百年國小─北港鎮南陽國小〉,《雲林文獻》,第四十三輯,1999年6月。
8. 施懿琳:〈日據時期台灣古典詩的抗議精神與比興諷喻傳統〉《古典文學》第十二集,台北:學生書局,1992年10月。
9. 莊永清:〈賴和漢詩詩觀及其漢文化意識〉,《高苑學報》第8卷,2002年7月。
10. 黃文博:〈金湖港牽水○─雲林縣口湖鄉蚶仔寮萬善爺的故事〉,《雲林文獻》第四十一輯,1997年12月。
11. 陳石柱:〈從北港朝天宮武文昌殿探討清代笨港社學「聚奎閣」〉,《雲林文獻》第三十五輯,1991年5月。
12. 鄭定國:〈四湖旋馬庭主人林友笛漢詩析論〉,《漢學論壇》第三輯,國立雲林科技大學漢學資料整理研究所,民92年6月。
13. 鄭定國:〈斗六張立卿漢詩的特色〉,《漢學論壇》第四輯,國立雲林科技大學漢學資料整理研究所,民93年6月。
14. 趙天儀:〈台灣文學研究的人事物〉,《文訊》214期,2002年11月。
15. 懷笨佬:〈笨港聚奎閣遷建朝天宮秘辛〉,《雲林文獻》第四十三輯,1999年6月。