啤酒酵母的轉譯起始因子eIF4G透過SSF1基因影響60S核醣體生合成

Abstract

eIF4G (eukaryotic initiation factor 4G)是真核生物中重要的轉譯起始因子，幫助40S核醣體結合至mRNA、預備進行蛋白質轉譯(translation)。除了蛋白質轉譯，核醣體的生合成亦是調控蛋白質總量的關鍵途徑；核糖體的生合成需要超過200多個組裝因子進行核醣體RNAs的裁切、修飾、穩定核醣體蛋白，及確認核醣體的組裝正確。已有研究指出，啤酒酵母的eIF4G缺失時，60S核醣體會出現生合成的缺失，但目前沒有研究探討eIF4G在60S核醣體生合成的分子機制，故仍無直接的證據說明eIF4G參與核醣體生合成途徑，及其扮演的角色、重要性。本研究針對啤酒酵母的eIF4G1(Tif4631)和eIF4G2(Tif4632)進行分析，發現兩者皆參與早期60S核醣體的生合成，過多或過少的eIF4G都會干擾60S的生合成。有趣的是，比較eIF4G1和eIF4G2之間差異，本研究發現雖然tif4631Δ中核醣體生合成的缺失比tif4632Δ嚴重，但高溫(37°C)下高量表現的eIF4G2會明顯改變Ssf1(60S的組裝因子)在細胞中的分布，及其與60S核醣體的結合，而高量eIF4G1不會；亦發現高量eIF4G1/2會自RNA層級開始下降SSF1的表現量，高量eIF4G2則對於SSF1啟動子調控的專一性較高。另一方面，在細胞失去其中一個eIF4G (tif4631Δ或tif4632Δ)，依序地，高溫下SSF (SSF1或SSF2)的啟動子運作會受到eIF4G2和eIF4G1不一樣的調控。因此，本研究證明eIF4G1/2透過SSF1影響細胞中60S核醣體生合成的進行，且兩者調控SSF1 mRNA表現量及其蛋白質交互作用的方式具有差異性。

eIF4G, an important eukaryotic translation initiation factor, recruites 40S ribosomal subunit to mRNA to start protein translation. In addition to translation, ribosome biogenesis is also a key pathway to regulate protein levels. In ribosome biogenesis, more than 200 trans-acting (assembly) factors are required for rRNA processing, modification, stabilization of ribosomal proteins, and to ensure the accuracy in ribosome assembly. In S. cerevisiae, it was reported that deletion of eIF4G1 or eIF4G2 caused defect in 60S biogenesis. However, no study has shown the molecular mechanism of eIF4G in 60S ribosome biogenesis, so it is unclear to depict the role or the importance of eIF4G in this pathway. In this study, both eIF4G1 (Tif4631) and eIF4G2 (Tif4632) in yeast were required for early 60S ribosome biogenesis. Excess or insufficient expression levels of eIF4G disturbed 60S biogenesis. Interestingly, there are differences between eIF4G1 and eIF4G2. Although the ribosome biogenesis defect in tif4631Δ was more severe than that of tif4632Δ, overexpression of eIF4G2 at high temperature (37°C) apparently changed the localization and interaction with 60S of Ssf1, a 60S assembly factor, but eIF4G1 didn’t. Overexpression of eIF4G1 or eIF4G2 decreased transcription of SSF1, and eIF4G2 showed higher specificity toward this regulation. On the other hand, at the higher temperature, SSF (SSF1 or SSF2) promoter was oppositely regulated by eIF4G2 (in tif4631Δ) and eIF4G1 (in tif4632Δ). These data indicate that eIF4G1 and eIF4G2 impact 60S ribosome biogenesis via SSF1, yet they may regulate SSF1 mRNA expression and Ssf1 protein interactions in a disparate way.