Search

Thyroid hormone (TH) regulates gene transcription by binding to the thyroid hormone receptor (TR) and plays a critical role in the regulation of development, growth and metabolism. The ligated TR activates many effector genes, which contributes to the orchestrated remodelling of the amphibian metamorphosis. However, the mechanisms regulating TRα protein level remain unknown. We determined the nucleotide sequences of the 5′-untranslated regions (5′-UTRs) in amphibian TRα mRNAs. The TRα 5′-UTR contains evolutionarily conserved regions. We demonstrated that a 161-nt stretch of the Xenopus TRα 5′-UTR strongly represses the translation of the downstream open reading frame in both frog and human cell lines, as well as in a cell-free translation system. An analysis using successive deletions of the TRα 5′-UTR revealed five elements possessing translational repressive activity. We analysed two elements, the 14-nt GC-rich region and the 15-nt upstream open reading frame (uORF), by introducing point mutations. This analysis showed that the GC-rich region, which shares its nucleotide sequence with the Sp1-binding site, requires stringent sequence specificity at a nucleotide level for translational repression to take place, whereas under our study conditions, the uORF does not. This study provides an example of complex translational regulation by multiple elements. [ABSTRACT FROM PUBLISHER]

Thermospermine is a structural isomer of spermine, which is one of the polyamines studied extensively in the past, and is produced from spermidine by the action of thermospermine synthase encoded by a gene named ACAULIS5 (ACL5) in plants. According to recent genome sequencing analyses, ACL5-like genes are widely distributed throughout the plant kingdom. In Arabidopsis, ACL5 is expressed specifically during xylem formation from procambial cells to differentiating xylem vessels. Loss-of-function mutants of ACL5 display overproliferation of xylem vessels along with severe dwarfism, suggesting that thermospermine plays a role in the repression of xylem differentiation. Studies of suppressor mutants of acl5 that recover the wild-type phenotype in the absence of thermospermine suggest that thermospermine acts on the translation of specific mRNAs containing upstream open reading frames (uORFs). Thermospermine is a novel type of plant growth regulator and may also serve in the control of wood biomass production. [ABSTRACT FROM AUTHOR]

The Alu element has been a major source of new exons during primate evolution. Thousands of human genes contain spliced exons derived from Alu elements. However, identifying Alu exons that have acquired genuine biological functions remains a major challenge. We investigated the creation and establishment of Alu exons in human genes, using transcriptome profiles of human tissues generated by high-throughput RNA sequencing (RNA-Seq) combined with extensive RT-PCR analysis. More than 25% of Alu exons analyzed by RNA-Seq have estimated transcript inclusion levels of at least 50% in the human cerebellum, indicating widespread establishment of Alu exons in human genes. Genes encoding zinc finger transcription factors have significantly higher levels of Alu exonization. Importantly, Alu exons with high splicing activities are strongly enriched in the 55′-UTR, and two-thirds (10/15) of 5′-UTR Alu exons tested by luciferase reporter assays significantly alter mRNA translational efficiency. Mutational analysis reveals the specific molecular mechanisms by which newly created 55′-UTR Alu exons modulate translational efficiency, such as the creation or elongation of upstream ORFs that repress the translation of the primary ORFs. This study presents genomic evidence that a major functional consequence of Alu exonization is the lineage-specific evolution of translational regulation. Moreover, the preferential creation and establishment of Alu exons in zinc finger genes suggest that Alu exonization may have globally affected the evolution of primate and human transcriptomes by regulating the protein production of master transcriptional regulators in specific lineages. [ABSTRACT FROM AUTHOR]

Abstract: The p13 gene is a group II nucleopolyhedroviruses (NPVs) specific gene and featured by containing upstream mini ORFs (uORF) in its 5′ UTR region. However, there are almost no reports published on the functions of the uORFs of p13 gene. In this study, the Luciferase Reporter Assay System was employed to investigate how the mini ORFs of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) p13 gene (Ha-p13) and its rare codons regulated the downstream gene expression. After the coding sequence of uORFs in the Ha-p13 gene was fused to the luciferase reporter gene in the expression vector pGL3 and the plasmid DNA was then transfected into the Hz-AM1 cells, the translation of the fusion protein could be initiated from the start codon of the uORFs. The uAUG and its context in uORF2 seemed to be more efficient for translation initiation than that in uORF1. Mutation of the start codons in one or both of uORFs (uORF1 or uORF2) could significantly increase the expression of the downstream reporter gene. The start codon mutation in uORF1 produced a higher reporter gene expression than that in uORF2, indicating that the uORF1 could be a stronger inhibitor than the uORF2, and the length of uORFs seemed not to be crucial for down-regulating translation. The expression of both uORFs could co-regulate the associated gene expression. Substituting the rare codons in uORF1, uORF2 or both with less rare codons dramatically increased the expression of the downstream reporter gene. Rare codon mutations in both uORFs were much more efficient in up-regulating the associate gene expression than mutations in either of the two uORFs alone. [ABSTRACT FROM AUTHOR]

Abstract: The RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5′ untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression. [ABSTRACT FROM AUTHOR]

The authors provide an overview of upstream open reading frame (uORF)-mediated translational control. They describe how aberrant uORF regulation can be translated into pathophysiology. They cite the role of contemporary sequencing technologies in addressing the implications of uORF-mediated translational control in human disease. The authors also conclude that uORF translation has pathophysiological significance.

Abstract: Mitochondrial ribosomal protein S29 (MRPS29) is a mitochondrial pro-apoptotic protein also known as death associated protein 3 (DAP3). Over-expression of MRPS29 has been reported to induce apoptosis in several different human cell lines while conferring resistance in glioma and Ataxia telangiectasia cells. These two contradictory reports led us to investigate the MRPS29-induced apoptosis further. Cyber searches of the EST databases revealed the presence of a splice variant of MRPS29 mRNA containing an upstream open reading frame (uORF) at the 5′ untranslated region (UTR). In this study, we confirmed the presence of this uORF using real-time RT-PCR and investigated its role in MRPS29 expression. [Copyright &y& Elsevier]

Background: Polyamines are small polycationic molecules found ubiquitously in all organisms and function in a wide variety of biological processes. In the past decade, molecular and genetic studies using mutants and transgenic plants with an altered activity of enzymes involved in polyamine biosynthesis have contributed much to a better understanding of the biological functions of polyamines in plants. [ABSTRACT FROM PUBLISHER]

Approximately 20% of plant genes possess upstream open-reading frames (uORFs). The effect of uORFs on gene expression has mainly been studied at the translational level. Very little is known about the impact of plant uORFs on transcript content through the nonsense-mediated mRNA decay (NMD) pathway, which degrades transcripts bearing premature termination codons (PTCs). Here we examine the impact of the uORF of the Arabidopsis AtMHX gene on transcript accumulation. The suggestion that this uORF exposes transcripts containing it to NMD is supported by (i) the increase in transcript levels upon eliminating the uORF from constructs containing it, (ii) experiments with a modified uORF-peptide, which excluded peptide-specific degradation mechanisms, (iii) the increase in levels of the native AtMHX transcript upon treatment with cycloheximide, which inhibits translation and blocks NMD, and (iv) the sensitivity of transcripts containing the uORF of AtMHX to the presence of introns. We also showed that introns can increase NMD efficiency not only in transcripts having relatively short 3′ untranslated regions (UTRs), but also in uORF-containing transcripts. AtMHX transcript levels were almost unaltered in mutants of the NMD factors UPF3 and UPF1. Possible reasons, including the existence of a NMD-compensatory mechanism, are discussed. Interestingly, the levels of UPF3 transcript were higher in upf1 mutants, suggesting a compensatory mechanism that links weak function of the NMD machinery to increased expression of UPF3. Our findings highlight that uORFs, which are abundant in plants, can not only inhibit translation but also strongly affect transcript accumulation. [ABSTRACT FROM AUTHOR]

Upstream open reading frames (uORFs) are frequently present in the 5′- leader regions of fungal mRNAs. They can affect translation by controlling the ability of ribosomes that scan from the mRNA 5′ end to reach the downstream genic reading frame. The translation of uORFs can also affect mRNA stability. For several genes, including Saccharomyces cerevisiae GCN4, S. cerevisiae CPA1, and Neurospora crassa arg-2, regulation by uORFs controls expression in response to specific physiological signals. The roles of many uORFs that are identified by genome-level approaches, as have been initiated for Saccharomyces, Aspergillus, and Cryptococcus species, remain to be determined. Some uORFs may have regulatory roles, while others may exist to insulate the genic reading frame from the negative impacts of upstream translation start sites in the mRNA 5′ leader. [ABSTRACT FROM AUTHOR]

H2B histone family, member W, testis-specific (H2BFWT) gene encodes a testis-specific histone that becomes incorporated into sperm chromatin. A male infertility-associated single nucleotide polymorphism (−9C > T) within the 5′ untranslated region (5′UTR) of the H2BFWT gene was identified by direct sequencing. Statistical association studies showed the polymorphism significantly associated with male infertility ( n= 442, P= 0.0157), especially in non-azoospermia ( n= 262, P= 0.018). Furthermore, this polymorphism is also associated with sperm parameters, especially sperm count ( n= 164, P= 0.0127) and vitality ( n= 164, P= 0.0076). We investigated how the genetic variant at 5′UTR confers susceptibility to non-azoospermia. Western blotting of His-tag H2BFWT revealed a difference at the translational level between -9T and the wild-type −9C in the absence of change at the transcriptional level. Reporter assays showed that this reducing translational change originated from an upstream open reading frame (uORF) generated by the −9C to −9T change. Finally, in vivo H2BFWT expression in sperm was significantly dependent on the −9C > T genotype from non-azoospermia ( P= 0.0061). Therefore, this polymorphism could affect the translational efficiency of a quantitatively important histone protein by the uORF. Our data implicate H2BFWT as a susceptibility factor for male infertility, possibly with other genetic and environmental factors. [ABSTRACT FROM AUTHOR]

Upstream ORFs (uORFs) are mRNA elements defined by a start codon in the 5' UTR that is out-of-frame with the main coding sequence. Although uORFs are present in approximately half of human and mouse transcripts, no study has investigated their global impact on protein expression. Here, we report that uORFs correlate with significantly reduced protein expression of the downstream ORF, based on analysis of 11,649 matched mRNA and protein measurements from 4 published mammalian studies. Using reporter constructs to test 25 selected uORFs, we estimate that uORFs typically reduce protein expression by 30-80%, with a modest impact on mRNA levels. We additionally identify polymorphisms that alter uORF presence in 509 human genes. Finally, we report that 5 uORF-altering mutations, detected within genes previously linked to human diseases, dramatically silence expression of the downstream protein. Together, our results suggest that uORFs influence the protein expression of thousands of mammalian genes and that variation in these elements can influence human phenotype and disease. [ABSTRACT FROM AUTHOR]

The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation. [ABSTRACT FROM AUTHOR]

Abstract: A translational regulatory mechanism for Tomato golden mosaic virus (TGMV) complementary-sense gene expression has been characterized. TGMV transcribes two mRNAs, AL-1935 and AL-1629 transcripts, both of which contain the AL2 and AL3 open reading frames. However, AL2 is only expressed from AL-1629 whereas AL3 is expressed from both. Three AUG translation initiation codons are located upstream of both the AL2 and AL3 coding regions, within the 5′-untranslated region (UTR) of the AL-1935 transcript. Translation can initiate at the first AUG, specifying the C-terminal 122 amino acids of the AL1 protein (cAL1). Initiation of translation at this AUG is inhibitory for the downstream expression of both AL2 and AL3. This is most likely due to the terminator codon of cAL1 being positioned after the AUG initiation codon for the AL2 ORF. The mechanism by which AL3 is expressed from AL-1935 is currently unknown but a gap between the cAL1 termination codon and the start of AL3 suggests that it may involve reinitiation and/or internal initiation. In contrast, expression of AL3 from AL-1629 most likely occurs via leaky ribosome scanning since the AL3 initiation codon occurs before the terminator codon of AL2. Mutation of the AUG encoding cAL1 in the curtovirus, Spinach curly top virus, leads to increased infectivity as measured by a shorter latent period. Together this suggests that geminiviruses use a post-translational regulatory mechanism to regulate the synthesis of viral proteins important for replication and suppression of host defenses. [Copyright &y& Elsevier]

Abstract: The HIV-1 Vpu and Env proteins are translated from 16 alternatively spliced bicistronic mRNA isoforms. Translation of HIV-1 mRNAs generally follows the ribosome scanning mechanism. However, by using subgenomic env expression vectors, we found that translation of glycoprotein from polycistronic mRNAs was inconsistent with leaky scanning. Instead a conserved minimal upstream open reading frame (uORF) consisting only of a start and stop codon that overlaps with the vpu start site, appears to augment access to the env start codon downstream. Mutating the translational start and stop codons of this uORF resulted in up to fivefold reduction in Env expression. Removing the vpu uORF and increasing the strength of the authentic vpu initiation sequence abolished Env expression from subgenomic constructs and replication of HIV-1, whereas an identical increase in the strength of the minimal uORF initiation site did not alter Env expression. [Copyright &y& Elsevier]

Abstract: The her-2 (neu, erbB-2) oncogene encodes a 185-kDa transmembrane receptor tyrosine kinase. HER2 overexpression occurs in numerous primary human tumors and contributes to 25–30% of breast and ovarian carcinomas. Synthesis of HER2 is controlled in part by an upstream open reading frame (uORF) present in the transcript. We used synthetic capped and polyadenylated mRNAs containing sequences derived from the 5′ region of the her-2 transcript fused to a firefly luciferase (LUC) reporter to examine this uORF’s effect on translation in cell-free systems derived from reticulocytes, wheat germ and Neurospora crassa, and in RNA-transfected HeLa cells. The uORF reduced translation of the downstream cistron in all systems. [35S]Met labeling of in vitro translation products obtained indicated that the uORF also affected downstream start-site selection. Primer extension inhibition (toeprint) assays of ribosomes loaded at initiation codons in reticulocyte lysates indicated that the uORF affected the interaction of ribosomes with the primary her-2 AUG codon. [Copyright &y& Elsevier]

The cAMP-dependent kinase (PKA) plays a crucial part in long-term memory formation in the honeybee ( Apis mellifera). One of the putative substrates of the PKA activity is the cAMP response element binding protein (CREB), a transcription factor in the bZIP protein family. We searched the honeybee genome to characterize genes from the CREB/CREM and the PKA families. We identified two genes that encode regulatory subunits and three genes encode catalytic subunits of PKA. Eight genes code for bZIP proteins, but only one gene was found that encodes a member of the CREB/CREM family. The phylogenetic relationship of these genes was analysed with their Drosophila and human counterparts. [ABSTRACT FROM AUTHOR]

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane. In vivo low expression of UCP2 contrasts with a high UCP2 mRNA level, and induction of UCP2 expression occurs without change in mRNA level, demonstrating a translational control. The UCP2 mRNA is characterized by a long 5′ untranslated region (5′UTR), in which an upstream open reading frame (uORF) codes for a 36-amino-acid sequence. The 5′UTR and uORF have an inhibitory role in the translation of UCP2. The present study demonstrates that the 3′ region of the uORF is a major determinant for this inhibitory role. In this 3′ region, a single-base substitution that kept the codon sense unchanged significantly modified UCP2 translation, whereas some important amino acid changes had no effect. We discuss our results within the framework of the existing models explaining initiation of translation downstream of a uORF. [ABSTRACT FROM AUTHOR]

Abstract: The sperm mitochondrial cysteine-rich protein (SMCP) is a rapidly evolving cysteine- and proline-rich protein that is localized in the mitochondrial capsule and enhances sperm motility. The sequences of the SMCP protein, gene, and mRNA in a variety of mammals have been compared to understand their evolution and regulation. SMCP can now be reliably identified by its tripartite structure including a short amino-terminal segment; a central segment containing short tandem repeats rich in cysteine, proline, glutamine, and lysine; and a C-terminal segment containing no repeats, few cysteines, and a C-terminal lysine. The SMCP gene is located in the epidermal differentiation complex (EDC), a large gene cluster that functions in forming epithelial barriers. Similarities in chromosomal location, molecular function, intron–exon structure, and protein organization argue that SMCP originated from an EDC gene and acquired spermatogenic cell-specific transcriptional and translational regulation and a novel cellular function in sperm motility. The SMCP 5′ UTR and 3′ UTR contain conserved elements and uORFs that may function in cytoplasmic regulation of gene expression, and the levels of SMCP mRNA in human are much lower than in other mammals, a feature of male-biased expression. The evolution of SMCP has been accompanied by changes in the sequence, number, and length of repeat units, including three alleles in dogs. The major proteins associated with the mitochondrial capsule, SMCP and phospholipid hydroperoxide glutathione peroxidase, provide outstanding examples of changes in cellular function driven by selective pressures on sperm motility, an important determinant of male reproductive success. [Copyright &y& Elsevier]

Abstract: Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation, remodeling, and stability. Tie2 expression is also upregulated in various cancers implicating a role in tumor angiogenesis. Its mRNA transcript contains an unusually long (372 nucleotides) 5′ untranslated region (UTR) with five upstream open reading frames (uORFs) and an internal ribosome entry site (IRES) that allows this mRNA to be translated under hypoxic conditions. This sets up an alternative initiation pathway with the potential to clash with 5′ end-mediated initiation from the same template. Herein, we define experimental conditions under which the Tie2 IRES is not active, allowing us to assess the contribution of the 5′ UTR to cap-dependent translation on the Tie2 transcript. We find that the Tie2 5′ UTR is inhibitory to translation initiation with ribosome flow decreasing following encounters with each uORF. No single uORF was found to harbor significant cis-acting inhibitory activity. Our results suggest that the uORFs within the Tie2 5′ UTR serve to decrease the percent of ribosomes competent for reinitiation as these traverse the mRNA 5′ UTR, thus minimizing interference with the IRES. [Copyright &y& Elsevier]

AtMHX is an Arabidopsis tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions. This transporter, which may play a role in ion homeostasis of plants, is encoded by a single gene in Arabidopsis. The molecular mechanisms that regulate the expression of this transporter are practically unknown. While AtMHX transcript can be easily visualized, expression of the corresponding protein is apparently low. To understand whether AtMHX expression is repressed at the translational level, the 5′ untranslated region (5′ UTR) of this gene was fused to reporter genes. In vitro analyses showed that the 5′ UTR of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation at an upstream open-reading-frame (uORF) included in the 5′ UTR. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavourable, it was recognized by over 90% of the scanning ribosomes both in vitro and in vivo. The inhibitory effect of the uORF was mediated by imposing the need for reinitiation and not by ribosome stalling, as the inhibition was not dependent on the amino-acid sequence of the uORF peptide. The efficiency of reinitiation was low. The in vivo studies, carried out with transiently transformed tobacco plants, indicated that alternations in the Mg2+ or Zn2+ levels did not affect the rate of translation. These data suggest that AtMHX expression is repressed by the 5′ UTR of its gene. [ABSTRACT FROM PUBLISHER]

Eukaryotic gene expression, reflected in the amount of steady-state mRNA, is regulated at the post-transcriptional level. The 5′-untranslated regions (5′-UTRs) of some transcripts contain cis-acting elements, including upstream open reading frames (uORFs), that have been identified as being fundamental in modulating translation efficiency and mRNA stability. Previously, we demonstrated that uORFs present in the 5′-UTR of cystic fibrosis transmembrane conductance regular (CFTR) transcripts expressed in the heart were able to modulate translation efficiency of the main CFTR ORF. Here, we show that the same 5′-UTR elements are associated with the differential stability of the 5′-UTR compared to the main coding region of CFTR transcripts. Furthermore, these post-transcriptional mechanisms are important factors governing regulated CFTR expression in the heart, in response to developmental and pathophysiological stimuli. [Copyright &y& Elsevier]

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5′ untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5′UTR of the mouse Tsc1 gene transcript involving exon 1 and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5′UTR of the TSC1/Tsc1 gene. A comparative study of the 5′UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5′ to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5′ and 3′ deletion analysis has suggested that a ~587 bp region, from position +77 to −510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. [Copyright &y& Elsevier]

Vigilin, a K homology (KH) protein has been found in all eukaryotic species studied. It has a unique structure of 14–15 consecutively arranged KH domains which apparently mediate RNA-protein binding. Cloning and sequencing of the mouse vigilin cDNA confirmed that the amino acid sequences of vertebrate vigilins are highly conserved and contain conserved sequence motifs of nuclear import and export sequences. The human and murine vigilin mRNAs carry two alternatively spliced 5′ exons. In the 5′ leader region of one of the splice variants, variant 1A, we found an upstream open reading frame (uORF) highly conserved between mouse and human. Here we present for the first time evidence that a 13 amino acid long peptide encoded by this uORF is an inhibitor of vigilin expression operating on a posttranscriptional level. We propose that the two structurally different 5′ leader sequences of the human vigilin mRNA are involved in the regulation of vigilin biosynthesis. [ABSTRACT FROM AUTHOR]

The rice myb7 mRNA contains in its long leader an upstream open reading frame (uORF) putatively coding for a 40 amino acid peptide. uORFs have been found in the leader of mRNAs encoding transcriptional factors or other proteins involved in cellular growth and development. They are thought to translationally regulate the expression of downstream ORFs. Here, we showed the ability of the myb7 uORF to inhibit the expression of downstream reporter genes both in homologous (rice) and heterologous (tobacco) systems. This effect seems partially related to its translation, as indicated by the comparison with the mutagenized uORF. In both systems most of the inhibitory effect was due to the presence of the intercistronic region, in disagreement with the Kozak model. Moreover, replacing the uORF or the intercistronic region with a different one, we demonstrated that the inhibitory effect strictly depends on their co-presence. Finally, in vitro assays showed that the myb7 uORF is translated and inhibits the downstream ORF translation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We identified a near-full-length cDNA clone encoding ornithine decarboxylase (ODC) from tomato (Lycopersicon esculentum Mill). It contained a small upstream open reading frame (uORF) within its 5 untranslated region. An in vitro translation assay demonstrated that the uORF repressed expression of downstream ORF. Neither nucleotide nor predicted peptide sequence of the uORF was responsible for the repression. The presence of upstream AUG codon was shown to be responsible. ODC expression appeared to be organ specific. The ODC gene was expressed in roots, hypocotyls and sink leaves but not in source leaves. ODC transcripts were observed in apical meristem of primary roots, and were distributed in cells of cortex layer preferentially. ODC expression responded immediately to sucrose availability via the sucrose-specific pathway independent of hexokinase. Sucrose induction of ODC gene was seen in roots, hypocotyls and flowers but not in mature leaves. Moreover, only the root apical meristem responded to sucrose availability. These observations indicate that the spatial pattern of ODC expression is closely associated with cell proliferation and that sucrose sensing plays a major role in the spatial pattern of ODC expression. Also, the differential regulation of ODC and arginine decarboxylase gene expression by factors modulating plant growth suggests that they would have different physiological roles in plant development. [ABSTRACT FROM AUTHOR]

SignificanceProteins are the machinery which execute essential cellular functions. However, measuring their abundance within an organism can be difficult and resource-intensive. Cells use a variety of mechanisms to control protein synthesis from mRNA, including short open reading frames (uORFs) that lie upstream of the main coding sequence. Ribosomes can preferentially translate uORFs instead of the main coding sequence, leading to reduced translation of the main protein. In this study, we show that uORF sequence variation between individuals can lead to different rates of protein translation and thus variable protein abundances. We also demonstrate that natural variation in uORFs occurs frequently and can be linked to whole-plant phenotypes, indicating that uORF sequence variation likely contributes to plant adaptation.

The regulation of protein synthesis plays an important role in the growth and development of all organisms. Upstream open reading frames (uORFs) are commonly found in eukaryotic messenger RNA transcripts and typically attenuate the translation of associated downstream main ORFs (mORFs). Conserved peptide uORFs (CPuORFs) are a rare subset of uORFs, some of which have been shown to conditionally regulate translation by ribosome stalling. Here, we show that Arabidopsis CPuORF19, CPuORF46 and CPuORF47, which are ancient in origin, regulate translation of any downstream ORF, in response to the agriculturally significant environmental signals, heat stress and water limitation. Consequently, these CPuORFs represent a versatile toolkit for inducible gene expression with broad applications. Finally, we note that different classes of CPuORFs may operate during distinct phases of translation, which has implications for the bioengineering of these regulatory factors., (© 2022 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S. cerevisiae HOL1 mRNA is under translational control by polyamines, and we reveal that the encoded membrane transporter Hol1 is a high-affinity polyamine transporter and is required for yeast growth under limiting polyamine conditions. Moreover, we show that polyamine inhibition of the translation factor eIF5A impairs translation termination at a Pro-Ser-stop motif in a conserved upstream open reading frame on the HOL1 mRNA to repress Hol1 synthesis under conditions of elevated polyamines. Our findings reveal that polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels. [Display omitted] • Hol1 is the major high-affinity polyamine transporter in S. cerevisiae • Polyamine levels autoregulate HOL1 mRNA translation through a conserved uORF • Polyamines inhibit eIF5A to impair translation termination on the uORF • Ribosome stalling on the uORF prevents HOL1 translation Vindu et al. identify Hol1 as the major high-affinity polyamine transporter in S. cerevisiae. Polyamines autoregulate HOL1 expression through feedback inhibition of HOL1 mRNA translation mediated by polyamine inhibition of eIF5A-dependent translation termination on a conserved upstream open reading frame in the HOL1 mRNA leader. [ABSTRACT FROM AUTHOR]

Background: Upstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood., Methods: We examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs., Results: We provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses., Conclusion: Our data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides., (© 2022. The Author(s).)

Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of mammalian genes, we identified five homeobox ( Hox ) gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. An analysis of published cap analysis of gene expression sequencing (CAGE-seq) data and generated CAGE-seq data for messenger RNAs (mRNAs) from mouse somites revealed that the 5' leaders of Hox mRNAs of interest contain conserved uORFs, are generally much shorter than reported, and lack previously proposed internal ribosome entry site elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila , the sole activating E2F, E2F1, is both required for and sufficient to promote G1→S progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. Here, we investigate cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. We show that small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 5'UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 5'UTR uORFs. Further, we mapped a region in the E2f1 mRNA that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Polyamines are small organic and basic polycations that perform essential regulatory functions in all living organisms. Fluctuations in polyamine content have been observed to occur during growth, development and under stress conditions, implying that polyamines play pivotal roles in diverse cellular and physiological processes. To achieve polyamine homeostasis, the entire metabolic pathway is subjected to a fine-tuned regulation of its biosynthetic and catabolic genes and enzymes. In this review, we describe and discuss the most important mechanisms implicated in the translational and post-translational regulation of polyamine metabolic enzymes in plants. At the translational level, we emphasize the role of polyamines in the modulation of upstream open reading frame (uORF) activities that control the translation of polyamine biosynthetic and catabolic mRNAs. At the post-translational level, different aspects of the regulation of polyamine metabolic proteins are depicted, such as the proteolytic activation of enzyme precursors, the importance of dimerization in protein stability as well as in protein intracellular localization., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sugar metabolism not only determines fruit sweetness and quality but also acts as signaling molecules to substantially connect with other primary metabolic processes and, therefore, modulates plant growth and development, fruit ripening, and stress response. The basic region/leucine zipper motif (bZIP) transcription factor family is ubiquitous in eukaryotes and plays a diverse array of biological functions in plants. Among the bZIP family members, the smallest bZIP subgroup, S1-bZIP, is a unique one, due to the conserved upstream open reading frames (uORFs) in the 5' leader region of their mRNA. The translated small peptides from these uORFs are suggested to mediate Sucrose-Induced Repression of Translation (SIRT), an important mechanism to maintain sucrose homeostasis in plants. Here, we review recent research on the evolution, sequence features, and biological functions of this bZIP subgroup. S1-bZIPs play important roles in fruit quality, abiotic and biotic stress responses, plant growth and development, and other metabolite biosynthesis by acting as signaling hubs through dimerization with the subgroup C-bZIPs and other cofactors like SnRK1 to coordinate the expression of downstream genes. Direction for further research and genetic engineering of S1-bZIPs in plants is suggested for the improvement of quality and safety traits of fruit., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Zhang, Norris and Jiang.)