Gu Wenyi, Li Mengrong, Zhao Wei Ming, Fang Ning Xia, Bu Shurui, Frazer Ian H, Zhao Kong-Nan
Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia.
Nucleic Acids Res. 2004 Aug 19;32(15):4448-61. doi: 10.1093/nar/gkh748. Print 2004.
Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972-4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA(Ser)(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA(Ser)(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA(Ser)(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2alpha in the tRNA(Ser)(CGA) transfected L1 cell lines. The tRNA(Ser)(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.
外源性转运RNA（tRNA）在体外翻译系统中有利于牛乳头瘤病毒1型野生型（wt）L1 mRNA的翻译（Zhou等人，1999年，《病毒学杂志》，73卷，4972 - 4982页）。因此，我们研究了在补充外源性tRNA后，乳头瘤病毒（PV）野生型L1蛋白在真核细胞中的表达是否能增强。用PV1野生型L1基因转染的中国仓鼠卵巢（CHO）细胞和Cos1细胞都能有效转录这些基因，但不能翻译它们。然而，在与L1基因和一个表达tRNA(Ser)(CGA)的基因共转染后，证明了L1蛋白的翻译。用牛乳头瘤病毒1型（BPV1）野生型L1表达构建体稳定转染的细胞系，在转染tRNA(Ser)(CGA)基因后产生了L1蛋白，但在转染基础载体后没有产生，这表明tRNA(Ser)(CGA)基因由于内源性tRNA的改变以及tRNA(Ser)(CGA)转染的L1细胞系中翻译起始因子elF4E和elF2α的磷酸化而增强了野生型L1的翻译。tRNA(Ser)(CGA)基因表达显著降低了利用哺乳动物偏好密码子的密码子修饰（HB）PV L1基因所表达的L1蛋白的翻译，但对六种丝氨酸绿色荧光蛋白（GFP）变体所表达的绿色荧光蛋白（GFP）的翻译有不同影响。tRNA库的变化似乎与PV野生型和HB L1基因以及丝氨酸GFP变体的密码子组成相匹配，以调节它们mRNA的翻译。这些发现首次在真核细胞中证明，提供单个tRNA表达构建体可以对靶基因的翻译产生不同影响。
