酵母菌核糖核酸結合蛋白之作用蛋白—Nrp1p之功能特性探討

Functional characterization of Rbp1p interacting protein, Nrp1p, in Saccharomyces cerevisiae

林亞慧(Ya-Hui Lin)

指導教授 ： 李芳仁

國立臺灣大學/醫學院/分子醫學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01587

摘要

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。

關鍵字

粒腺體 ； 核糖核酸 ； 酵母菌

並列摘要

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.

並列關鍵字

mitochondria ； porin ； PUB1 ； NRP1 ； RBP1 ； RNA-binding protein

參考文獻

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