Disrupting upstream translation in mRNAs is associated with human disease.
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ABSTRACT:
SUBMITTER: Lee DSM
PROVIDER: S-EPMC7943595 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To find novel short coding sequences from accumulated full-length cDNA sequences, proteomic analysis of small proteins expressed in human leukemia K562 cells was performed using high-resolution nanoflow liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Our analysis led to the identification of 54 proteins not more than 100 amino acids in length, including four novel ones. These novel short coding sequences were all located upstream of the longest open reading frame (ORF) of the corresponding cDNA. Our findings indicate that the translation of short ORFs occurs in vivo whether or not there exists a longer coding region in the downstream of the mRNA. This investigation provides the first direct evidence of translation of upstream ORFs in human cells, which could greatly change the current outline of the human proteome.
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Project description:BACKGROUND:RNA secondary structures in the 5'-untranslated regions (5'-UTR) of mRNAs are key to the post-transcriptional regulation of gene expression. While it is evident that non-canonical Hoogsteen-paired G-quadruplex (rG4) structures somehow contribute to the regulation of translation initiation, the nature and extent of human mRNAs that are regulated by rG4s is not known. Here, we provide new insights into a mechanism by which rG4 formation modulates translation. RESULTS:Using transcriptome-wide ribosome profiling, we identify rG4-driven mRNAs in HeLa cells and reveal that rG4s in the 5'-UTRs of inefficiently translated mRNAs associate with high ribosome density and the translation of repressive upstream open reading frames (uORF). We demonstrate that depletion of the rG4-unwinding helicases DHX36 and DHX9 promotes translation of rG4-associated uORFs while reducing the translation of coding regions for transcripts that comprise proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites shows that reduced translation is mediated through direct physical interaction between the helicase and its rG4 substrate. CONCLUSION:This study identifies human mRNAs whose translation efficiency is modulated by the DHX36- and DHX9-dependent folding/unfolding of rG4s within their 5'-UTRs. We reveal a previously unknown mechanism for translation regulation in which unresolved rG4s within 5'-UTRs promote 80S ribosome formation on upstream start codons, causing inhibition of translation of the downstream main open reading frames. Our findings suggest that the interaction of helicases with rG4s could be targeted for future therapeutic intervention.
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Project description:Platinum resistance is a major cause of treatment failure and mortality in epithelial ovarian cancer. mTORC1/2 inhibitors, which impair mRNA translation, can re-sensitize resistant ovarian cancer cells to platinum chemotherapy but the mechanism remains poorly described. Using platinum-resistant OVCAR-3 cells treated with the selective mTORC1/2 inhibitor INK128/MLN128, we conducted genome-wide transcription and translation studies and analyzed the effect on cell proliferation, AKT-mTOR signaling and cell survival, to determine whether carboplatin resistance involves selective mRNA translational reprogramming, and whether it is sensitive to mTORC1/2 inhibition. Gene ontology and Ingenuity Pathway Analysis (IPA) were used to categorize gene expression changes into experimentally authenticated biochemical and molecular networks. We show that carboplatin resistance involves increased mTORC1/2 signaling, resulting in selective translation of mRNAs involved in DNA damage and repair responses (DDR), cell cycle and anti-apoptosis (survival) pathways. Re-sensitization of ovarian cancer cell killing by carboplatin required only modest mTORC1/2 inhibition, with downregulation of protein synthesis by only 20-30%. Genome-wide transcriptomic and translatomic analyses in OVCAR-3 cells revealed that the modest downregulation of global protein synthesis by dual mTORC1/2 inhibition is associated with greater selective inhibition of DDR, cell cycle and survival mRNA translation, which was confirmed in platinum-resistant SKOV-3 cells. These data suggest a clinical path to re-sensitize platinum resistant ovarian cancer to platinum chemotherapy through partial inhibition of mTORC1/2, resulting in selective translation inhibition of DDR and anti-apoptosis protective mRNAs.
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Project description:BackgroundFHIT is a genome caretaker/tumor suppressor that is silenced in >50% of cancers. Although it was identified more than 20 years ago, questions remain as to how FHIT loss contributes to cancer, and conversely, how FHIT acts to maintain genome integrity and suppress malignancy. Fhit belongs to the histidine triad family of enzymes that catalyze the degradation of nucleoside 5',5'-triphosphates, including the m7GpppN 'caps' that are generated when mRNAs undergo 3'-5' decay. This raised the possibility that Fhit loss might affect changes in the translation of cancer-associated mRNAs, possibly as a consequence of increased intracellular concentrations of these molecules.ResultsRibosome profiling identified several hundred mRNAs for which coding region ribosome occupancy changed as a function of Fhit expression. While many of these changes could be explained by changes in mRNA steady-state, a subset of these showed changes in translation efficiency as a function of Fhit expression. The onset of malignancy has been linked to changes in 5'-UTR ribosome occupancy and this analysis also identified ribosome binding to 5'-untranslated regions (UTRs) of a number of cancer-associated mRNAs. 5'-UTR ribosome occupancy of these mRNAs differed between Fhit-negative and Fhit-positive cells, and in some cases these differences correlated with differences in coding region ribosome occupancy.ConclusionsIn summary, these findings show Fhit expression impacts the translation of a number of cancer associated genes, and they support the hypothesis that Fhit's genome protective/tumor suppressor function is associated with post-transcriptional changes in expression of genes whose dysregulation contributes to malignancy.
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Project description:The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP-43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP-43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5'UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3'UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease.
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Project description:Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein.
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Project description:Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability.
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Project description:A key player in translation initiation is eIF4E, the mRNA 5' cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4L site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4L, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.
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Project description:G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5' and 3'-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed.
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Project description:Type I collagen is the most abundant protein in the human body and is composed of two α1(I) and one α2(I) polypeptides which assemble into a triple helix. For the proper assembly of the collagen triple helix, the individual polypeptides must be translated in coordination. Here, we show that serine-threonine kinase receptor-associated protein (STRAP) is tethered to collagen mRNAs by interaction with LARP6. LARP6 is a protein which directly binds the 5' stem-loop (5'SL) present in collagen α1(I) and α2(I) mRNAs, but it interacts with STRAP with its C-terminal domain, which is not involved in binding 5'SL. Being tethered to collagen mRNAs, STRAP prevents unrestricted translation, primarily that of collagen α2(I) mRNAs, by interacting with eukaryotic translation initiation factor 4A (eIF4A). In the absence of STRAP, more collagen α2(I) mRNA can be pulled down with eIF4A, and collagen α2(I) mRNA is unrestrictedly loaded onto the polysomes. This results in an imbalance of synthesis of α1(I) and α2(I) polypeptides, in hypermodifications of α1(I) polypeptide, and in inefficient assembly of the polypeptides into a collagen trimer and their secretion as monomers. These defects can be partially restored by supplementing STRAP. Thus, we discovered STRAP as a novel regulator of the coordinated translation of collagen mRNAs.
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