Translational efficiency of mRNA in yeast cells is not increased by plant viral leader sequences

Summary Synthetic oligonucleotides encoding the 5-non-translated (leader) sequence of the coat protein mRNA of alfalfa mosaic virus RNA 4 or the leader sequence of tobacco mosaic virus RNA were used to replace the natural leader region of the yeast phosphoglycerate kinase (PGK1) mRNAs and the translational efficiency of the chimeric mRNA was determined in yeast cells. In neither case did we observed a significant increase compared to the translational efficiency shown by the wild-type PGK mRNA, in contrast to the known stimulatory effect of these leader sequences on translation in mammalian, plant and bacterial in-vivo and/or in-vitro systems. The same result was obtained when the translational efficiencies in yeast cells of Escherichia coli -galactosidase mRNAs carrying the PGK or either of the two viral leader sequences were compared. Offprint requests to: H. A. Raué     

Identification of short untranslated regions that sufficiently enhance translation in high-quality wheat germ extract

High-quality wheat germ extract (hqWGE) is very useful for the high-yield production of various types of protein. The most important key to high productivity is the design of mRNA templates. Although the design has been refined for straightforward and efficient translation in hqWGE, there is still room for improvement in untranslated regions (UTRs), especially the 3′ UTR length, because a long, cumbersome 3′ UTR is commonly used for translation enhancement. Here we examined some short viral 3′ cap-independent translation enhancers (3′ CITEs) to identify effective ones for efficient translation in hqWGE. We then combined the most effective 3′ CITE and a 5′ enhancer to further increase the translation efficiency. mRNA with the optimal short 3′ and 5′ UTRs, both of whose length was less than 150 nt, exhibited a productivity of 1.4 mg/mL in prolonged large-scale protein synthesis in hqWGE, which was comparable to that of control mRNA with a commonly-used long 3′ UTR (∼1200 nt).     

Mutations in the rpIJ leader of Escherichia coli that abolish feedback regulation

We have isolated mutants that fail to exhibit biosynthetic feedback regulation of a rpIJ-lacZ fusion. Analysis of these mutants and of others that were isolated earlier indicates that crucial sequences for both translational feedback regulation and efficient translation lie closely intermingled in the central region of the rpIJ mRNA leader 70-195 bases upstream from the translation start of rpIJ. We suggest that our point mutations define a region of the rpIJ leader mRNA to which L10 binds in effecting autogenous translational regulation.     

Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough

The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.     

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.     

At least two nuclear gene products are specifically required for translation of a single yeast mitochondrial mRNA.

Mitochondrial translation of the oxi2 mRNA, encoding yeast cytochrome c oxidase subunit III (coxIII), has previously been shown to specifically require the mitochondrially located protein product of the nuclear gene PET494. We show here that this specific translational activation involves at least one other newly identified gene termed PET54. Mutations in PET54 cause an absence of the coxIII protein despite the presence of normal levels of its mRNA. pet494 mutations are known to be suppressible by mitochondrial gene rearrangements that replace the normal 5'-untranslated leader of the oxi2 mRNA with the leaders of other mitochondrial mRNAs. In this study we show that pet54, pet494 double mutants are suppressed by the same mitochondrial gene rearrangements, showing that the PET54 product is specifically required, in addition to the PET494 protein, for translation of the oxi2 mRNA. Since, as we show here, PET54 is not an activator of PET494 gene expression, our results suggest that the products of both of these genes may act together to stimulate coxIII translation.     

The adenovirus type 5 i-leader open reading frame functions in cis to reduce the half-life of L1 mRNAs.

The 440-nucleotide adenovirus type 5 i-leader sequence, encoding a 13.6-kilodalton protein, is located between the second and third components of the tripartite leader sequence. It appears primarily on the L1 family of mRNAs. To study its function, we constructed two point mutations within the i leader. pm382 lacks the wild-type i-leader splice acceptor and failed to splice the leader onto L1 mRNAs. pm383 lacks the ATG used for translation of the i-leader protein; it synthesized i-leader-containing mRNAs, but failed to produce detectable levels of the polypeptide. Both mutants exhibited modestly reduced yields in some but not all cell lines tested and accumulated slightly elevated levels of L1 mRNA and L1 52- and 55-kilodalton proteins in infected cells. Mutant phenotypes were consistently more pronounced in pm382- than in pm383-infected cells. In wild-type virus-infected cells, L1 mRNAs lacking the i leader displayed a half-life of about 26 h, whereas L1 mRNAs containing the leader were much less stable, with a half-life of less than 4 h. In pm383-infected cells (ATG mutant), L1 mRNAs containing the i leader exhibited a half-life of 26 h. The abnormally long half-life of pm383-encoded L1 mRNAs containing a mutant i leader was not reduced by coinfection with wild-type virus, suggesting that synthesis of the i-leader protein leads to destabilization of the i-leader-containing L1 mRNA undergoing translation.     

RNA secondary structure and translation inhibition: analysis of mutants in the rplJ leader

We have carried out measurements of the stable binding of the ribosomal protein (r-protein) complex L10-L7/L12 to mutant forms of the mRNA leader of the rplJ operon of Escherichia coli. One of the point mutations, base 1548, which lies within the L10-L7/L12-protected region, almost completely abolishes in vitro formation of a stable complex of L10-L7/L12 with rplJ mRNA leader, and a second point mutation, base 1634, strongly reduces it. These observations constitute strong support for the proposition that L10-L7/L12 binds to the rplJ leader in bringing about translational feedback. To account for the action of these and other mutations, and to explain the mechanism of translation feedback inhibition, we suggest a secondary structure model involving alternate forms of the rplJ mRNA leader.     

Sequence and structural features associated with translational initiator regions in yeast--a review

We have compared the translational initiator regions of 131 yeast genes. 95% utilize the first AUG from the 5' end of the message as the start codon for translation. Yeast leader regions in general are rich in adenine nucleotides (nt), have an average length of 52 nt, and are void of significant secondary structure. Sequences immediately adjacent to AUG start codons are preferred, however, the bias in nucleotide distribution (5'-A-YAA-UAAUGUCU-3') does not reflect a higher eukaryotic consensus (5'-CACCAUGG-3') with the exception of an adenine nucleotide preference at the -3 position. A minority of yeast mRNAs that contain AUG codons in the leader region that do not serve as the start codon for the primary gene product differ from the majority of mRNAs by one or more of these general properties. This analysis appears to indicate that basic features associated with yeast leader regions are consistent with a general mechanism of initiation of protein synthesis in eukaryotes, as proposed by the ribosomal 'scanning' model, but perhaps only basic features associated with ribosomal recognition of an AUG start codon are intact.     

Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae.

We have mutated various features of the 5' noncoding region of the HIS4 mRNA in light of established Saccharomyces cerevisiae and mammalian consensus translational initiator regions. Our analysis indicates that insertion mutations that introduce G + C-rich sequences in the leader, particularly those that result in stable stem-loop structures in the 5' noncoding region of the HIS4 message, severely affect translation initiation. Mutations that alter the length of the HIS4 leader from 115 to 39 nucleotides had no effect on expression, and sequence context changes both 5' and 3' to the HIS4 AUG start codon resulted in no more than a twofold decrease of expression. Changing the normal context at HIS4 5'-AAUAAUGG-3' to the optimal sequence context proposed for mammalian initiator regions 5'-CACCAUGG-3' did not result in stimulation of HIS4 expression. These studies, in conjunction with comparative and genetic studies in S. cerevisiae, support a general mechanism of initiation of protein synthesis as proposed by the ribosomal scanning model.     

Suppression of a defect in the 5'' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein.

Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.