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Science (New York, N.Y.) May 2024In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible...
In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.
Topics: Humans; Apoptosis; Cell Line, Tumor; Codon; DNA Damage; Leucine; Nuclear Proteins; Protein Biosynthesis; Protein Serine-Threonine Kinases; Ribosomes; Signal Transduction; Tumor Suppressor Protein p53; MAP Kinase Kinase Kinases
PubMed: 38753784
DOI: 10.1126/science.adh7950 -
Science (New York, N.Y.) Dec 2023The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase...
The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.
Topics: Animals; Mice; Aging; MAP Kinase Kinase Kinase 3; Obesity; Protein Biosynthesis; Reactive Oxygen Species; Ribosomes; Stress, Physiological; Zebrafish; Mice, Knockout
PubMed: 38060659
DOI: 10.1126/science.adf3208 -
Nature Methods Nov 2023Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of...
Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected messenger RNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with ordered two-template relay, a single-tube protocol for sequencing library preparation that incorporates adaptors by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling and termination. Our optimized methods for mRNA footprint generation and capture provide a richer translatome profile with low input and fewer technical challenges.
Topics: Humans; Saccharomyces cerevisiae; Protein Biosynthesis; Ribosome Profiling; Ribosomes; Transcriptome; RNA, Messenger; High-Throughput Nucleotide Sequencing
PubMed: 37783882
DOI: 10.1038/s41592-023-02028-1 -
Nature Aug 2023An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot. Here, we demonstrate that rapid activation of...
An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.
Topics: Animals; Humans; Mice; Ambystoma mexicanum; Amino Acid Sequence; Extremities; Regeneration; RNA, Messenger; TOR Serine-Threonine Kinases; Wound Healing; Mechanistic Target of Rapamycin Complex 1; Biological Evolution; Species Specificity; Protein Biosynthesis; Antioxidants; Nutrients; Polyribosomes
PubMed: 37495694
DOI: 10.1038/s41586-023-06365-1 -
Science (New York, N.Y.) Feb 2024We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes...
We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of , , and . We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.
Topics: Anti-Bacterial Agents; Erythromycin; Microbial Sensitivity Tests; Drug Resistance, Multiple, Bacterial; Staphylococcus aureus; Escherichia coli; Pseudomonas aeruginosa; Bridged-Ring Compounds; Oxepins; Lincosamides; Animals; Mice; Drug Design; Ribosomes
PubMed: 38359125
DOI: 10.1126/science.adk8013 -
Science Advances Jun 2023Quantifying aging rate is important for evaluating age-associated decline and mortality. A blood single-cell RNA sequencing dataset for seven supercentenarians (SCs) was...
Quantifying aging rate is important for evaluating age-associated decline and mortality. A blood single-cell RNA sequencing dataset for seven supercentenarians (SCs) was recently generated. Here, we generate a reference 28-sample aging cohort to compute a single-cell level aging clock and to determine the biological age of SCs. Our clock model placed the SCs at a blood biological age to between 80.43 and 102.67 years. Compared to the model-expected aging trajectory, SCs display increased naive CD8 T cells, decreased cytotoxic CD8 T cells, memory CD4 T cells, and megakaryocytes. As the most prominent molecular hallmarks at the single-cell level, SCs contain more cells and cell types with high ribosome level, which is associated with and, according to Bayesian network inference, contributes to a low inflammation state and slow aging of SCs. Inhibiting ribosomal activity or translation in monocytes validates such translation against inflammation balance revealed by our single-cell aging clock.
Topics: Humans; Aged, 80 and over; Longevity; CD8-Positive T-Lymphocytes; Leukocytes, Mononuclear; Bayes Theorem; Single-Cell Gene Expression Analysis; Aging; Cellular Senescence; Inflammation; Ribosomes
PubMed: 37379396
DOI: 10.1126/sciadv.abq7599 -
Molecular Cell Jul 2023Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics...
Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.
Topics: Humans; Tubulin; Ribosomes; Microtubules; Homeostasis; RNA, Messenger; RNA Stability; Carrier Proteins; Transcription Factors
PubMed: 37295431
DOI: 10.1016/j.molcel.2023.05.020 -
Cell Jul 2023Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but...
Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.
Topics: Animals; Protein Biosynthesis; Ribosomes; Proteins; RNA, Messenger; Codon, Terminator; Mammals
PubMed: 37339632
DOI: 10.1016/j.cell.2023.05.035 -
PloS One 2023The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such...
The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 μM) with little concomitant cytotoxicity (CC50 ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 μM) but also the viral inhibition (IC50 ~ 1 μM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.
Topics: Humans; COVID-19; Lysine; SARS-CoV-2; Alanine; HIV Seropositivity; HIV-1; Momordica charantia; Ribosome Inactivating Proteins; Ribosomes; COVID-19 Drug Treatment
PubMed: 37384752
DOI: 10.1371/journal.pone.0286370 -
Science (New York, N.Y.) Jul 2023During the early stages of human large ribosomal subunit (60) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers...
During the early stages of human large ribosomal subunit (60) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60 particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60 assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60 particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.
Topics: Humans; Cell Nucleus; Cryoelectron Microscopy; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; RNA, Ribosomal; Saccharomyces cerevisiae; Protein Conformation
PubMed: 37410842
DOI: 10.1126/science.adh3892