Puf6與Loc1對Rpl43蛋白穩定性及轉錄後調控之研究

The study of Puf6 and Loc1 in protein stability and post-transcriptional regulation of Rpl43

越樂昀(Le-Yun Yueh)

指導教授 ： 羅凱尹

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903279

摘要

啤酒酵母 (Saccharomyces cerevisiae)是個適合研究核醣體生合成的真核模式生物。核醣體是由rRNA與核醣體蛋白所構成，在快速分裂生長期間，為了維持正常核醣體的生理機能，核醣體蛋白會高度表現，同時這些核醣體蛋白表現後需要在細胞中受到調控及保護。本實驗所研究的目標核醣體蛋白，Rpl43，在核醣體的位置上位於約E-site附近。在先前的研究中，我們發現Puf6、Loc1及Rpl43之間在酵母菌中會有蛋白質的交互作用，形成三元複合體。當細胞缺乏Puf6、Loc1的時候，Rpl43核醣蛋白的含量會下降，而在實驗結果顯示出Puf6與Loc1可能會參與並維持Rpl43的穩定性與核醣體的組裝。
為了了解Puf6、Loc1會如何影響Rpl43，我們先去分析新生成的Rpl43在細胞中的穩定性，分析的結果顯示在puf6Δ 及loc1Δ細胞內，Rpl43的穩定性會顯著地降低。且RPL43 mRNA的量在puf6Δ 及loc1Δ皆不會有改變的情形，顯示在RNA層次上，會有其他機制來調控RPL43 生成。以帶有不同RPL43片段的報導基因分析不同片段對表現蛋白質與RNA的影響，實驗結果發現，RPL43B的3’UTR會抑制蛋白質與RNA的生成，而帶有RPL43B 內含子能夠抵銷3’UTR的抑制；RPL43A的3’UTR會增加蛋白質與RNA的生成，而帶有RPL43A 內含子能夠抵銷3’UTR的活化。Puf6的存在與否會影響內含子的調控；Loc1和Rpl43會影響3’UTR的調控。接著，我們以核醣體圖譜分析RPL43 mRNA的轉譯作用，發現puf6Δ 及loc1Δ造成Rpl43蛋白質生成上的影響。這些結果顯示， Puf6與Loc1會結合RPL43 mRNA調控其RNA 並增加其轉譯，並和Rpl43結合，形成複合體保護其穩定性。

關鍵字

核醣體生合成 ； RNA 結合蛋白 ； 輔助因子 ； 轉錄後調控 ； 轉譯調控

並列摘要

Saccharomyces cerevisiae is a good model organism to study ribosome biogenesis. Ribosome is composed of rRNAs and ribosomal proteins. During rapid growth, ribosomal proteins are highly expressed. In order to build up a functional ribosome, the qualities of ribosomal proteins need to be rigorously controlled. Ribosomal protein large subunit 43 (Rpl43) is located nearby the E-site of ribosome. In our previous study, we found Puf6, Loc1, and Rpl43 formed a trimeric complex in Saccharomyces cerevisiae. In the absence of PUF6 or LOC1, Rpl43 protein level was under-accumulated. The data suggests that the functions of Puf6 and Loc1 may correlate with the stability and assembly of Rpl43.
In this study, we further dissected the connections among these three proteins. The stability of free Rpl43 protein decreased significantly both in puf6Δ and loc1Δ. While the level of mature RPL43 mRNA did not change in puf6Δ and loc1Δ, there might be other mechanisms to regulate its mRNA. Different RPL43 reporter genes were constructed to detect the expression of proteins and RNA. We found 3’UTR of RPL43B could repress the expression of RNA and protein, and intron could counteract the repression of 3’UTR in the reporter assay. The 3’UTR of RPL43A could enhance the expression of protein, and intron could remove the enhancement of 3’UTR in the reporter assay. The presence of Puf6 could interfere the regulation of intron. Loc1 and Rpl43 could regulate the 3’UTR. In addition, the translation of RPL43 mRNA decreased in puf6Δ and loc1Δ mutants. The results in this study suggest that Puf6 and Loc1 bound RPL43 mRNA to regulate its transcription and translation. In addition, they formed a complex with free Rpl43 protein to protect its stability.

並列關鍵字

ribosome biogenesis ； RNA binding proteins ； transacting factors ； post-transcriptional regulation ； translational control

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