李芳仁臺灣大學：分子醫學研究所林亞慧Lin, Ya-HuiYa-HuiLin2007-11-262018-07-092007-11-262018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/51379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 62778250 bytesapplication/pdfen-US粒腺體核糖核酸酵母菌mitochondriaporinPUB1NRP1RBP1RNA-binding proteinotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51379/1/ntu-93-R91448008-1.pdf