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DENR-regulated reinitiation events uncover predictive uORF features and links to circadian timekeeping via Clock regulation

ABSTRACT: DENR-regulated reinitiation events uncover predictive uORF features and links to circadian timekeeping via Clock regulation

PROVIDER: PRJNA513490 | ENA |

REPOSITORIES: ENA

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Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock.

Castelo-Szekely Violeta V De Matos Mara M Tusup Marina M Pascolo Steve S Ule Jernej J Gatfield David D

Nucleic acids research 20190601 10

The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncover ...[more]

PMID: 30982898

Similar Datasets

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock.

Project description:The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncovered 240 transcripts with altered translation rate, and used linear regression analysis to extract 5' UTR features predictive of DENR dependence. Among core clock genes, we identified Clock as a DENR target. Using Clock 5' UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments revealed an alternative downstream start codon, likely representing the bona fide CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.

2019-01-01 | S-EPMC6547434 | BioStudies

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

Project description:The non-canonical initiation factor DENR promotes translation reinitiation on uORF-containing mRNAs. Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting that uORF usage and reinitiation regulate clock function. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We found 240 transcripts with altered translation rate, and used linear regression analysis to extract uORF features predictive of DENR dependance. Among core clock genes, we identified Clock as a DENR target. Using Clock 5'UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments identified an alternative downstream start codon, which likely represents the true CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large. Overall design: Denr knock-down or control NIH3t3 cells were grown to subconfluency in 15 cm plates/replicate. Cells were pre-treated with cycloheximide for 2 min, prior to lysate preparation. Two different sh-RNAs were used for knock-down experiments, and Ribosome profiling (RPF-seq) and parallel RNA-seq libraries were prepared in triplicate for each. For control, non-targeting scramble sh-RNA in triplicate, and Gfp-shRNA in duplicate was used.

2019-04-01 | GSE116221 | GEO

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

Project description:The non-canonical initiation factor DENR promotes translation reinitiation on uORF-containing mRNAs. Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting that uORF usage and reinitiation regulate clock function. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We found 240 transcripts with altered translation rate, and used linear regression analysis to extract uORF features predictive of DENR dependance. Among core clock genes, we identified Clock as a DENR target. Using Clock 5'UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments identified an alternative downstream start codon, which likely represents the true CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large. Overall design: iCLIP experiments: Denr-overexpressing (OE) or non-transfected (NT) NIH3t3 cells were grown to subconfluency in 15 cm plates/replicate. Cells were crosslinked with UV-C (254 nm) at 150 mJ/cm2,prior to lysate preparation. RNA was partially digested, and DENR-RNA complexes were pulled down with anti-DENR antibody (pre-immobilised in protein G dynabeads). An infrarred adaptor was ligated in the 3' end of RNAs and complexes were visualised after PAGE and nitrocellulose transfer in a Licor machine. RNA-protein complexes of 60-130 Kda were excised and used for library preparation. 24 PCR cycles were used for final library amplification. An IgG immunoprecipitation was used as IP control.

2019-04-01 | GSE124790 | GEO

DENR-MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation.

Project description:The succession of molecular events leading to eukaryotic translation reinitiation-whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA-is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR-MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR-MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.

2018-01-01 | S-EPMC6013234 | BioStudies

DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4.

Project description:Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.

2020-01-01 | S-EPMC7494916 | BioStudies

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.

Project description:Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.

2013-01-01 | S-EPMC3630359 | BioStudies

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

Project description:Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

| PRJNA513492 | ENA

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

Project description:Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

| PRJNA477799 | ENA

Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1.

Project description:The repertoire of the density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein was recently expanded to include translational control of a specific set of cancer-related mRNAs. DENR and MCT-1 form the heterodimer, which binds to the ribosome and operates at both translation initiation and reinitiation steps, though by a mechanism that is yet unclear. Here, we determined the crystal structure of the human small ribosomal subunit in complex with DENR-MCT-1. The structure reveals the location of the DENR-MCT-1 dimer bound to the small ribosomal subunit. The binding site of the C-terminal domain of DENR on the ribosome has a striking similarity with those of canonical initiation factor 1 (eIF1), which controls the fidelity of translation initiation and scanning. Our findings elucidate how the DENR-MCT-1 dimer interacts with the ribosome and have functional implications for the mechanism of unconventional translation initiation and reinitiation.

2017-01-01 | S-EPMC5551485 | BioStudies

Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation.

Project description:The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR's MCT-1-binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

2019-01-01 | S-EPMC6329987 | BioStudies

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