Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia.
tRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes
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.
Nucleic Acids Res. 32(15): 4448-61.
To determine if the expression of the tRNASer (CGA) gene construct could enhance translation of wt PV L1 mRNAs in eukaryotic cell lines, CHO cells and Cos1 cells were transfected with both PV wt L1 expression construct and codon-optimized HB PV L1 expression construct. In HB PV L1 gene, L1 ORFs are substituted with codons preferentially used in the mammalian genome. Experimental results showed that the transcriptions of both PV wt L1 and HB PV L1 were not affected by co-transfection with tRNASer (CGA) gene. However, expression of L1 protein encoded by PV wt L1 gene required co- transfection with tRNASer (CGA) gene. For codon-optimized gene, HB PV L1, tRNASer (CGA) gene resulted in a reduction in the levels of the L1 protein expression. So the hypothesis made could be approved that the matching between tRNAs and codon usage of the genes regulates expression of the cellular proteins in the cells. Futher hypothesis was made that the tRNASer (CGA) gene may favor the expression of the target genes in which UCG or other codons from the six serine codons are used more frequently. The cells were transfected solely with gene and the six serine GFP variants or co-transfected with tRNASer (CGA) gene and study the transcription and translation were study to test the hypothesis. According to the results, the hypothesis was approved.