mRNA structures influencing translation in the yeast Saccharomyces cerevisiae.

The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYC1 alleles having alterations in the 5' leader region. Measurements of levels of CYC1 mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two- to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.     

Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

Translation initiation of alphavirus subgenomic mRNA (sgmRNA) can occur in the absence of several initiation factors (eIFs) in infected cells; however, the precise translation mechanism is still poorly understood. In this study, we have examined the mechanism of initiation and AUG selection in Sindbis virus (SINV) sgmRNA. Our present findings suggest that sgmRNA is translated via a scanning mechanism, since the presence of a hairpin structure before the initiation codon hampers protein synthesis directed by this mRNA. In addition, translation is partially recovered when an in-frame AUG codon is placed upstream of this hairpin. This scanning process takes place without the participation of eIF4A and active eIF2. These results, combined with our findings through modifying the SINV sgmRNA leader sequence, do not support the possibility of a direct initiation from the start codon without previous scanning, or a shunting mechanism. Moreover, studies carried out with sgmRNAs containing two alternative AUG codons within a good context for translation reveal differences in AUG selection which are dependent on the cellular context and the phosphorylation state of eIF2α. Thus, initiation at the additional AUG is strictly dependent on active eIF2, whereas the genuine AUG codon can start translation following eIF2α inactivation. Collectively, our results suggest that SINV sgmRNA is translated by a scanning mechanism without the potential participation of crucial eIFs. A model is presented that explains the mechanism of initiation of mRNAs bearing two alternative initiation codons.     

Downstream sequence adjacent to AUG affects translation of chloramphenicol acetyl transferase in eukaryotic cells

The CAT gene is widely used as a reporter in eukaryotic systems because of the efficient translation of its mRNA. We report here that a sequence occurring in the CAT mRNA at +15 nucleotides from CAT AUG is essential for translation. This sequence includes a stem-loop structure, which, however, exhibits a calculated stability significantly lower than that required for a hairpin to act as an enhancer of translation in vitro. Replacement of this region with the corresponding sequence from mRNAs that are normally translated in eukaryotic systems drastically reduced translation of CAT in COS cells, although the consensus sequence around the AUG, known to be required for high-level translation initiation, was conserved. These observations may be relevant for the exploitation of the CAT reporter system for analysis of the mechanisms of translation initiation by means of fusion constructs.     

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

We determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5'-AGGA-3' to 5'-UUUU-3' results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon–anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.     

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.     

The flatworm spliced leader 3'-terminal AUG as a translation initiator methionine

Spliced leader (SL) RNA trans-splicing contributes the 5' termini to mRNAs in a variety of eukaryotes. In contrast with some transsplicing metazoan groups (e.g. nematodes), flatworm spliced leaders are variable in both sequence and length in different flatworm taxa. However, an absolutely conserved and unique feature of all flatworm spliced leaders is the presence of a 3'-terminal AUG. We previously suggested that the Schistosoma mansoni spliced leader AUG might contribute a required translation initiator methionine to recipient mRNAs. Here we identified and examined trans-spliced cDNAs from a large set of newly available schistosome cDNAs. 28% of the trans-spliced cDNAs have the SL AUG in-frame with the major open reading frame of the mRNA. We identified over 40 cDNAs (40% of the SL AUG in-frame clones) that require the SL AUG as an initiator methionine to synthesize phylogenetically conserved N-terminal residues characteristic of orthologous proteins. RNA transfection experiments using several schistosome stages demonstrated that the flatworm SL AUG can serve as a translation initiator methionine in vivo. We also present in vivo translation studies of the schistosome initiator methionine context and the effect of the spliced leader AUG added upstream and out-of-frame with the main open reading of recipient mRNAs. Overall, our data have provided evidence that another function of flatworm spliced leader trans-splicing is to provide some recipient mRNAs with an initiator methionine for translation initiation.     

Effect of tandem repeated AUG codons on translation efficiency of eukaryotic mRNA carrying a short leader sequence

The effect on translation of multiple copies of the initiation codon AUG at the initiation site in a eukaryotic mRNA carrying a short leader sequence was tested in translation experiments in vitro. DNA, corresponding to a chimeric mRNA sequence consisting of the 5 leader region of brome mosaic virus (BMV) RNA4 and the goat pre--lactalbumin mRNA sequence, was prepared and transcribed in vitro using SP6 RNA polymerase. Site-directed mutagenesis was carried out to change the sequence around the initiation codon AUG. In a wheat germ translation system, the yield of protein obtained using the mRNA with a duplication of the AUG codons at the initiation site was 1.6 times that achieved when only one AUG was present. The rate of formation of the 80S initiation complex was measured by the ribosome binding assay using cycloheximide. A good correlation was observed between the ability to form the complex and translation efficiency.     

Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation.

Alphaviruses, particularly Sinbis virus and Semliki Forest virus, are proving to be useful vectors for the expression of heterologous genes. In infected cells, these self-replicating vectors (replicons) transcribe a subgenomic mRNA that codes for a heterologous protein instead of the structural proteins. We reported recently that translation of the reporter gene lacZ is enhanced 10-fold when the coding sequences of this gene are fused downstream of and in frame with the 5' half of the capsid gene (I. Frolov and S. Schlesinger, J. Virol. 68:8111-8117, 1994). The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin structure. We have mutated this region by making changes in the codons which do not affect the protein sequence but should destabilize the putative hairpin structure. These changes caused a decrease in the accumulation of the capsid-beta-galactosidase fusion protein. When these alterations were inserted into the capsid gene in the context of the intact Sindbis virus genome, they led to a decrease in the rate of virus formation but did not affect the final yield. We also altered the original sequence to one that has 12 contiguous G.C base pairs and should form a stable hairpin. The new sequence was essentially as effective as the original had been in enhancement of translation and in the rate of virus formation. The position of the predicted hairpin structure is important for its function; an insertion of 9 nucleotides or a deletion of 9 nucleotides decreased the level of translation. The insertion of a hairpin structure at a particular location downstream of the initiating AUG appears to be a way that alphaviruses have evolved to enhance translation of their mRNA, and, as a consequence, they produce high levels of the structural proteins which are needed for virus assembly. This high level of translation requires an intracellular environment in which host cell protein synthesis is inhibited.     

Structure and expression of the mRNA for murine granulocyte-macrophage colony stimulating factor.

A cDNA containing a virtually complete copy of the mRNA for the haemopoietic growth regulator, granulocyte-macrophage colony stimulating factor (GM-CSF), has been isolated from a murine T lymphocyte cDNA library. When a eukaryotic expression vector with this cDNA coupled to the SV40 late promoter was introduced into simian COS cells, significant quantities of GM-CSF were secreted. Since all of the biological activities previously ascribed to highly purified GM-CSF were exhibited in the COS cell-derived GM-CSF, all of these activities are intrinsic to the product of a single gene. There are two potential translational initiation codons in the GM-CSF mRNA; the first is buried in the stem and the second located in the loop of a very stable hairpin structure. Expression studies using deletion derivatives of the cDNA indicated that the second AUG is able to initiate the translation and secretion of GM-CSF. The amino acid sequence of the leader peptide is rather atypical for a secreted protein and we speculate that molecules which initiate at the first AUG might exist as integral membrane proteins whereas those initiating at the second are secreted.     

Translation of the Saccharomyces cerevisiae tcm1 gene in the absence of a 5'-untranslated leader.

The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other. The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3). Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced. Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet. A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin. This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader. We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency.     

Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes.

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.     

The positive regulatory function of the 5''-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.     

Potential open reading frames in 5′-untranslated regions of eukaryotic mRNA

It is known that 5′-untranslated sequences of eukaryotic mRNA often contain AUG triplets, which can serve as translation initiation sites. It is assumed that such leader open reading frames can perform regulatory functions and code functionally active proteins; however, their characteristics have been studied insufficiently. In the article, the context organization of leader open reading frames of eukaryotic mRNA was considered. It was shown that their characteristics correlate with their position with respect to the protein-coding sequence, which may be related to the translation initiation efficiency.     

An adenovirus agnogene.

The nucleotide sequence of a 550 base pairs long segment, located between map positions 21 and 22.5 in the adenovirus type 2 genome has been determined. S1 nuclease mapping and sequence analysis of cDNA copies of adenovirus mRNA demonstrated that the established sequence includes the i-leader which is spliced to the 5'-end of certain adenovirus mRNAs (1). The i-leader which is 440 nucleotides long contains an open translational reading frame which is preceded by an AUG triplet and which terminates in the third segment of the tripartite leader. A polypeptide with the same molecular weight as predicted from the DNA sequence was identified by in vitro translation of mRNA which had been selected by hybridization to DNA fragments, containing sequences from the i-leader. The results thus suggest that the i-leader, unlike other adenovirus leader segments, is used for translation.