Sequences 5' of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation.

Translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is modulated by amino acid levels in the cell. Ribosomes which translate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream. Under non-starvation conditions reinitiation occurs at one of the remaining three uORFs and GCN4 is repressed. Under starvation conditions, in contrast, ribosomes bypass the uORFs and reinitiate at GCN4 instead. The high frequency of reinitiation following uORF1 translation depends on an adequate distance to the next start codon and particular sequences surrounding the uORF1 stop codon. We present evidence that sequences 5' to uORF1 also strongly enhance reinitiation. First, reinitiation was severely inhibited when uORF1 was transplanted into the position of uORF4, even though the native sequence environment of the uORF1 stop codon was maintained, and this effect could not be accounted for by the decreased uORF1-GCN4 spacing. Second, insertions and deletions in the leader preceding uORF1 greatly reduced reinitiation at GCN4. Sequences 5' to uORF1 may influence the probability of ribosome release following peptide termination at uORF1. Alternatively, they may facilitate rebinding of an initiation factor required for reinitiation prior to resumption of the scanning process.     

A dual upstream open reading frame-based autoregulatory circuit controlling polyamine-responsive translation

A novel form of translational regulation is described for the key polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC). Plant AdoMetDC mRNA 5' leaders contain two highly conserved overlapping upstream open reading frames (uORFs): the 5' tiny and 3' small uORFs. We demonstrate that the small uORF-encoded peptide is responsible for constitutively repressing downstream translation of the AdoMetDC proenzyme ORF in the absence of increased polyamine levels. This first example of a sequence-dependent uORF to be described in plants is also functional in Saccharomyces cerevisiae. The tiny uORF is required for normal polyamine-responsive AdoMetDC mRNA translation, and we propose that this is achieved by control of ribosomal recognition of the occluded small uORF, either by ribosomal leaky scanning or by programmed -1 frameshifting. In vitro expression demonstrated that both the tiny and the small uORFs are translated. This tiny/small uORF configuration is highly conserved from moss to Arabidopsis thaliana, and a more diverged tiny/small uORF arrangement is found in the AdoMetDC mRNA 5' leader of the single-celled green alga Chlamydomonas reinhardtii, indicating an ancient origin for the uORFs.     

A quantitative model for translational control of the GCN4 gene of Saccharomyces cerevisiae

Expression of the GCN4 gene of Saccharomyces cerevisiae is regulated at the translational level by short open reading frames (uORFs) present in the leader sequence of its mRNA. Under conditions of amino acid sufficiency, these sequences restrict the flow of initiating ribosomes to the GCN4 AUG start codon. Mutational analysis of GCN4 has led to a model in which ribosomes must translate the 5'-proximal uORF1 and reassemble an initiation complex in order to translate GCN4. This reassembly process is thought to be rapid when amino acids are abundant, such that reinitiation occurs at uORF2, uORF3, or uORF4. Reinitiation at these sites prevents translation of GCN4, presumably because ribosomes dissociate from the mRNA following termination at uORFs 2 to 4. Because of reduced initiation factor activity under starvation conditions, a substantial fraction of ribosomal subunits scanning downstream from uORF1 are not ready to reinitiate when they reach uORFs 2 to 4, but become competent to do so while scanning the additional sequences between uORF4 and GCN4. Examination of the effects of point mutations in the ATG codons of the different uORFs suggests a quantitative model for this control mechanism that describes the probability of reinitiation as a function of the distance scanned downstream from uORF1. This model accounts for the phenotypes of a number of deletion and insertion mutations that alter the intercistronic spacing between the uORFs and GCN4. The correspondence between observed and predicted results implies that the differential rates of reinitiation at GCN4 versus uORFs 2 to 4 are determined largely by the different scanning times required to reach each of these start sites following translation of uORF1. In addition, it supports the notion that an increased scanning-time requirement for reinitiation in amino acid-starved cells forms the basis for translational derepression of GCN4 expression.     

Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

Upstream open reading frames (uORFs) are often found in the 5′-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression.     

The h subunit of eIF3 promotes reinitiation competence during translation of mRNAs harboring upstream open reading frames

Upstream open reading frames (uORFs) are protein coding elements in the 5′ leader of messenger RNAs. uORFs generally inhibit translation of the main ORF because ribosomes that perform translation elongation suffer either permanent or conditional loss of reinitiation competence. After conditional loss, reinitiation competence may be regained by, at the minimum, reacquisition of a fresh methionyl-tRNA. The conserved h subunit of Arabidopsis eukaryotic initiation factor 3 (eIF3) mitigates the inhibitory effects of certain uORFs. Here, we define more precisely how this occurs, by combining gene expression data from mutated 5′ leaders of Arabidopsis AtbZip11 (At4g34590) and yeast GCN4 with a computational model of translation initiation in wild-type and eif3h mutant plants. Of the four phylogenetically conserved uORFs in AtbZip11, three are inhibitory to translation, while one is anti-inhibitory. The mutation in eIF3h has no major effect on uORF start codon recognition. Instead, eIF3h supports efficient reinitiation after uORF translation. Modeling suggested that the permanent loss of reinitiation competence during uORF translation occurs at a faster rate in the mutant than in the wild type. Thus, eIF3h ensures that a fraction of uORF-translating ribosomes retain their competence to resume scanning. Experiments using the yeast GCN4 leader provided no evidence that eIF3h fosters tRNA reaquisition. Together, these results attribute a specific molecular function in translation initiation to an individual eIF3 subunit in a multicellular eukaryote.     

Physical evidence for distinct mechanisms of translational control by upstream open reading frames.

The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF. To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation. Ribosomes predominantly loaded first at GCN4 uORF1. Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF. Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.     

Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs

Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5' and 3' cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5' cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5' sequences making up the 5' enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5' enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5' enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5' enhancer's stimulatory activity is strictly dependent on and thus follows the 3' enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5' leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.     

cis-acting sequences involved in the translational control of GCN4 expression

Four short upstream open reading frames (uORFs) in the mRNA leader are required for the translational control of GCN4 expression in response to amino acid availability. Data are reviewed demonstrating that the fourth (3' proximal) uORF is sufficient to establish the repressed levels of GCN4 expression, while the first uORF functions as a positive regulatory element under starvation conditions to stimulate GCN4 translation. Furthermore, positive and negative trans-acting regulatory factors, the activities of which appear to be modulated according to amino acid availability, exert their effects on GCN4 expression through the uORFs. Direct comparison of the uORFs indicates that there are important nucleotide sequence differences between uORF1 and 4, and that these are located primarily around the termination codons of these elements. Recent findings suggest that the sequences that mediate repression of GCN4 expression are complex, but can be overcome under starvation conditions by ribosomes that have previously translated uORF1.     

Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region

Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR beta 2 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111- 1119). To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions. uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major ORF.     

Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation

Background  

The translational efficiency of an mRNA can be modulated by upstream open reading frames (uORFs) present in certain genes. A uORF can attenuate translation of the main ORF by interfering with translational reinitiation at the main start codon. uORFs also occur by chance in the genome, in which case they do not have a regulatory role. Since the sequence determinants for functional uORFs are not understood, it is difficult to discriminate functional from spurious uORFs by sequence analysis.     

The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging.

Rous sarcoma virus (RSV) RNA leader contains three short upstream open reading frames. We have shown recently that both uORFs 1 and 3 influence in vivo translation of the downstream gag gene and are involved in the virus RNA packaging process. In this report, we have studied the translational events occurring at the upstream AUGs in vivo. We show that (i) the first and third AUGs are efficient translational initiation sites; (ii) ribosomes reinitiate efficiently at AUG3; and (iii) deletions in the intercistronic distance between uORF1 and 3 (which is well conserved among avian strains) prevent ribosome initiation at AUG3, thus increasing translation efficiency at the downstream AUGgag. The roles of the uORFs in translation and packaging are discussed.     

Translational regulation of human methionine synthase by upstream open reading frames

Methionine synthase is a key enzyme poised at the intersection of folate and sulfur metabolism and functions to reclaim homocysteine to the methionine cycle. The 5' leader sequence in human MS is 394 nucleotides long and harbors two open reading frames (uORFs). In this study, regulation of the main open reading frame by the uORFs has been elucidated. Both uORFs downregulate translation as demonstrated by mutation of the upstream AUG codons (uAUG) either singly or simultaneously. The uAUGs are capable of recruiting the 40S ribosomal complex as revealed by their ability to drive reporter expression in constructs in which the luciferase is fused to the uORFs. uORF2, which is predicted to encode a 30 amino acid long polypeptide, has a clustering of rare codons encoding arginine and proline. Mutation of a tandemly repeated rare codon for arginine at positions 3 and 4 in uORF2 to either common codons for the same amino acid or common codons for alanine results in complete alleviation of translation inhibition. This suggests a mechanism for ribosome stalling and demonstrates that the cis-effects on translation by uORF2 is dependent on the nucleotide sequence but is apparently independent of the sequence of the encoded peptide. This study reveals complex regulation of the essential housekeeping gene, methionine synthase, by the uORFs in its leader sequence.     

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.