Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition.
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ABSTRACT:
SUBMITTER: Alghoul F
PROVIDER: S-EPMC8172242 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation reinitiation. However, termination in the 5' UTR at the end of uORFs resembles premature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation reinitiation has been proposed as a method for mRNAs to prevent NMD. Here, we test how uORF length influences translation reinitiation and mRNA stability in HeLa cells. Using custom 5' UTRs and uORF sequences, we show that reinitiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives in HeLa cells and mining available mRNA half-life data sets for cumulative predicted uORF length, we conclude that translation reinitiation after uORFs is not a robust method for mRNAs to prevent NMD. Together, these data suggest that the decision of whether NMD ensues after translating uORFs occurs before reinitiation in mammalian cells.
| S-EPMC10187673 | biostudies-literature
Project description: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.
| S-EPMC8892498 | biostudies-literature
Project description: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 human genes, we identified 5 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. We analyzed published CAGE-seq data and generated CAGE-seq data from mRNAs from mouse somites. These data demonstrate that the 5’ leaders of Hox mRNAs of interest contain conserved uORFs, are much shorter than reported, and lack previously proposed IRES 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.
2022-02-21 | GSE184515 | GEO
Project description:Gene expression is regulated at many levels, including mRNA transcription, translation, and post-translational modification. Compared with transcriptional regulation, mRNA translational control is a more critical step in gene expression and allows for more rapid changes of encoded protein concentrations in cells. Translation is highly regulated by complex interactions between cis-acting elements and trans-acting factors. Initiation is not only the first phase of translation, but also the core of translational regulation, because it limits the rate of protein synthesis. As potent cis-regulatory elements in eukaryotic mRNAs, upstream open reading frames (uORFs) generally inhibit the translation initiation of downstream major ORFs (mORFs) through ribosome stalling. During the past few years, with the development of RNA-seq and ribosome profiling, functional uORFs have been identified and characterized in many organisms. Here, we review uORF identification, uORF classification, and uORF-mediated translation initiation. More importantly, we summarize the translational regulation of uORFs in plant metabolic pathways, morphogenesis, disease resistance, and nutrient absorption, which open up an avenue for precisely modulating the plant growth and development, as well as environmental adaption. Additionally, we also discuss prospective applications of uORFs in plant breeding.
| S-EPMC7503886 | biostudies-literature
Project description:Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity. Disease-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic technology. Nevertheless, enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental affects. Upstream open reading frames (uORFs) are translational control elements located in the 5' untranslated region (UTR) of eukaryotic mRNAs and may repress the translation of downstream genes. To investigate the function of three uORFs from the 5'-UTR of ACCELERATED CELL 11 (uORFsACD11), we develop a fluorescent reporter system and find uORFsACD11 function in repressing downstream gene translation. Individual or simultaneous mutations of the three uORFsACD11 lead to repression of downstream translation efficiency at different levels. Importantly, uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant leaves. When coupled with the PATHOGENESIS-RELATED GENE 1 (PR1) promoter, the uORFsACD11 cassettes can upregulate accumulation of Arabidopsis thaliana LECTIN RECEPTOR KINASE-VI.2 (AtLecRK-VI.2) during pathogen attack and enhance plant resistance to Phytophthora capsici. These findings indicate that the uORFsACD11 cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack, while for ensuring lower levels of protein expression at normal conditions.
| S-EPMC10521741 | biostudies-literature
Project description:Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5' and 3' cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5' cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5' sequences making up the 5' enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5' enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5' enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5' enhancer's stimulatory activity is strictly dependent on and thus follows the 3' enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5' leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.
| S-EPMC3131280 | biostudies-literature
Project description:Most of our knowledge about translation regulatory mechanisms comes from studies on lower organisms. However, the translation control system of higher organisms is less understood. Here we find that in 5' untranslated region (5'UTR) of human Annexin II receptor (AXIIR) mRNA, there are two upstream open reading frames (uORFs) acting in a fail-safe manner to inhibit the translation from the main AUG. These uORFs are unfavorable for re-initiation after termination of uORF translation. Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), hnRNPA0 and ELAV like RNA binding protein 1 (ELAVL1) bind to the 5'UTR of AXIIR mRNA. They focus the translation of uORFs on uORF1 and attenuate leaky scanning that bypasses uORFs. The cooperation between the two uORFs and the three proteins formed a multiple fail-safe system that tightly inhibits the translation of downstream AXIIR. Such cooperation between multiple molecules and elements reflects that higher organism develops a complex translation regulatory system to achieve accurate and flexible gene expression control.
| S-EPMC5389705 | biostudies-literature
Project description:BackgroundA large fraction of genes contains upstream ORFs (uORFs) in the 5' untranslated region (5'UTR). The translation of uORFs can inhibit the translation of the main coding sequence, for example by causing premature dissociation of the two ribosomal units or ribosome stalling. However, it is currently unknown if most uORFs are inhibitory or if this activity is restricted to specific cases. Here we interrogate ribosome profiling data from three different stress experiments in yeast to gain novel insights into this question.ResultsBy comparing ribosome occupancies in different conditions and experiments we obtain strong evidence that, in comparison to primary coding sequences (CDS), which undergo translational arrest during stress, the translation of uORFs is mostly unaffected by changes in the environment. As a result, the relative abundance of uORF-encoded peptides increases during stress. In general, the changes in the translational efficiency of regions containing uORFs do not seem to affect downstream translation. The exception are uORFs found in a subset of genes that are significantly up-regulated at the level of translation during stress; these uORFs tend to be translated at lower levels in stress conditions than in optimal growth conditions, facilitating the translation of the CDS during stress. We find new examples of uORF-mediated regulation of translation, including the Gcn4 functional homologue fil1 and ubi4 genes in S. pombe.ConclusionWe find evidence that the relative amount of uORF-encoded peptides increases during stress. The increased translation of uORFs is however uncoupled from the general CDS translational repression observed during stress. In a subset of genes that encode proteins that need to be rapidly synthesized upon stress uORFs act as translational switches.
| S-EPMC8126119 | biostudies-literature
Project description:Ribosome biogenesis (RiBi) is an extremely energy intensive process that is critical for gene expression. It is thus highly regulated, including through the tightly coordinated expression of over 200 RiBi genes by positive and negative transcriptional regulators. We investigated RiBi regulation as cells initiated meiosis in budding yeast and noted early transcriptional activation of RiBi genes, followed by their apparent translational repression 1 hour (h) after stimulation to enter meiosis. Surprisingly, in the representative genes examined, measured translational repression depended on their promoters rather than mRNA regions. Further investigation revealed that the signature of this regulation in our data depended on pre-treating cells with the translation inhibitor, cycloheximide (CHX). This treatment, at 1 h in meiosis, but not earlier, rapidly resulted in accumulation of RiBi mRNAs that were not translated. This effect was also seen in with CHX pre-treatment of cells grown in media lacking amino acids. For NSR1, this effect depended on the -150 to -101 region of the promoter, as well as the RiBi transcriptional repressors Dot6 and Tod6. Condition-specific RiBi mRNA accumulation was also seen with translation inhibitors that are dissimilar from CHX, suggesting that this phenomenon might represent a feedback response to global translation inhibition.
| S-EPMC6547411 | biostudies-literature
Project description:A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.
| S-EPMC5766168 | biostudies-literature