Plants utilise ancient conserved peptide upstream open reading frames in stress-responsive translational regulation.
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ABSTRACT:
SUBMITTER: Causier B
PROVIDER: S-EPMC9305500 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundUpstream open reading frames (uORFs) can down-regulate the translation of the main open reading frame (mORF) through two broad mechanisms: ribosomal stalling and reducing reinitiation efficiency. In distantly related plants, such as rice and Arabidopsis, it has been found that conserved uORFs are rare in these transcriptomes with approximately 100 loci. It is unclear how prevalent conserved uORFs are in closely related plants.ResultsWe used a homology-based approach to identify conserved uORFs in five cereals (monocots) that could potentially regulate translation. Our approach used a modified reciprocal best hit method to identify putative orthologous sequences that were then analysed by a comparative R-nomics program called uORFSCAN to find conserved uORFs.ConclusionThis research identified new genes that may be controlled at the level of translation by conserved uORFs. We report that conserved uORFs are rare (<150 loci contain them) in cereal transcriptomes, are generally short (less than 100 nt), highly conserved (50% median amino acid sequence similarity), position independent in their 5'-UTRs, and their start codon context and the usage of rare codons for translation does not appear to be important.
| S-EPMC2527020 | biostudies-literature
Project description:Upstream open reading frames (uORFs) are present in the 5'-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1517 (1373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.
| S-EPMC7530721 | biostudies-literature
Project description:Post-transcriptional control has emerged as a major regulatory event in gene expression and often occurs at the level of translation initiation. Although overexpression or constitutive activation of tyrosine kinases (TKs) through gene amplification, translocation or mutation are well-characterized oncogenic events, current knowledge about translational mechanisms of TK activation is scarce. Here, we report the presence of translational cis-regulatory upstream open reading frames (uORFs) in the majority of transcript leader sequences of human TK mRNAs. Genetic ablation of uORF initiation codons in TK transcripts resulted in enhanced translation of the associated downstream main protein-coding sequences (CDSs) in all cases studied. Similarly, experimental removal of uORF start codons in additional non-TK proto-oncogenes, and naturally occurring loss-of-uORF alleles of the c-met proto-oncogene (MET) and the kinase insert domain receptor (KDR), was associated with increased CDS translation. Based on genome-wide sequence analyses we identified polymorphisms in 15.9% of all human genes affecting uORF initiation codons, associated Kozak consensus sequences or uORF-related termination codons. Together, these data suggest a comprehensive role of uORF-mediated translational control and delineate how aberrant induction of proto-oncogenes through loss-of-function mutations at uORF initiation codons may be involved in the etiology of cancer. We provide a detailed map of uORFs across the human genome to stimulate future research on the pathogenic role of uORFs.
| S-EPMC4820681 | biostudies-literature
Project description: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.
| S-EPMC125715 | biostudies-literature
Project description:Gene expression is prone to burst production, making it a highly noisy process that requires additional controls. Upstream open reading frames (uORFs) are widely present in the 5' leader sequences of 30-50% of eukaryotic messenger RNAs1-3. The translation of uORFs can repress the translation efficiency of the downstream main coding sequences. Whether the low translation efficiency leads to a different variation, or noise, in gene expression has not been investigated, nor has the direct biological impact of uORF-repressed translation. Here we show that uORFs achieve low but precise protein production in plant cells, possibly by reducing the protein production rate. We also demonstrate that, by buffering a stable TIMING OF CAB EXPRESSION 1 (TOC1) protein production level, uORFs contribute to the robust operation of the plant circadian clock. Our results provide both an action model and the biological impact of uORFs in translational control to mitigate transcriptional noise for precise protein production.
| S-EPMC9122824 | biostudies-literature
Project description:Macroautophagy/autophagy, a highly conserved catabolic pathway that maintains proper cellular homeostasis is stringently regulated by numerous autophagy-related (Atg) proteins. Many studies have investigated autophagy regulation at the transcriptional level; however, relatively little is known about translational control. Here, we report the upstream open reading frame (uORF)-mediated translational control of multiple Atg proteins in Saccharomyces cerevisiae and in human cells. The translation of several essential autophagy regulators in yeast, including Atg13, is suppressed by canonical uORFs under nutrient-rich conditions, and is activated during nitrogen-starvation conditions. We also found that the predicted human ATG4B and ATG12 non-canonical uORFs suppress downstream coding sequence translation. These results demonstrate that uORF-mediated translational control is a widely used mechanism among ATG genes from yeast to human and suggest a model for how some ATG genes bypass the general translational suppression that occurs under stress conditions to maintain a proper level of autophagy.Abbreviations: 5' UTR, 5' untranslated region; Atg, autophagy-related; CDS, coding sequence; Cvt, cytoplasm-to-vacuole targeting; HBSS, Hanks' balanced salt solution; PA, protein A; PE, phosphati-dylethanolamine; PIC, preinitiation complex; PtdIns3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative reverse transcription PCR; Ubl, ubiquitin-like; uORF, upstream open reading frame; WT, wild-type.
| S-EPMC9851245 | biostudies-literature
Project description:BackgroundSome upstream open reading frames (uORFs) regulate gene expression (i.e., they are functional) and can play key roles in keeping organisms healthy. However, how uORFs are involved in gene regulation is not yet fully understood. In order to get a complete view of how uORFs are involved in gene regulation, it is expected that a large number of experimentally verified functional uORFs are needed. Unfortunately, wet-experiments to verify that uORFs are functional are expensive.ResultsIn this paper, a new computational approach to predicting functional uORFs in the yeast Saccharomyces cerevisiae is presented. Our approach is based on inductive logic programming and makes use of a novel combination of knowledge about biological conservation, Gene Ontology annotations and genes' responses to different conditions. Our method results in a set of simple and informative hypotheses with an estimated sensitivity of 76%. The hypotheses predict 301 further genes to have 398 novel functional uORFs. Three (RPC11, TPK1, and FOL1) of these 301 genes have been hypothesised, following wet-experiments, by a related study to have functional uORFs. A comparison with another related study suggests that eleven of the predicted functional uORFs from genes LDB17, HEM3, CIN8, BCK2, PMC1, FAS1, APP1, ACC1, CKA2, SUR1, and ATH1 are strong candidates for wet-lab experimental studies.ConclusionsLearning based prediction of functional uORFs can be done with a high sensitivity. The predictions made in this study can serve as a list of candidates for subsequent wet-lab verification and might help to elucidate the regulatory roles of uORFs.
| S-EPMC2813248 | biostudies-literature
Project description:We engineered short ORFs and used them to control the expression level of recombinant proteins. These short ORFs, encoding a two-amino acid peptide, were placed upstream of an ORF encoding a protein of interest. Insertion of these upstream ORFs (uORFs) resulted in suppression of protein expression. By varying the base sequence preceding the uORF, we sought to vary the translation initiation rate of the uORF and subsequently control the degree of this suppression. Using this strategy, we generated a library of RNA sequence elements that can specify protein expression over a broad range of levels. By also using multiple uORFs in series and non-AUG start codons, we were able to generate particularly low expression levels, allowing us to achieve expression levels spanning three orders of magnitude. Modeling supported a mechanism where uORFs shunt the flow of ribosomes away from the downstream protein-coding ORF. With a lower translation initiation rate at the uORF, more ribosomes "leak" past the uORF; consequently, more ribosomes are able to reach and translate the downstream ORF. We report expression control by engineering uORFs and translation initiation to be robust, predictable, and reproducible across all cell types tested. We propose control of translation initiation as a primary method of choice for tuning expression in mammalian systems.
| S-EPMC3710870 | biostudies-literature
Project description:In many eukaryotic mRNAs one or more short 'upstream' open reading frames, uORFs, precede the initiator of the main coding sequence. Upstream ORFs are functionally diverse as illustrated by their variety of features in polyamine pathway biosynthetic mRNAs. Their propensity to act as sensors for regulatory circuits and to amplify the signals likely explains their occurrence in most polyamine pathway mRNAs. The uORF-mediated polyamine responsive autoregulatory circuits found in polyamine pathway mRNAs exemplify the translationally regulated dynamic interface between components of the proteome and metabolism.
| S-EPMC2811004 | biostudies-literature
Project description:Upstream open reading frames (uORFs) initiate translation within mRNA 5’ leaders,and have the potential to altermain coding sequence(CDS) translationontranscripts in which they reside. Ribosome profiling(RP)studies suggest that translating ribosomes are pervasive within 5’ leadersacross model systems. However, the significance of this observationremains unclear. To explore a role for uORF usage in neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Using a spectral coherence algorithm (SPECtre),we identify4,954uORFsacross31%of all neuroblastoma transcripts. These uORFspredominantly utilize non-AUG initiationcodonsand exhibit translational efficiencies(TE)comparable to annotated coding regions. Usage of both AUG initiated uORFs and aconservedandconsistently translated subset of non-AUG initiated uORFs correlateswith repressed CDS translation. Ribosomal protein transcripts are enriched in uORFs, and select uORFs on such transcripts were validated for expression. Withneuronal differentiation, we observedan overall positive correlation between translational shifts in uORF/CDSpairs. However,a subset of transcripts exhibit inverse shifts in translation of uORF/CDSpairs. TheseuORFsare enriched in AUG initiation sites, non-overlapping, and shorterin length. Cumulatively, CDSs downstream of uORFs characterized by persistent translation show smaller shifts in TE withneuronal differentiation relative to CDSs without a predicted uORF, suggesting that fluctuations in CDS translation are buffered by uORF translation. In sum, this work provides insights into the dynamic relationshipsand potential regulatory functionsof uORF/CDS pairs in a model of neuronal differentiation.
2018-09-07 | GSE119615 | GEO