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Molecular Cell Dec 2023Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to...
Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.
Topics: Humans; RNA; Ubiquitination; Ubiquitin; Ribosomes; Ubiquitin-Protein Ligases; Aldehydes; Protein Biosynthesis
PubMed: 37951216
DOI: 10.1016/j.molcel.2023.10.012 -
Nature Structural & Molecular Biology Sep 2023Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear....
Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4-NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4-NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4-NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.
Topics: Humans; Animals; Rabbits; Saccharomyces cerevisiae; Mammals; Ribosomes; Ubiquitination; Mass Spectrometry; Transcription Factors; Receptors, CCR4; Ribonucleases; Saccharomyces cerevisiae Proteins
PubMed: 37653243
DOI: 10.1038/s41594-023-01075-8 -
Nature Sep 2023Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles...
Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.
Topics: Humans; Arabidopsis; Codon, Initiator; Innate Immunity Recognition; Open Reading Frames; Protein Biosynthesis; Ribosomes; RNA, Double-Stranded; RNA, Messenger; Transcriptome; DEAD-box RNA Helicases; Deep Learning; Nucleic Acid Conformation
PubMed: 37674078
DOI: 10.1038/s41586-023-06500-y -
Cell Reports Oct 2023Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in...
Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.
Topics: Cell Nucleolus; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ribosomes; RNA, Ribosomal, 18S; Humans; Animals; Rats; DNA-Binding Proteins; Transcription Factors; Protein Biosynthesis
PubMed: 37851577
DOI: 10.1016/j.celrep.2023.113280 -
Cell Death & Disease Jul 2023Impairment of protein translation can cause stalling and collision of ribosomes and is a signal for the activation of ribosomal surveillance and rescue pathways. Despite...
Impairment of protein translation can cause stalling and collision of ribosomes and is a signal for the activation of ribosomal surveillance and rescue pathways. Despite clear evidence that ribosome collision occurs stochastically at a cellular and organismal level, physiologically relevant sources of such aberrations are poorly understood. Here we show that a burst of the cellular signaling molecule nitric oxide (NO) reduces translational activity and causes ribosome collision in human cell lines. This is accompanied by activation of the ribotoxic stress response, resulting in ZAKα-mediated activation of p38 and JNK kinases. In addition, NO production is associated with ZNF598-mediated ubiquitination of the ribosomal protein RPS10 and GCN2-mediated activation of the integrated stress response, which are well-described responses to the collision of ribosomes. In sum, our work implicates a novel role of NO as an inducer of ribosome collision and activation of ribosomal surveillance mechanisms in human cells.
Topics: Humans; Nitric Oxide; Ribosomes; Protein Biosynthesis; Ribosomal Proteins; Ubiquitination; Carrier Proteins
PubMed: 37495584
DOI: 10.1038/s41419-023-05997-5 -
Cell Reports Nov 2023Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation,...
Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation, however, is unclear. We therefore perform Ribo-seq and Disome-seq in Saccharomyces cerevisiae and show that oxidative stress causes ribosome pausing at specific amino acid motifs, which also leads to ribosome collisions. However, these redox-pausing signatures are lost in the absence of Rad6 and do not depend on the ribosome-associated quality control (RQC) pathway. We also show that Rad6 is needed to inhibit overall translation in response to oxidative stress and that its deletion leads to increased expression of antioxidant genes. Finally, we observe that the lack of Rad6 leads to changes during translation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.
Topics: Ubiquitin; Saccharomyces cerevisiae Proteins; gamma-Glutamyl Hydrolase; Saccharomyces cerevisiae; Ribosomes; Oxidative Stress
PubMed: 37917585
DOI: 10.1016/j.celrep.2023.113359 -
Science Advances Aug 2023Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation...
Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and, subsequently, CDK5RAP3, an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation, the E3 complex associated with ufmylated RPL26 on the 60 subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results provide insights into not only the molecular basis of the ufmylation but also its role in proteostasis.
Topics: Endoplasmic Reticulum-Associated Degradation; Ribosomes; Ubiquitin-Protein Ligases; Ubiquitination; Ubiquitins; Humans; HEK293 Cells
PubMed: 37595036
DOI: 10.1126/sciadv.adh3635 -
Molecular & Cellular Proteomics : MCP Sep 2023Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of...
Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of ribosome translation outside the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7000 noncanonical ORFs are translated, which, at first glance, has the potential to expand the number of human protein CDSs by 30%, from ∼19,500 annotated CDSs to over 26,000 annotated CDSs. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of noncanonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome but searching for guidance on how to proceed. Here, we discuss the current state of noncanonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein coding."
Topics: Humans; Proteome; Protein Biosynthesis; Proteomics; Ribosome Profiling; Ribosomes; Open Reading Frames
PubMed: 37572790
DOI: 10.1016/j.mcpro.2023.100631 -
Cell Reports Mar 2024Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hinders host gene expression, curbing defenses and licensing viral protein synthesis and virulence. During...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hinders host gene expression, curbing defenses and licensing viral protein synthesis and virulence. During SARS-CoV-2 infection, the virulence factor non-structural protein 1 (Nsp1) targets the mRNA entry channel of mature cytoplasmic ribosomes, limiting translation. We show that Nsp1 also restrains translation by targeting nucleolar ribosome biogenesis. SARS-CoV-2 infection disrupts 18S and 28S ribosomal RNA (rRNA) processing. Expression of Nsp1 recapitulates the processing defects. Nsp1 abrogates rRNA production without altering the expression of critical processing factors or nucleolar organization. Instead, Nsp1 localizes to the nucleolus, interacting with precursor-rRNA and hindering its maturation separately from the viral protein's role in restricting mature ribosomes. Thus, SARS-CoV-2 Nsp1 limits translation by targeting ribosome biogenesis and mature ribosomes. These findings revise our understanding of how SARS-CoV-2 Nsp1 controls human protein synthesis, suggesting that efforts to counter Nsp1's effect on translation should consider the protein's impact from ribosome manufacturing to mature ribosomes.
Topics: Humans; SARS-CoV-2; RNA, Ribosomal; COVID-19; Ribosomes; Viral Proteins; Viral Nonstructural Proteins
PubMed: 38427561
DOI: 10.1016/j.celrep.2024.113891 -
Cell Reports Dec 2023Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons...
Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.
Topics: Animals; Humans; Codon Usage; Ribosomes; Protein Biosynthesis; Codon; RNA, Messenger; Proteins; Drosophila; Transcription Factors
PubMed: 38096059
DOI: 10.1016/j.celrep.2023.113413