啤酒酵母的轉譯起始因子eIF4G的突變及在60S核醣體生合成上影響的研究

Abstract

eIF4G為真核生物中相當重要的轉譯起始因子，且能影響細胞的生長、發育，以及其壽命。除了在轉譯起始上所扮演的角色，eIF4G亦曾被報導能在細胞核中參與mRNA的裁切及降解。在本篇研究中，發現eIF4G具有參與60S核醣體生合成的功能。當eIF4G缺失時，發現pre-60S核醣體上27S rRNA的裁切，以及早期的組裝過程會受到阻礙。目前在eIF4G上已知的重要功能區域有PAB1、4E、4A，分別能與Pab1 (poly-A binding protein)、eIF4E、eIF4A進行結合。實驗中，以在啤酒酵母中表現量較大的eIF4G1為主要研究對象，將其各重要功能區域分別進行去除，發現位於eIF4G C端區域上的eIF4E binding region及eIF4A binding region為主要負責參與60S核醣體生合成的功能性區域，且能與核醣體輔助因子Ssf1進行結合。Ssf1為Brix 蛋白家族的其中一個成員，此家族中還包含了Ssf2、Brx1、Rpf1、Rpf2，以及Imp4，這些輔助因子皆擁有一段保守的Brix domain，並且都能參與核醣體生合成，實驗中也發現它們能與eIF4G有蛋白質以及基因上的交互作用。在eIF4G缺失的突變株中，Ssf1與60S核醣體的結合量會發生異常，且會更進一步的影響核醣體PET domain周圍Rpl31及Arx1的結合。本篇研究的結果說明eIF4G與60S核醣體的潛在關係，以及其在60S核醣體生合成上可能扮演的功能性角色。

eIF4G is an essential translation initiation factor and is important in controlling cell growth, development, and lifespan. Apart from its role in translation, eIF4G has also been shown to involve in degradation and splicing of mRNAs in the nucleus. In this study, eIF4G was shown to have additional functional role in 60S ribosome biogenesis. Under depletion of eIF4G, defects in 27S rRNA processing and assembly of pre-60S ribosome could be detected. Several important domains have been identified in eIF4G, like Pab1, 4E, 4A, to interacts with Pab1 (poly-A binding protein), eIF4E, and eIF4A, respectively. This study focused on eIF4G1 as it has higher expression in Saccharomyces cerevisiae compared to eIF4G2. The individual domain was mutated in eIF4G1 and found that eIF4E and eIF4A binding region on C fragment of eIF4G1 was important in 60S biogenesis and interaction with the transacting factor, Ssf1. Ssf1 was a member of Brix family protein, which also includes Ssf2, Brx1, Rpf1, Rpf2, and Imp4. These factors have a conserved Brix domain and are all involved in ribosome biogenesis. This study also showed strong genetic and protein interactions between eIF4G and Brix family protein. In eIF4G mutant, the binding of Ssf1 with 60S ribosome was incorrect and further altered the loading of Rpl31 and Arx1 at the PET (polypeptide exit tunnel) site. The results in this study showed how eIF4G associated with 60S subunits potentially and its potential functional role in ribosome biogenesis.