The impact of AUG start codon context on maize gene expression in vivo

Summary In mammals, the sequence context surrounding an AUG start codon can alter the efficiency at which translation is initiated. Less is known about the AUG context requirements for translation initiation in plants. Using a maize transient assay, we present evidence that the naturally occurring AUG start codon of the Alcohol dehydrogenase-1 is efficiently used in vivo. We have also tested the effects of upstream, out-of-frame AUGs on the translation of firefly luciferase reporter gene mRNAs. The presence of an upstream out-of-frame AUG, even when surrounded by a poor context, eliminated most luciferase expression, suggesting efficient translation initiation at the upstream AUG. The relaxed requirements for AUG context in maize suggest that plants and mammals may differ in their requirements for efficient translation initiation.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Summary Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5 to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5 to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.     

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     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Evidence for endonucleolytic cleavage at the 5''-proximal segment of the trp messenger RNA in Escherichia coli.

Summary The 5-proximal trp leader RNA segment (about 5S) decays at 2 to 3 times slower rates than the distal trp mRNA sequence. This has been demonstrated by employing the deletion mutants which lack a large portion of the structural genes but retain the promoter-proximal region of the trp operon. Relative stability of the leader RNA is not merely due to the presence of an untranslatable region in the segment; the internal untranslatable segment of trp mRNA downstream from the nonsense alteration site of a double mutant trpAD28·trpE9758 decays as fast as the normal trp mRNA sequence. These results suggest that the trp mRNA is endonucleolytically cleaved to yield the small 5-proximal leader RNA segment before the distal mRNA decays and that the leader RNA sequence is not subject to usual mode of mRNA decay in the 5 to 3 direction.     

Indirect effect of salmon carcasses on growth of a freshwater amphipod, <Emphasis Type="Italic">Jesogammarus jesoensis</Emphasis> (Gammaridea): An experimental study

The effect of salmon carcasses on the growth of a freshwater amphipod, Jesogammarus jesoensis (Schellenberg) (Gammaridea, Anisogammaridae), was examined experimentally. Growth rate was determined by rearing juvenile amphipods in four food treatments (leaves, salmon, leaves and salmon and leaves with salmon leachate) for 20days. Mass loss and oxygen consumption of the leaves were also measured in the three treatments with leaves. The oxygen consumption rate of leaves was lower in the leaves treatment than in either the leaves and salmon or leaves with salmon leachate treatments, indicating that microbial activity on leaves was enhanced by the presence of salmon carcasses. Mass loss of leaves did not differ between the three treatments with leaves. The growth rate of the amphipods did not differ between the leaves and salmon treatments, or between the leaves and salmon and leaves with salmon leachate treatments. Growth rates in the two latter treatments were higher than rates in the leaves treatment, but not higher than the rates in the salmon treatment. Therefore, it appears likely that consuming leaves fertilized with nutrients from salmon carcasses facilitates growth in this amphipod.     

A 43 kD light-regulated chloroplast RNA-binding protein interacts with the psbA 5′ non-translated leader RNA

Expression of the chloroplast psbA gene coding for the D1 protein of Photosystem II is subject to regulation at different levels in higher plants, including control of mRNA accumulation and translation. In dicots, the conserved 5 non-translated leader (5-UTR) of the psbA mRNA is sufficient to direct the light-dependent translation of the D1 protein. In this report we show that the psbA mRNA 5-UTR forms a stem-loop structure and binds a 43 kD chloroplast protein (43RNP). Binding of the 43RNP is sensitive to competition with poly(U), but insensitive to high concentrations of tRNA, the RNA homopolymers poly(A), poly(G), poly(C), or poly(A):poly(U) as a double-strand RNA. The 43RNP does not bind efficiently to the psbA mRNA 3 non-translated region, although the RNA sequence is U-rich and folds into a stem-loop. A deletion mutant of the psbA 5-UTR RNA in which 5 sequences of the stem-loop are removed does not affect 43RNP binding. Together, these properties suggest that the 43RNP binds most effectively to a specific single-strand U-rich sequence preceding the AUG start codon in the psbA mRNA. Binding of the 43RNP is not detectable in plastid protein extracts from 5-day-old dark-grown seedlings, but is detectable in light-grown seedlings as well as mature plants in the light and after shifted to the dark. The 43RNP is therefore a candidate for a regulatory RNA-binding protein that may control the accumulation and/or translation of the psbA mRNA during light-dependent seedling development.Abbreviations DMS dimethylsulfate - psb Photosystem II genes - RNP ribonucleoprotein - UTR non-translated leader - UV crosslinking ultra-violet light crosslinking     

The short 5′ untranslated region of the βA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning

Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. A3- and A1-crystallin, two members of the -crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the A3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of A1-crystallin. It has been suggested that the very short leader (5–7 nucleotides) of the A3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the A1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both A3- and A1-crystallin at very similar relative levels.     

Interrelations between the efficiency of translation start sites and other sequence features of yeast mRNAs

The translation start site (TSS) plays an important role in the control of the translational efficiency and cytoplasmic stability of eukaryotic mRNAs. The efficiency of TSS recognition is known to be influenced by sequence context, and mRNAs with weak TSSs are relatively abundant. We analyzed a sample of 4113 yeast genes in a search for features that might serve to compensate for the inefficient recognition of weak TSSs by initiating ribosomes. The first feature found to correlate with variations in TSS strength is differences in the stability of secondary structure upstream and downstream of the start AUG codon. The second feature concerns the characteristics of AUG triplets found at the beginning of the coding sequence, i.e., downstream of the predicted TSS. In particular, the proximal downstream AUG lies in frame with the CDS significantly more often if the TSS itself is located in a weak context. The accuracy of TSS annotation, the possibility of polypeptide heterogeneity due to the use of alternative downstream AUGs, and the influence of related features of mRNA sequences are discussed.Communicated by C. P. Hollenberg     

Regulation of protein synthesis by mRNA structure

In addition to the m⁷G cap structure, the length of the 5 UTR and the position and context of the AUG initiator codon (which have been discussed elsewhere in this volume), higher order structures within mRNA represent a critical parameter for translation. The role of RNA structure in translation initiation will be considered primarily, although structural elements have also been found to affect translation elongation and termination. We will first describe the different effects of higher order RNA structuresper se, and then consider specific examples of RNA structural elements which control translation initiation by providing binding sites for regulatory proteins.     

The role of alternative translation start sites in the generation of human protein diversity

According to the scanning model, 40S ribosomal subunits initiate translation at the first (5 proximal) AUG codon they encounter. However, if the first AUG is in a suboptimal context, it may not be recognized, and translation can then initiate at downstream AUG(s). In this way, a single RNA can produce several variant products. Earlier experiments suggested that some of these additional protein variants might be functionally important. We have analysed human mRNAs that have AUG triplets in 5 untranslated regions and mRNAs in which the annotated translational start codon is located in a suboptimal context. It was found that 3% of human mRNAs have the potential to encode N-terminally extended variants of the annotated proteins and 12% could code for N-truncated variants. The predicted subcellular localizations of these protein variants were compared: 31% of the N-extended proteins and 30% of the N-truncated proteins were predicted to localize to subcellular compartments that differed from those targeted by the annotated protein forms. These results suggest that additional AUGs may frequently be exploited for the synthesis of proteins that possess novel functional properties.Electronic Supplementary Material Supplementary material is available for this article at     

Translational control of cellular and viral mRNAs

We are becoming increasingly aware of the role that translational control plays in regulating gene expression in plants. There are now many examples in which specific mechanisms have evolved at the translational level that directly impact the amount of protein produced from an mRNA. All regions of an mRNA, i.e., the 5 leader, the coding region, and the 3-untranslated region, have the potential to influence translation. The 5-terminal cap structure and the poly(A) tail at the 3 terminus serve as additional elements controlling translation. Many viral mRNAs have evolved alternatives to the cap and poly(A) tail that are functionally equivalent. Nevertheless, for both cellular and viral mRNAs, a co-dependent interaction between the terminal controlling elements appears to be the universal basis for efficient translation.     

Exploration of RNA 3′ terminal locations in rat ribosome

The locations of the 3 ends of RNAs in rat ribosome were studied by a fluorescencelabeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3 ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe – fluorescein 5thiosemicarbazide (FTSC), indicating that the 3 termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3 terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3 end of 5 S RNA was located on the interface of two subunits. However, no fluorescencelabeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3 end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3 ends of ribosomal RNAs including oxidation with NaIO₄, reduction with KBH₄ and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3 ends of RNAs were not involved in the translation activity of ribosome.     

Organ-specific modulation of gene expression in transgenic plants using antisene RNA

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS gene dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense. RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially down mutations in essential genes where only a short 5 sequence of the mRNA is required. They also suggest that the position effect on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.     

The sup8 tRNALeu gene of Schizosaccharomyces pombe has an unusual intervening sequence and reduced pairing in the anticodon stem

Summary We have cloned and sequenced the wild-type and suppressor alleles of the S. pombe sup8 tRNA gene. The wild-type allele has a leucine UAA anticodon and the suppressor (sup8-e) carries the opal suppressor anticodon UCA. The gene has a 16 base pair intervening sequence that, in the RNA, is predicted to form a secondary structure which involves base pairing to the 5, rather than the usual 3 side of the 5 splice site. When incubated in Saccharomyces cerevisiae cell-free extracts both alleles are efficiently transcribed, the 5 leader and 3 trailer sequences are removed and CCA is added to the 3 processed end; however, the intervening sequence is not excised. This finding implies that the structural requirements of the splicing endonucleases in the two yeasts have diverged. No other tRNA genes with related sequences were detected in S. pombe DNA by hybridization, suggesting that other UUA isoacceptors may be structurally dissimilar to sup8 or that the UUA codon may be decoded by a UUG leucine isoacceptor.     

IGF-I activates the eIF4F system in cardiac muscle in vivo

IGF-I acutely stimulates protein synthesis in cardiac muscle through acceleration of mRNA translation. In the present study, we examined the regulatory signaling pathways and translation protein factors that potentially contribute to the myocardial responsiveness of protein synthesis to IGF-I in vivo. IGF-I was injected IV into rats and 20 min later the hearts were excised and homogenized for assay of regulatory proteins. IGF-I increased assembly of the translationally active eukaryotic initiation factor (eIF)4GeIF4E complex. The increased assembly of eIF4GeIF4E was associated with an enhanced eIF4G phosphorylation and increased availability of eIF4E. Increased availability of eIF4E occurred as a consequence of diminished abundance of the inactive 4E-BP1eIF4E complex following IGF-I. The assembly of the 4E-BP1eIF4E complex appeared to be decreased through an IGF-I-induced phosphorylation of 4E-BP1. IGF-I also caused an increase in the phosphorylation of S6K1. Activation of the potential upstream regulators of 4E-BP1 and S6K1 phosphorylation via PKB and mTOR was also observed. In contrast, there was no effect of IGF-I on phosphorylation of elongation factor (eFE)2. The results suggest the major impact of IGF-I in cardiac muscle occurred via stimulation of translation initiation rather than elongation. Furthermore, the results are consistent with a role for assembly of active eIF4GeIF4E complex and activation of S6K1 in mediating the stimulation of mRNA translation initiation by IGF-I through a PKB/mTOR signaling pathway.     

Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1 (EF-1), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1. An intron is located in the 5 untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1tef-c (97%) than to the P. purpurea EF-1tef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.     

Detection of cellular proteins and viral core protein interacting with the 5′ untranslated region of hepatitis C virus RNA

Hepatitis C virus (HCV) is a pesti- and flavi-like virus, which contains a highly conserved 5-untranslated region (UTR). This region is implicated in the regulation of both translation and RNA replication. To examine the possible cellular factors involved in HCV replication, we performed UV cross-linking experiments to detect cellular proteins binding to 5-UTR of HCV RNA. No cytoplasmic proteins were found to cross-link to 5-UTR. Surprisingly, when nuclear extracts were used for UV cross-linking, a major protein of 110 kD and several other minor proteins were detected. Competition assays confirmed that the binding of the 110-kD protein was specific to the 5-UTR. The protein-binding site was mapped within the 78-nt region between nucleotides 199 and 277 from the 5 end of the viral RNA. This protein was present in several different cell lines tested. No cellular proteins specifically bound to the complementary strands of the 5-UTR. We have also shown by an RNA-protein blotting assay that 5-UTR bound to the HCV core protein, which can be translocated to the nuclei. These findings suggest that HCV RNA may enter nuclei by complexing with the viral core protein and interact with nuclear proteins that are involved in the regulation of RNA replication or translation. It is thus possible that HCV employs a replication strategy distinct from its related pestiviruses or flaviviruses.     

The sequence and secondary structure of the 3′-UTR affect 3′-end maturation,RNA accumulation,and translation in tobacco chloroplasts

RNA maturation and modulation of RNA stability play important roles in chloroplast gene expression. In vitro and in vivo studies have shown that both the 5- and 3-untranslated regions (UTRs) contain sequence and structural elements that guide these processes, and interact with specific proteins. We have previously characterized the spinach chloroplast petD 3-UTR in detail by in vitro approaches. This stem-loop forming sequence is a weak terminator but is required for RNA maturation and also exhibits sequence-specific protein binding. To test petD 3-UTR function in vivo, tobacco chloroplast transformants were generated containing uidA reporter genes flanked by variants of the petD 3-UTR, including one which does not form an RNA-protein complex in vitro, and one which lacks a stem-loop structure. Analysis of uidA mRNA indicated that a stable secondary structure is required to accumulate a discrete mRNA, and that changes in the 3-UTR sequence which affect protein binding in vitro can also affect RNA metabolism in vivo. The 3-UTR also influenced -glucuronidase protein accumulation, but not in proportion to RNA levels. These results raise the possibility that in tobacco chloroplasts, the 3-UTR may influence translational yield.