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Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translational regulation of mRNAs encoding ribosomal proteins

ABSTRACT: Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translational regulation of mRNAs encoding ribosomal proteins

PROVIDER: PRJNA485510 | ENA |

REPOSITORIES: ENA

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Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translational regulation of mRNAs encoding ribosomal proteins

Project description:The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it remains unclear whether mTOR-4EBP1/2 is the exclusive translational regulator of this group of genes, and furthermore, systematic searches for novel translational modulators have been immensely challenging due to difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for novel translational modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translational effects in a manner that is dependent upon GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a novel translational assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a non-canonical, mTOR-independent mode for translational regulation of ribosomal proteins.

2018-09-21 | GSE118422 | GEO

Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

Project description:Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

2020-05-31 | GSE136091 | GEO

Assessment of mTOR-Dependent Translational Regulation of Interferon Stimulated Genes

Project description:Type-I interferon (IFN)-induced activation of the mammalian target of rapamycin (mTOR) signaling pathway has been implicated in translational control of mRNAs encoding interferon-stimulated genes (ISGs). However, mTOR-sensitive translatomes commonly include mRNAs with a 5’ terminal oligopyrimidine tract (TOP), such as those encoding ribosomal proteins, but not ISGs. Because these translatomes were obtained under conditions when ISG expression is not induced, we examined the mTOR-sensitive translatome in human WISH cells stimulated with IFNβ. The mTOR inhibitor Torin1 resulted in a repression of global protein synthesis, including that of ISG products, and translation of all but 3 ISG mRNAs (TLR3, NT5C3A, and RNF19B) was not selectively more sensitive to mTOR inhibition. Detailed studies of NT5C3A revealed an IFN-induced change in transcription start site resulting in a switch from a non-TOP to a TOP-like transcript variant and mTOR sensitive translation. Thus, we show that, in the cell model used, translation of the vast majority of ISG mRNAs is not selectively sensitive to mTOR activity and describe an uncharacterized mechanism wherein the 5’-UTR of an mRNA is altered in response to a cytokine, resulting in a shift from mTOR-insensitive to mTOR-sensitive translation.

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Analysis of HIF and mTOR dependent translational regulation of mRNAs resolved by their transcription start sites, using HP5 technology

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Transcription profiling of mouse embryonic stem cells transfected with miR-10a or miR-10a-MUT shows that miR-10a interacts with the 5 prime untranslated region of mRNAs encoding ribosomal proteins and enhances their translation

Project description:MicroRNAs (miRNAs) are small RNAs that function as post-transcriptional regulators of gene expression. miRNAs affect a variety of signaling pathways and impaired miRNA regulation may contribute to the development of cancer and other diseases. We show that miRNA miR-10a interacts with the 5' untranslated region of mRNAs encoding ribosomal proteins and enhances their translation. miR-10a alleviates translational repression of the ribosomal protein mRNAs during amino acid starvation and is required for their stress-mediated activation following anisomycin treatment. miR-10a binds immediately downstream of the regulatory 5' TOP motif and the 5Â´TOP is necessary for miR-10a translational enhancement. The results indicate that miR-10a may positively control global protein synthesis via stimulation of ribosomal protein mRNA translation and that the 5' TOP regulatory complex and miR-10a are functionally interconnected.

2008-11-08 | E-MEXP-1375 | biostudies-arrayexpress

Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins.

Project description: Not available

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Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

Project description:Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

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Translational control of mTOR/4E-BP1 axis in MiaPaca-2 cells

Project description:Pancreatic ductal adenocarcinoma (PDAC) relies on hyper-activated protein synthesis. Consistently, human and mouse PDAC lose expression of the translational repressor and mTOR target 4E-BP1. Using genome-wide polysome-profiling, we here explore mRNAs whose translational efficiencies depend on the mTOR/4E-BP1 axis in Miapaca-2 cells. This was performed by isolating cytoplasmic and efficiently translated (heavy polysome-associated) mRNAs from MiaPaca-2 cells upon PP242-mediated mTOR inhibition

2019-09-18 | GSE137553 | GEO

nanoCAGE reveals 5â UTR features that define specific modes of translation of functionally related mTOR-sensitive mRNAs

Project description:mTOR regulates mRNA translation. Whereas ribosome-profiling suggested that mTOR exclusively stimulates translation of TOP (containing a 5â-terminal oligopyrimidine [5âTOP] motif) and TOP-like mRNAs, polysome-profiling implied that mTOR also modulates translation of non-TOP mRNAs. We show that ribosome-, but not polysome-profiling, is biased towards identification of TOP mRNAs as differentially translated while obscuring detection of changes in non-TOP mRNA translation. Transcription start site profiling by Nano-Cap Analysis of Gene Expression (nanoCAGE) revealed that many mTOR-sensitive mRNAs do not have 5âTOP motifs. Moreover, nanoCAGE showed that 5â UTR features distinguish two functionally and translationally distinct subsets of mTOR-sensitive mRNAs: i) those with short 5â UTRs enriched for mitochondrial functions such as respiration, that are translated in an eIF4E, but not eIF4A1-dependent manner and ii) mRNAs encoding proliferation- and survival-promoting proteins, that harbor long 5â UTRs, and require both eIF4E and eIF4A1 for their efficient translation. Selective inhibition of translation of mRNAs harboring long 5â UTRs via suppression of eIF4A leads to uncoupling of expression of proteins involved in respiration (e.g. ATP5O) from those protecting mitochondrial integrity (e.g. BCL-2) ultimately resulting in apoptosis. Conversely, simultaneous translational downregulation of both long and short 5â UTR mRNAs by mTOR inhibitors results in suppression of mitochondrial respiration and predominantly cytostatic effects. Therefore, 5â UTR features define differential modes of translation of functionally distinct mTOR-sensitive mRNAs, which explains discrepancies between the effects of mTOR and eIF4A inhibitors on neoplastic cells. Determination of 5'UTR lengths using nanoCAGE in MCF7 cells

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Tau drives translational selectivity by interacting with ribosomal proteins

Project description:Tau drives translational selectivity by interacting with ribosomal proteins

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