Phylogeny-linked occurrence of ribosome stalling on the mRNAs of Arabidopsis unfolded protein response factor bZIP60 orthologs in divergent plant species.

The bZIP60, XBP1 and HAC1 mRNAs encode transcription factors that mediate the unfolded protein response (UPR) in plants, animals and yeasts, respectively. Upon UPR, these mRNAs undergo unconventional cytoplasmic splicing on the endoplasmic reticulum (ER) to produce active transcription factors. Although cytoplasmic splicing is conserved, the ER targeting mechanism differs between XBP1 and HAC1. The ER targeting of HAC1 mRNA occurs before translation, whereas that of XBP1 mRNA involves a ribosome-nascent chain complex that is stalled when a hydrophobic peptide emerges from the ribosome; the corresponding mechanism is unknown for bZIP60. Here, we analyzed ribosome stalling on bZIP60 orthologs of plants. Using a cell-free translation system, we detected nascent peptide-mediated ribosome stalling during the translation elongation of the mRNAs of Arabidopsis, rice and Physcomitrium (moss) orthologs, and the termination-step stalling in the Selaginella (lycopod) ortholog, all of which occurred ∼50 amino acids downstream of a hydrophobic region. Transfection experiments showed that ribosome stalling contributes to cytoplasmic splicing in bZIP60u orthologs of Arabidopsis and Selaginella. In contrast, ribosome stalling was undetectable for liverwort, Klebsormidium (basal land plant), and green algae orthologs. This study highlights the evolutionary diversity of ribosome stalling and its contribution to ER targeting in plants.

Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels.

KEY MESSAGE: This study identified four novel regulatory non-AUG-initiated upstream ORFs (uORFs) with evolutionarily conserved sequences in Arabidopsis and elucidated the mechanism by which a non-AUG-initiated uORF promotes main ORF translation. Upstream open reading frames (uORFs) are short ORFs found in the 5'-untranslated regions (5'-UTRs) of eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that hundreds or thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. In this study, to identify physiologically important regulatory non-AUG uORFs in Arabidopsis, we took an approach that combined bioinformatics and experimental analysis. Since physiologically important non-AUG uORFs are likely to be conserved across species, we first searched the Arabidopsis genome for non-AUG-initiated uORFs with evolutionarily conserved sequences. Then, we examined the effects of the conserved non-AUG uORFs on the expression of the downstream mORFs using transient expression assays. As a result, three inhibitory and one promotive non-AUG uORFs were identified. Among the inhibitory non-AUG uORFs, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.

Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ß-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ß-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

Comprehensive genome-wide identification of angiosperm upstream ORFs with peptide sequences conserved in various taxonomic ranges using a novel pipeline, ESUCA.

BACKGROUND: Upstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups. RESULTS: To efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 89 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved across wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides. CONCLUSIONS: This study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.

Boron-Dependent Translational Suppression of the Borate Exporter BOR1 Contributes to the Avoidance of Boron Toxicity.

Boron (B) is an essential element for plants; however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis (Arabidopsis thaliana) that facilitates B translocation into shoots under B deficiency conditions. When the B supply is sufficient, BOR1 expression is down-regulated by selective degradation of BOR1 protein, while additional BOR1 regulatory mechanisms are proposed to exist. In this study, we identified a novel B-dependent BOR1 translational suppression mechanism. In vivo and in vitro reporter assays demonstrated that BOR1 translation was reduced in a B-dependent manner and that the 5'-untranslated region was both necessary and sufficient for this process. Mutational analysis revealed that multiple upstream open reading frames in the 5'-untranslated region were required for BOR1 translational suppression, and this process depended on the efficiency of translational reinitiation at the BOR1 open reading frame after translation of the upstream open reading frames. To understand the physiological significance of BOR1 regulation, we characterized transgenic plants defective in either one or both of the BOR1 regulation mechanisms. BOR1 translational suppression was induced at higher B concentrations than those triggering BOR1 degradation. Plants lacking both regulation mechanisms exhibited more severe shoot growth reduction under high-B conditions than did plants lacking BOR1 degradation alone, thus demonstrating the importance of BOR1 translational suppression. This study demonstrates that two mechanisms of posttranscriptional BOR1 regulation, each induced under different B concentrations, contribute to the avoidance of B toxicity in plants.

The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation.

Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.

Identification of Arabidopsis thaliana upstream open reading frames encoding peptide sequences that cause ribosomal arrest.

Specific sequences of certain nascent peptides cause programmed ribosomal arrest during mRNA translation to control gene expression. In eukaryotes, most known regulatory arrest peptides are encoded by upstream open reading frames (uORFs) present in the 5'-untranslated region of mRNAs. However, to date, a limited number of eukaryotic uORFs encoding arrest peptides have been reported. Here, we searched for arrest peptide-encoding uORFs among Arabidopsis thaliana uORFs with evolutionarily conserved peptide sequences. Analysis of in vitro translation products of 22 conserved uORFs identified three novel uORFs causing ribosomal arrest in a peptide sequence-dependent manner. Stop codon-scanning mutagenesis, in which the effect of changing the uORF stop codon position on the ribosomal arrest was examined, and toeprint analysis revealed that two of the three uORFs cause ribosomal arrest during translation elongation, whereas the other one causes ribosomal arrest during translation termination. Transient expression assays showed that the newly identified arrest-causing uORFs exerted a strong sequence-dependent repressive effect on the expression of the downstream reporter gene in A. thaliana protoplasts. These results suggest that the peptide sequences of the three uORFs identified in this study cause ribosomal arrest in the uORFs, thereby repressing the expression of proteins encoded by the main ORFs.

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.

Ribosomes in a stacked array: elucidation of the step in translation elongation at which they are stalled during S-adenosyl-L-methionine-induced translation arrest of CGS1 mRNA.

Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.

The N-terminal cleavable pre-sequence encoded in the first exon of cystathionine γ-synthase contains two different functional domains for chloroplast targeting and regulation of gene expression.

Chloroplast transit peptide sequences (cTPs) located in the N-terminal region of nuclear-encoded chloroplast proteins are essential for their sorting, and are generally cleaved from the proteins after their import into the chloroplasts. The Arabidopsis thaliana cystathionine γ-synthase (CGS), the first committed enzyme of methionine biosynthesis, is a nuclear-encoded chloroplast protein. Arabidopsis CGS possesses an N-terminal extension region that is dispensable for enzymatic activity. This N-terminal extension contains the cTP and several functional domains including an MTO1 region, the cis-element for post-transcriptional feedback regulation of CGS1 that codes for CGS. A previous report suggested that the cTP cleavage site of CGS is located upstream of the MTO1 region. However, the region required for protein sorting has not been analyzed. In this study, we carried out functional analyses to elucidate the region required for chloroplast targeting by using a chimeric protein, Ex1:GFP, in which the CGS1 exon 1 coding region containing the N-terminal extension was tagged with green fluorescent protein. The sequence upstream of the MTO1 region was responsible for efficient chloroplast targeting and for avoidance of missorting to the mitochondria. Our data also showed that the major N-terminus of Ex1:GFP is Ala91, which is located immediately downstream of the MTO1 region, and the MTO1 region is not retained in the mature Ex1:GFP accumulated in the chloroplast. These findings suggest that the N-terminal cleavable pre-sequence harbors dual functions in protein sorting and in regulating gene expression. Our study highlights the unique properties of Arabidopsis CGS cTP among chloroplast-targeted proteins.

Polyamine-responsive ribosomal arrest at the stop codon of an upstream open reading frame of the AdoMetDC1 gene triggers nonsense-mediated mRNA decay in Arabidopsis thaliana.

During mRNA translation, nascent peptides with certain specific sequences cause arrest of ribosomes that have synthesized themselves. In some cases, such ribosomal arrest is coupled with mRNA decay. In yeast, mRNA quality control systems have been shown to be involved in mRNA decay associated with ribosomal arrest. However, a link between ribosomal arrest and mRNA quality control systems has not been found in multicellular organisms. In this study, we aimed to explore the relationship between ribosomal arrest and mRNA decay in plants. For this purpose, we used an upstream open reading frame (uORF) of the Arabidopsis thaliana AdoMetDC1 gene, in which the uORF-encoded peptide is involved in polyamine-responsive translational repression of the main coding sequence. Our in vitro analyses revealed that the AdoMetDC1 uORF-encoded peptide caused ribosomal arrest at the uORF stop codon in response to polyamine. Using transgenic calli harboring an AdoMetDC1 uORF-containing reporter gene, we showed that polyamine promoted mRNA decay in a uORF sequence-dependent manner. These results suggest that the polyamine-responsive ribosomal arrest mediated by the uORF-encoded peptide is coupled with mRNA decay. Our results also showed that the polyamine-responsive acceleration of mRNA decay was compromised by defects in factors that are essential for nonsense-mediated mRNA decay (NMD), an mRNA quality control system that degrades mRNAs with premature stop codons, suggesting that NMD is involved in AdoMetDC1 uORF peptide-mediated mRNA decay. Collectively, these findings suggest that AdoMetDC1 uORF peptide-mediated ribosomal arrest at the uORF stop codon induces NMD.

Boron-dependent degradation of NIP5;1 mRNA for acclimation to excess boron conditions in Arabidopsis.

Boron (B) is an essential plant micronutrient that is toxic at higher levels. NIP5;1 is a boric acid channel required for B uptake and growth under B deficiency. Accumulation of the NIP5;1 transcript is upregulated under B deficiency in Arabidopsis thaliana roots. To elucidate the mechanism of regulation, the 5' untranslated region (UTR) of NIP5;1 was tested for its ability to confer B-dependent regulation using ß-glucuronidase and green fluorescent protein as reporters. This analysis showed that the 5' UTR was involved in NIP5;1 transcript accumulation in response to B conditions. We also found that high-B conditions trigger NIP5;1 mRNA degradation and that the sequence from +182 to +200 bp in the 5' UTR is required for this mRNA destabilization. In the nip5;1-1 mutant background, a NIP5;1 complementation construct without the 5' UTR produced high levels of mRNA accumulation, increased B concentrations in tissues, and reduced growth under high-B conditions. These data suggest that the 5' UTR controls B-dependent NIP5;1 mRNA degradation and that NIP5;1 mRNA degradation is important for plant acclimation to high-B conditions.

In vitro co-expression chromatin assembly and remodeling platform for plant histone variants.

Histone variants play a central role in shaping the chromatin landscape in plants, yet, how their distinct combinations affect nucleosome properties and dynamics is still largely elusive. To address this, we developed a novel chromatin assembly platform for Arabidopsis thaliana, using wheat germ cell-free protein expression. Four canonical histones and five reported histone variants were used to assemble twelve A. thaliana nucleosome combinations. Seven combinations were successfully reconstituted and confirmed by supercoiling and micrococcal nuclease (MNase) assays. The effect of the remodeling function of the CHR11-DDR4 complex on these seven combinations was evaluated based on the nucleosome repeat length and nucleosome spacing index obtained from the MNase ladders. Overall, the current study provides a novel method to elucidate the formation and function of a diverse range of nucleosomes in plants.

Changes in mRNA stability associated with cold stress in Arabidopsis cells.

Control of mRNA half-life is a powerful strategy to adjust individual mRNA levels to various stress conditions, because the mRNA degradation rate controls not only the steady-state mRNA level but also the transition speed of mRNA levels. Here, we analyzed mRNA half-life changes in response to cold stress in Arabidopsis cells using genome-wide analysis, in which mRNA half-life measurements and transcriptome analysis were combined. Half-lives of average transcripts were determined to be elongated under cold conditions. Taking this general shift into account, we identified more than a thousand transcripts that were classified as relatively stabilized or relatively destabilized. The relatively stabilized class was predominantly observed in functional categories that included various regulators involved in transcriptional, post-transcriptional and post-translational processes. On the other hand, the relatively destabilized class was enriched in categories related to stress and hormonal response proteins, supporting the idea that rapid decay of mRNA is advantageous for swift responses to stress. In addition, pentatricopeptide repeat, cyclin-like F-box and Myb transcription factor protein families were significantly over-represented in the relatively destabilized class. The global analysis presented here demonstrates not only the importance of mRNA turnover control in the cold stress response but also several structural characteristics that might be important in the control of mRNA stability.

SS-mPMG and SS-GA: tools for finding pathways and dynamic simulation of metabolic networks.

Metabolomics analysis tools can provide quantitative information on the concentration of metabolites in an organism. In this paper, we propose the minimum pathway model generator tool for simulating the dynamics of metabolite concentrations (SS-mPMG) and a tool for parameter estimation by genetic algorithm (SS-GA). SS-mPMG can extract a subsystem of the metabolic network from the genome-scale pathway maps to reduce the complexity of the simulation model and automatically construct a dynamic simulator to evaluate the experimentally observed behavior of metabolites. Using this tool, we show that stochastic simulation can reproduce experimentally observed dynamics of amino acid biosynthesis in Arabidopsis thaliana. In this simulation, SS-mPMG extracts the metabolic network subsystem from published databases. The parameters needed for the simulation are determined using a genetic algorithm to fit the simulation results to the experimental data. We expect that SS-mPMG and SS-GA will help researchers to create relevant metabolic networks and carry out simulations of metabolic reactions derived from metabolomics data.

BAIUCAS: a novel BLAST-based algorithm for the identification of upstream open reading frames with conserved amino acid sequences and its application to the Arabidopsis thaliana genome.

MOTIVATION: Upstream open reading frames (uORFs) are often found in the 5'-untranslated regions of eukaryotic messenger RNAs. Some uORFs have been shown to encode functional peptides involved in the translational regulation of the downstream main ORFs. Comparative genomic approaches have been used in genome-wide searches for uORFs encoding bioactive peptides, and by comparing uORF sequences between a few selected species or among a small group of species, uORFs with conserved amino acid sequences (UCASs) have been identified in plants, mammals and insects. Regulatory regions within uORF-encoded peptides that are involved in translational control are typically 10-20 amino acids long. Detection of homology between such short regions largely depends on the selection of species for comparison. To maximize the chances of identifying UCASs with short conserved regions, we devised a novel algorithm for homology search among a large number of species and the automatic selection of uORFs conserved in a wide range of species. RESULTS: In this study, we developed the BAIUCAS (BLAST-based algorithm for identification of UCASs) method and identified 18 novel Arabidopsis uORFs whose amino acid sequences are conserved across diverse eudicot species, which include uORFs not found in previous comparative genomic studies due to low sequence conservation among species. Therefore, BAIUCAS is a powerful method for the identification of UCASs, and it is particularly useful for the detection of uORFs with a small number of conserved amino acid residues.

Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways.

Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5;1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5;1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5;1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.

Upstream open reading frame-mediated upregulation of ANAC082 expression in response to nucleolar stress in Arabidopsis.

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5'-untranslated region (5'-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 expression in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5'-UTR were treated with reagents that induced nucleolar stress, expression of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent repressive effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent regulation was lost in ANAC103 during evolution.

S-adenosyl-L-methionine induces compaction of nascent peptide chain inside the ribosomal exit tunnel upon translation arrest in the Arabidopsis CGS1 gene.

Expression of the Arabidopsis CGS1 gene, encoding the first committed enzyme of methionine biosynthesis, is feedback-regulated in response to S-adenosyl-L-methionine (AdoMet) at the mRNA level. This regulation is first preceded by temporal arrest of CGS1 translation elongation at the Ser-94 codon. AdoMet is specifically required for this translation arrest, although the mechanism by which AdoMet acts with the CGS1 nascent peptide remained elusive. We report here that the nascent peptide of CGS1 is induced to form a compact conformation within the exit tunnel of the arrested ribosome in an AdoMet-dependent manner. Cysteine residues introduced into CGS1 nascent peptide showed reduced ability to react with polyethyleneglycol maleimide in the presence of AdoMet, consistent with a shift into the ribosomal exit tunnel. Methylation protection and UV cross-link assays of 28 S rRNA revealed that induced compaction of nascent peptide is associated with specific changes in methylation protection and UV cross-link patterns in the exit tunnel wall. A 14-residue stretch of amino acid sequence, termed the MTO1 region, has been shown to act in cis for CGS1 translation arrest and mRNA degradation. This regulation is lost in the presence of mto1 mutations, which cause single amino acid alterations within MTO1. In this study, both the induced peptide compaction and exit tunnel change were found to be disrupted by mto1 mutations. These results suggest that the MTO1 region participates in the AdoMet-induced arrest of CGS1 translation by mediating changes of the nascent peptide and the exit tunnel wall.

Arabidopsis cell-free extract, ACE, a new in vitro translation system derived from Arabidopsis callus cultures.

The analysis of post-transcriptional regulatory mechanisms in plants has benefited greatly from the use of cell-free extract systems. Arabidopsis as a model system provides extensive genetic resources; however, to date a suitable cell-free translation system from Arabidopsis has not been available. In this study, we devised an Arabidopsis cell-free extract (ACE) to be used for in vitro translation studies. Protoplasts were prepared from callus cultures derived from Arabidopsis seedlings, and cell-free extracts were prepared after evacuolation of the protoplasts by Percoll gradient centrifugation. The new ACE system exhibits translation activity comparable with that of the wheat germ extract system. We demonstrated that ACE prepared from the 5'-3' exoribonuclease-deficient mutant of Arabidopsis, xrn4-5, exhibited increased stability of an uncapped mRNA as compared with that from wild-type Arabidopsis. We applied the ACE system to study post-transcriptional regulation of AtCGS1. AtCGS1 codes for cystathionine γ-synthase (CGS) that catalyzes the first committed step of methionine and S-adenosyl-l-methionine (AdoMet) biosynthesis in plants, and is feedback regulated by mRNA degradation coupled with translation elongation arrest. The ACE system was capable of reproducing translation elongation arrest and subsequent AtCGS1 mRNA degradation that are induced by AdoMet. The ACE system described here can be prepared in a month after seed sowing and will make it possible to study post-transcriptional regulation of plant genes while taking advantage of the genetics of Arabidopsis.