A gut-derived hormone regulates cholesterol metabolism.

The reciprocal coordination between cholesterol absorption in the intestine and de novo cholesterol synthesis in the liver is essential for maintaining cholesterol homeostasis, yet the mechanisms governing the opposing regulation of these processes remain poorly understood. Here, we identify a hormone, Cholesin, which is capable of inhibiting cholesterol synthesis in the liver, leading to a reduction in circulating cholesterol levels. Cholesin is encoded by a gene with a previously unknown function (C7orf50 in humans; 3110082I17Rik in mice). It is secreted from the intestine in response to cholesterol absorption and binds to GPR146, an orphan G-protein-coupled receptor, exerting antagonistic downstream effects by inhibiting PKA signaling and thereby suppressing SREBP2-controlled cholesterol synthesis in the liver. Therefore, our results demonstrate that the Cholesin-GPR146 axis mediates the inhibitory effect of intestinal cholesterol absorption on hepatic cholesterol synthesis. This discovered hormone, Cholesin, holds promise as an effective agent in combating hypercholesterolemia and atherosclerosis.

Automated profiling of gene function during embryonic development.

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes.

Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.

Evolutionary Dynamics and Molecular Mechanisms of HORMA Domain Protein Signaling.

Controlled assembly and disassembly of multi-protein complexes is central to cellular signaling. Proteins of the widespread and functionally diverse HORMA family nucleate assembly of signaling complexes by binding short peptide motifs through a distinctive safety-belt mechanism. HORMA proteins are now understood as key signaling proteins across kingdoms, serving as infection sensors in a bacterial immune system and playing central roles in eukaryotic cell cycle, genome stability, sexual reproduction, and cellular homeostasis pathways. Here, we describe how HORMA proteins' unique ability to adopt multiple conformational states underlies their functions in these diverse contexts. We also outline how a dedicated AAA+ ATPase regulator, Pch2/TRIP13, manipulates HORMA proteins' conformational states to activate or inactivate signaling in different cellular contexts. The emergence of Pch2/TRIP13 as a lynchpin for HORMA protein action in multiple genome-maintenance pathways accounts for its frequent misregulation in human cancers and highlights TRIP13 as a novel therapeutic target.

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.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Synthetic Genomes.

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

How Does the Ribosome Fold the Proteome?

Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.

ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome.

Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1-/y mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1-/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase ß subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.

Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism.

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.

Local Fatty Acid Channeling into Phospholipid Synthesis Drives Phagophore Expansion during Autophagy.

Autophagy is a conserved catabolic homeostasis process central for cellular and organismal health. During autophagy, small single-membrane phagophores rapidly expand into large double-membrane autophagosomes to encapsulate diverse cargoes for degradation. It is thought that autophagic membranes are mainly derived from preformed organelle membranes. Instead, here we delineate a pathway that expands the phagophore membrane by localized phospholipid synthesis. Specifically, we find that the conserved acyl-CoA synthetase Faa1 accumulates on nucleated phagophores and locally activates fatty acids (FAs) required for phagophore elongation and autophagy. Strikingly, using isotopic FA tracing, we directly show that Faa1 channels activated FAs into the synthesis of phospholipids and promotes their assembly into autophagic membranes. Indeed, the first committed steps of de novo phospholipid synthesis at the ER, which forms stable contacts with nascent autophagosomes, are essential for autophagy. Together, our work illuminates how cells spatially tune synthesis and flux of phospholipids for autophagosome biogenesis during autophagy.

A Small Molecule Targeting Mutagenic Translesion Synthesis Improves Chemotherapy.

Intrinsic and acquired drug resistance and induction of secondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and in vivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-molecule inhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.

Error-Prone Replication through UV Lesions by DNA Polymerase θ Protects against Skin Cancers.

Cancers from sun-exposed skin accumulate "driver" mutations, causally implicated in oncogenesis. Because errors incorporated during translesion synthesis (TLS) opposite UV lesions would generate these mutations, TLS mechanisms are presumed to underlie cancer development. To address the role of TLS in skin cancer formation, we determined which DNA polymerase is responsible for generating UV mutations, analyzed the relative contributions of error-free TLS by Polη and error-prone TLS by Polθ to the replication of UV-damaged DNA and to genome stability, and examined the incidence of UV-induced skin cancers in Polθ-/-, Polη-/-, and Polθ-/- Polη-/- mice. Our findings that the incidence of skin cancers rises in Polθ-/- mice and is further exacerbated in Polθ-/- Polη-/- mice compared with Polη-/- mice support the conclusion that error-prone TLS by Polθ provides a safeguard against tumorigenesis and suggest that cancer formation can ensue in the absence of somatic point mutations.

Spatially Stable Mitochondrial Compartments Fuel Local Translation during Plasticity.

Local translation meets protein turnover and plasticity demands at synapses, however, the location of its energy supply is unknown. We found that local translation in neurons is powered by mitochondria and not by glycolysis. Super-resolution microscopy revealed that dendritic mitochondria exist as stable compartments of single or multiple filaments. To test if these mitochondrial compartments can serve as local energy supply for synaptic translation, we stimulated individual synapses to induce morphological plasticity and visualized newly synthesized proteins. Depletion of local mitochondrial compartments abolished both the plasticity and the stimulus-induced synaptic translation. These mitochondrial compartments serve as spatially confined energy reserves, as local depletion of a mitochondrial compartment did not affect synaptic translation at remote spines. The length and stability of dendritic mitochondrial compartments and the spatial functional domain were altered by cytoskeletal disruption. These results indicate that cytoskeletally tethered local energy compartments exist in dendrites to fuel local translation during synaptic plasticity.

HMCES Maintains Genome Integrity by Shielding Abasic Sites in Single-Strand DNA.

Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template.

Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.

Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design.

Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.