The ribotoxic stress response drives UV-mediated cell death.

While ultraviolet (UV) radiation damages DNA, eliciting the DNA damage response (DDR), it also damages RNA, triggering transcriptome-wide ribosomal collisions and eliciting a ribotoxic stress response (RSR). However, the relative contributions, timing, and regulation of these pathways in determining cell fate is unclear. Here we use time-resolved phosphoproteomic, chemical-genetic, single-cell imaging, and biochemical approaches to create a chronological atlas of signaling events activated in cells responding to UV damage. We discover that UV-induced apoptosis is mediated by the RSR kinase ZAK and not through the DDR. We identify two negative-feedback modules that regulate ZAK-mediated apoptosis: (1) GCN2 activation limits ribosomal collisions and attenuates ZAK-mediated RSR and (2) ZAK activity leads to phosphodegron autophosphorylation and its subsequent degradation. These events tune ZAK's activity to collision levels to establish regimes of homeostasis, tolerance, and death, revealing its key role as the cellular sentinel for nucleic acid damage.

Ribosome ADP-ribosylation inhibits translation and maintains proteostasis in cancers.

Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.

Mechanisms of Cotranslational Maturation of Newly Synthesized Proteins.

The timely production of functional proteins is of critical importance for the biological activity of cells. To reach the functional state, newly synthesized polypeptides have to become enzymatically processed, folded, and assembled into oligomeric complexes and, for noncytosolic proteins, translocated across membranes. Key activities of these processes occur cotranslationally, assisted by a network of machineries that transiently engage nascent polypeptides at distinct phases of translation. The sequence of events is tuned by intrinsic features of the nascent polypeptides and timely association of factors with the translating ribosome. Considering the dynamics of translation, the heterogeneity of cellular proteins, and the diversity of interaction partners, it is a major cellular achievement that these processes are temporally and spatially so precisely coordinated, minimizing the generation of damaged proteins. This review summarizes the current progress we have made toward a comprehensive understanding of the cotranslational interactions of nascent chains, which pave the way to their functional state.

Magnesium Flux Modulates Ribosomes to Increase Bacterial Survival.

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.

mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

Quantifying Protein-mRNA Interactions in Single Live Cells.

Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to ß-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with ß-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.

Codon optimality-mediated mRNA degradation: Linking translational elongation to mRNA stability.

Messenger RNA (mRNA) translation by the ribosome represents the final step of a complicated molecular dance from DNA to protein. Although classically considered a decipherer that translates a 64-word genetic code into a proteome of astonishing complexity, the ribosome can also shape the transcriptome by controlling mRNA stability. Recent work has discovered that the ribosome is an arbiter of the general mRNA degradation pathway, wherein the ribosome transit rate serves as a major determinant of transcript half-lives. Specifically, members of the degradation complex sense ribosome translocation rates as a function of ribosome elongation rates. Central to this notion is the concept of codon optimality: although all codons impact translation rates, some are deciphered quickly, whereas others cause ribosome hesitation as a consequence of relative cognate tRNA concentration. These transient pauses induce a unique ribosome conformational state that is probed by the deadenylase complex, thereby inducing an orchestrated set of events that enhance both poly(A) shortening and cap removal. Together, these data imply that the coding region of an mRNA not only encodes for protein content but also impacts protein levels through determining the transcript's fate.

Sensing of individual stalled 80S ribosomes by Fap1 for nonfunctional rRNA turnover.

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.

Mimicry of Canonical Translation Elongation Underlies Alanine Tail Synthesis in RQC.

Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.

TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes.

Target of rapamycin complex 1 (TORC1) promotes biogenesis and inhibits the degradation of ribosomes in response to nutrient availability. To ensure a basal supply of ribosomes, cells are known to preserve a small pool of dormant ribosomes under nutrient-limited conditions. However, the regulation of these dormant ribosomes is poorly characterized. Here, we show that upon inhibition of yeast TORC1 by rapamycin or nitrogen starvation, the ribosome preservation factor Stm1 mediates the formation of nontranslating, dormant 80S ribosomes. Furthermore, Stm1-bound 80S ribosomes are protected from proteasomal degradation. Upon nutrient replenishment, TORC1 directly phosphorylates and inhibits Stm1 to reactivate translation. Finally, we find that SERBP1, a mammalian ortholog of Stm1, is likewise required for the formation of dormant 80S ribosomes upon mTORC1 inhibition in mammalian cells. These data suggest that TORC1 regulates ribosomal dormancy in an evolutionarily conserved manner by directly targeting a ribosome preservation factor.

Expanded profiling of WD repeat domain 5 inhibitors reveals actionable strategies for the treatment of hematologic malignancies.

WD40 Repeat Domain 5 (WDR5) is a highly conserved nuclear protein that recruits MYC oncoprotein transcription factors to chromatin to stimulate ribosomal protein gene expression. WDR5 is tethered to chromatin via an arginine-binding cavity known as the "WIN" site. Multiple pharmacological inhibitors of the WDR5-interaction site of WDR5 (WINi) have been described, including those with picomolar affinity and oral bioavailability in mice. Thus far, however, WINi have only been shown to be effective against a number of rare cancer types retaining wild-type p53. To explore the full potential of WINi for cancer therapy, we systematically profiled WINi across a panel of cancer cells, alone and in combination with other agents. We report that WINi are unexpectedly active against cells derived from both solid and blood-borne cancers, including those with mutant p53. Among hematologic malignancies, we find that WINi are effective as a single agent against leukemia and diffuse large B cell lymphoma xenograft models, and can be combined with the approved drug venetoclax to suppress disseminated acute myeloid leukemia in vivo. These studies reveal actionable strategies for the application of WINi to treat blood-borne cancers and forecast expanded utility of WINi against other cancer types.

Translational control through ribosome heterogeneity and functional specialization.

The conceptual origins of ribosome specialization can be traced back to the earliest days of molecular biology. Yet, this field has only recently begun to gather momentum, with numerous studies identifying distinct heterogeneous ribosome populations across multiple species and model systems. It is proposed that some of these compositionally distinct ribosomes may be functionally specialized and able to regulate the translation of specific mRNAs. Identification and functional characterization of specialized ribosomes has the potential to elucidate a novel layer of gene expression control, at the level of translation, where the ribosome itself is a key regulatory player. In this review, we discuss different sources of ribosome heterogeneity, evidence for ribosome specialization, and also the future directions of this exciting field.

Mitochondrial protein synthesis quality control.

Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical barrier to separate transcription and translation, nor is there evidence that quality control surveillance pathways are active to prevent translation on faulty mRNA transcripts. Mitochondrial ribosomes synthesize 13 hydrophobic proteins that require co-translational insertion into the inner membrane of the organelle. To maintain the integrity of the inner membrane, which is essential for organelle function, requires responsive quality control mechanisms to recognize aberrations in protein synthesis. In this review, we explore how defects in mitochondrial protein synthesis can arise due to the culmination of inherent mistakes that occur throughout the steps of gene expression. In turn, we examine the stepwise series of quality control processes that are needed to eliminate any mistakes that would perturb organelle homeostasis. We aim to provide an integrated view on the quality control mechanisms of mitochondrial protein synthesis and to identify promising avenues for future research.

High-Resolution Reconstruction of a C. elegans Ribosome Sheds Light on Evolutionary Dynamics and Tissue Specificity.

Caenorhabditis elegans is an important model organism for human health and disease, with foundational contributions to the understanding of gene expression and tissue patterning in animals. An invaluable tool in modern gene expression research is the presence of a high-resolution ribosome structure, though no such structure exists for C. elegans. Here we present a high-resolution single-particle cryogenic electron microscopy (cryoEM) reconstruction and molecular model of a C. elegans ribosome, revealing a significantly streamlined animal ribosome. Many facets of ribosome structure are conserved in C. elegans, including overall ribosomal architecture and the mechanism of cycloheximide, while other facets such as expansion segments and eL28 are rapidly evolving. We identify uL5 and uL23 as two instances of tissue-specific ribosomal protein paralog expression conserved in Caenorhabditis, suggesting that C. elegans ribosomes vary across tissues. The C. elegans ribosome structure will provide a basis for future structural, biochemical, and genetic studies of translation in this important animal system.

The Discovery of Ribosome Heterogeneity and Its Implications for Gene Regulation and Organismal Life.

The ribosome has recently transitioned from being viewed as a passive, indiscriminate machine to a more dynamic macromolecular complex with specialized roles in the cell. Here, we discuss the historical milestones from the discovery of the ribosome itself to how this ancient machinery has gained newfound appreciation as a more regulatory participant in the central dogma of gene expression. The first emerging examples of direct changes in ribosome composition at the RNA and protein level, coupled with an increased awareness of the role individual ribosomal components play in the translation of specific mRNAs, is opening a new field of study centered on ribosome-mediated control of gene regulation. In this Perspective, we discuss our current understanding of the known functions for ribosome heterogeneity, including specialized translation of individual transcripts, and its implications for the regulation and expression of key gene regulatory networks. In addition, we suggest what the crucial next steps are to ascertain the extent of ribosome heterogeneity and specialization and its importance for regulation of the proteome within subcellular space, across different cell types, and during multi-cellular organismal development.

Ribosomal dormancy at the nexus of ribosome homeostasis and protein synthesis.

Dormancy or hibernation is a non-proliferative state of cells with low metabolic activity and gene expression. Dormant cells sequester ribosomes in a translationally inactive state, called dormant/hibernating ribosomes. These dormant ribosomes are important for the preservation of ribosomes and translation shut-off. While recent studies attempted to elucidate their modes of formation, the regulation and roles of the diverse dormant ribosomal populations are still largely understudied. The mechanistic details of the formation of dormant ribosomes in stress and especially their disassembly during recovery remain elusive. In this review, we discuss the roles of dormant ribosomes and their potential regulatory mechanisms. Furthermore, we highlight the paradigms that need to be answered in the field of ribosomal dormancy.

Entropic control of the free-energy landscape of an archetypal biomolecular machine.

Biomolecular machines are complex macromolecular assemblies that utilize thermal and chemical energy to perform essential, multistep, cellular processes. Despite possessing different architectures and functions, an essential feature of the mechanisms of action of all such machines is that they require dynamic rearrangements of structural components. Surprisingly, biomolecular machines generally possess only a limited set of such motions, suggesting that these dynamics must be repurposed to drive different mechanistic steps. Although ligands that interact with these machines are known to drive such repurposing, the physical and structural mechanisms through which ligands achieve this remain unknown. Using temperature-dependent, single-molecule measurements analyzed with a time-resolution-enhancing algorithm, here, we dissect the free-energy landscape of an archetypal biomolecular machine, the bacterial ribosome, to reveal how its dynamics are repurposed to drive distinct steps during ribosome-catalyzed protein synthesis. Specifically, we show that the free-energy landscape of the ribosome encompasses a network of allosterically coupled structural elements that coordinates the motions of these elements. Moreover, we reveal that ribosomal ligands which participate in disparate steps of the protein synthesis pathway repurpose this network by differentially modulating the structural flexibility of the ribosomal complex (i.e., the entropic component of the free-energy landscape). We propose that such ligand-dependent entropic control of free-energy landscapes has evolved as a general strategy through which ligands may regulate the functions of all biomolecular machines. Such entropic control is therefore an important driver in the evolution of naturally occurring biomolecular machines and a critical consideration for the design of synthetic molecular machines.

Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues.

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.

Utilizing high-resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas.

Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas) for generating high-resolution data covering more than 10 000 different transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5' and 3' sections of transcripts and characteristics of eukaryotic translation termination, which are fundamentally distinct for chloroplast translation. In chloroplasts, a heterogeneous RPF size distribution along the coding sequence indicates specific regulatory phases during protein synthesis. For example, local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly uncovering an uncharacterized regulatory step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light-harvesting complex proteins, LHCs. By providing high-quality datasets for all subcellular genomes and attaching our data to the Chlamydomonas reference genome, we aim to make ribosome profiles easily accessible for the broad research community. The data can be browsed without advanced bioinformatic background knowledge for translation output levels of specific genes and their splice variants and for monitoring genome annotation.