The EMBO Journal

mRNAs can form stable structures that need to be resolved to facilitate translation. During translation initiation in mammals, the scanning 48S complex requires the helicase activity of DHX29 to unwind stable mRNA structures that cannot be resolved by eIF4A. Here, we show that the yeast DHX29 homolog, Ylr419w (Dhx29), has a similar function during translation on elongating 80S ribosomes. Cryo-EM analyses show that the Dhx29 helicase module is positioned at the mRNA entry channel to engage mRNA, while its double-stranded RNA-binding domain (dsRBD) senses hairpin-forming mRNA in the ribosomal A-site. By selective ribosome profiling, we observed that Dhx29 is associated with transcripts that form RNA structures, such as stable tetraloops. Dhx29 mutants with perturbed helicase activity enrich 80S with hairpins in the A-site, as well as ribosome collisions, while a mutant lacking the N-terminal dsRBD sensor domain loses the specificity for such ribosomes. We thus propose that Dhx29 functions in translation elongation by resolving structured mRNA formed in the ribosomal A-site through its 3'-5' helicase activity and pulling on the mRNA from its 3' end.

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole writer for intracellular O-GlcNAcylation. It catalyzes O-GlcNAcylation of thousands of protein substrates, but relatively less is known about the post-translational modifications that occur on OGT itself. Herein, we demonstrate that OGT is S-palmitoylated at Cys-472 and Cys-477, which is mediated by the S-acyltransferase Zinc Finger DHHC-Type Palmitoyl transferase 14 (zDHHC14) and removed by acyl protein thioesterase 2 (APT2). S-Palmitoylation stabilizes OGT by shunting it away from the lysosomal chaperone-mediated autophagy (CMA) pathway, as S-palmitoylation decreases the interaction between OGT and heat shock cognate 70 kDa protein (HSC70), the CMA chaperone. Via label-free quantitative mass spectrometry, we find that S- palmitoylation elevates the affinity between OGT and protein phosphatase 1 catalytic subunit gamma (PPP1CC), but not PPP1CB. We further demonstrate that S-palmitoylation of OGT augments binding with Yes-associated protein-1 (YAP), a protein that associates with PPP1CC, and subsequently enhances YAP O-GlcNAcylation. Our work unearths S-palmitoylation of OGT and CMA-mediated degradation of lysosomal OGT, the orchestration of which finetunes the activity of key OGT complexes, such as OGT-PPP1CC, and contributes to OGT substrate selectivity.

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N1-methyladenosine at position 58 (m1A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m1A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNAiMet) as selectively vulnerable to m1A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNAiMet showed that m1A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m1A58 promotes RNase P-mediated 5'-leader removal and RNase Z-mediated 3'-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m1A58-modified tRNAiMet. Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m1A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

Replication-associated errors can cause DNA damage to accumulate on the newly synthesized strand over time. In specific cases such as stem cells, retention of the immortal strand used as template preserves one daughter cell into pluripotency while correlating with terminal differentiation of the damage one. In somatic cells, DNA damage distribution after mitosis remains unclear. Here, we uncovered a mechanism of non-random segregation of the DNA damage marker gH2AX occurring during a single cell division cycle. Replication stress using hydroxyurea (HU) upon release into S phase in RPE-1, BJ, hCEC D29 and fibroblasts showed reproducible Non-Random Segregation (NRS) of gH2AX in the ensuing G1, a phenotype not observed in any of the cancer cell lines analyzed. Notably, removal of R-loops led to a reduction of cells with NRS, whether RNaseH1 was over-expressed globally or exclusively targeted to centromeres, indicating that centromeric DNA-RNA hybrids contribute to NRS of the damage. In line with our previous evidence of centromeric chromatin disruption leading to R-loops, rapid removal of the histone H3 variant CENP-A causes damage and NRS, although to a lower extent than HU alone. This implies that additional mechanisms contribute to centromeric R-loops and NRS of damage in the daughter cells upon mitotic exit. Mechanistically, chemical inhibition of the catalytic activity of Rad51 led to a significant drop in NRS without a change in the total amount of damaged cells, implying involvement of the Homologous Recombination (HR) pathway to accumulation of gH2AX to only one chromatid. In turn, this affects the spindle-kinetochore with a measurable length asymmetry, inducing mechanical and/or epigenetic signals that affect the orientation of the sister chromatids on the metaphase plate to bias segregation. Altogether, we found replication-induced asymmetric segregation of DNA damage during mitosis that is influenced by centromeric R-loops, Rad51 activity and spindle dynamics, with implications on cell fate, chromosome and genome stability in the daughter cells.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of infecting a wide range of tissues and organs. Its persistence during chronic infection is strongly associated with biofilm formation, which depends on extracellular polysaccharides such as alginate. The HtrA-like periplasmic serine protease MucD is a key regulator of bacterial virulence, stress response, and alginate production, yet its molecular mechanism has remained largely unclear. Here, we discovered the alginate acetylation and export proteins AlgX and AlgK as MucD substrates, and characterized their degradation by mass spectrometry and bioinformatic analysis. We further determined the cryo-EM structure of MucD bound to an AlgK-derived substrate peptide, offering atomic insights into MucD oligomerization assembly, substrate recognition, and specificity. Together with structure-guided mutagenesis and biochemical assays, our results revealed that MucD proteolytic activity is governed by an equilibrium between a resting 12-mer and an active trimer. Crucially, we demonstrate that MucD represses alginate biosynthesis post-translationally, in addition to its previously implicated role in transcriptional regulation. These findings define a distinct activation mechanism and regulatory function for MucD and provide new insight into bacterial HtrA-like serine proteases.

Plant-specific RNA Polymerase V (Pol V) transcribes noncoding RNAs in the RNA-directed DNA methylation pathway, thereby influencing gene expression and genome stability by controlling de novo DNA methylation. However, the mechanisms governing precise chromatin localization and transcriptional activities of Pol V remain elusive. Here we show that Pol V localization is spatially constrained by the chromatin regulators microrchidia (MORC) and MORPHEUS' MOLECULE 1 (MOM1). MORC and MOM1 promote Pol V occupancy at sites near active chromatin, whereas their loss leads to redistribution of Pol V into CMT3-enriched heterochromatin, accompanied by noncoding RNA transcription, small RNA production and DNA methylation. Our findings reveal a combinatorial model in which recruitment, spatial constraint and DNA methylation feedback collectively define Pol V chromatin distribution and epigenetic function.

Proliferating cells maintain their cytoplasmic density within a narrow range but deviate when entering quiescence or experiencing stress, suggesting active regulation. The mechanisms driving these density adjustments and their impact on cellular function remain unclear. Here, we demonstrate that the conserved cAMP-activated protein kinase A (PKA) is a key regulator of cytoplasmic properties. Inactivation of PKA leads to a drastic increase in cytoplasmic dry mass density and reduced diffusion that depends on the environmental stress response (ESR) transcription factors Msn2/4. This change is mediated by the accumulation of glycogen and trehalose, which have opposing effects on intracellular diffusion. Importantly, the accumulation of these carbohydrates confers stress resistance in distinct ways and independently of their roles as energy sources. Our findings highlight the importance of the biophysical properties of the cytoplasm in stress resistance and the role of glycogen and trehalose in regulating these properties.

Circadian rhythms in transcription are facilitated by well-defined genetic circuits, but how molecular clocks drive daily rhythms in mammalian physiology is poorly understood. The mechanistic target-of-rapamycin (mTOR) complex integrates daily systemic and circadian intracellular timing cues for input into the cellular timekeeping machinery. Here we demonstrate that mTOR is a major clock output pathway whose activity is required for most daily variation in cellular and organismal physiology, with PERIOD2 shown to interact directly with mTORC1. Acute mTOR inhibition abolishes functional rhythms in cells and most daily variation in mouse liver physiology. mTOR activity is not required for clock protein or locomotor rhythms, indicating that mTOR is not part of the cellular or central circadian timekeeping mechanism. In the forebrain, mTOR activity is required for most detectable daily rhythms in protein abundance and phosphorylation; however, the daily architecture of the sleep/wake cycle is remarkably preserved in mice and zebrafish under mTOR blockade, with a significant increase in wakefulness. Clock outputs in Arabidopsis (plant) and Neurospora (fungus) are also more sensitive to mTOR inhibition than core clock mechanisms indicating evolutionary conservation of mTOR as a circadian effector. We conclude that most but not all daily physiological rhythms in mammalian cells and tissues depend on rhythmic regulation by the mTOR pathway.

Apoptosis-inducing factor is a mitochondrial flavoprotein that links redox metabolism to mitochondrial homeostasis through its interaction with the disulfide relay protein CHCHD4. Although NADH-dependent AIF dimerization has been proposed as the activated state mediating CHCHD4 engagement, whether it is strictly required for productive AIF-CHCHD4 function remains unclear. Here, combining cellular, biochemical and biophysical approaches, we show that disruption of the AIF dimer interface compromises oxidative phosphorylation, respiratory-chain organization and CHCHD4-dependent mitochondrial homeostasis, yet preserves partial AIF function. Our data reveal that the AIF-CHCHD4 system operates as a conformational dynamic redox module in which distinct AIF oligomeric and redox states sustain CHCHD4 activity with different efficiencies. Mechanistically, dimerization is coupled to NADH-dependent conformational changes that regulate coenzyme binding, charge-transfer complex stabilization and catalytic efficiency. In turn, CHCHD4 binding remodels AIF conformational and redox properties, partially compensating for defects in dimer stabilization or redox coupling. Consistently, a peptide derived from the CHCHD4 N-terminus partially restores redox function in a pathogenic AIF variant defective in dimer stabilization, supporting partner-assisted allosteric regulation as a potential therapeutic strategy.

Zinc is an essential micronutrient at low concentrations, yet it becomes toxic at slightly higher ones. This is exploited by plants as an effective defensive strategy. However, the molecular components that are involved zinc-mediated immunity remain poorly defined. Here, we show that mixed-linked {beta}-1,3/1,4-glucans naturally occurring in microbial and grass cell walls and used as an agrobiological solution, trigger zinc accumulation in the Arabidopsis apoplast and upregulate the expression of the zinc transporters HMA2 and HMA4. This response occurs independently of salicylic acid, jasmonic acid and ethylene-mediated signalling pathways, but it requires the LysM receptor kinases CERK1, LYK4 and LYK5, indicating a specific pattern triggered immunity-associated mechanism. We further demonstrate that hma2hma4 mutants display constitutive activation of a broad set of defence-related genes, yet this transcriptional reprogramming is insufficient to confer resistance against the necrotrophic fungus Plectosphaerella cucumerina BMM. Moreover, metabolomic profiling highlights the contribution of specialized metabolites to this defective defence output. Altogether, our findings reveal that zinc-mediated toxicity constitutes a defence mechanism integrated into the immune response triggered by specific microbial or damage associated molecular patterns.

Initiator-methionine excision is among the earliest protein modifications, yet its relationship to assembly geometry is unknown. Burial of the mature first residue was measured across 7,246 deposited human biological assemblies (22,291 chain-level observations; 1,191 proteins). Among 1,143 analyzable proteins, termini in MetAP-permissive penultimate-residue sequence classes were less often interface-engaged than termini in MetAP-nonpermissive classes (37.4% versus 47.4%; adjusted odds ratio 0.65, p = 7.2e-4). Curated processing annotations did not show a corresponding burial difference, and correlated residue properties preclude attributing the sequence-class association specifically to iMet removal. The analysis identified 264 interface-engaged MetAP-permissive candidates concentrated in cellular machines. In a fully recomputed conformer scan of deeply buried proteasome positions, modeled methionine accommodation was less favorable than at observed-methionine controls (median overlap -0.30 versus -1.12 angstrom, p = 0.0049), although most scoreable sites permitted a nonoverlapping placement. The census therefore reveals a graded structural constraint - not universal steric failure - and prioritizes complexes in which altered packing, assembly kinetics, lipidation or N-terminal methylation can be tested.

Proteins that distinguish quiescent cells from other non-proliferative states and actively regulate their return to proliferation remain poorly understood. Here, we combined quantitative proteomics with functional image-based screening to identify regulators of the quiescence-to-proliferation transition. Amongst the functional quiescence signature proteins we identified, we focussed on integrin 11 (ITGA11) which is induced across multiple models of reversible quiescence in distinct cell types and that has low expression in proliferating and senescent cells. Although ITGA11 is dispensable for proliferation of asynchronously cycling cells, it is required for efficient cell-cycle re-entry from quiescence. Mechanistically, ITGA11 promotes YAP accumulation and nuclear localization, thereby sustaining SKP2 expression and p27 degradation during cell cycle re-entry. Depletion of p27, or pharmacological activation of YAP signalling rescues the cell-cycle re-entry defect caused by ITGA11 depletion. Together, these findings identify ITGA11 as a functional quiescence signature protein that couples extracellular matrix sensing to YAP-dependent regulation of the Skp2-p27 axis, revealing a mechanism that controls the transition from quiescence to proliferation.

The Gram-negative bacterial cell envelope features an asymmetric outer membrane, that confers intrinsic resistance to toxins. Maintenance of this barrier relies on the Mla system, which mediates retrograde transport of mislocalized phospholipids. In Escherichia coli, this system comprises the lipoprotein MlaA, the periplasmic shuttle protein MlaC, and the ABC transporter complex MlaFEDB. Intriguingly, in Campylobacter jejuni, mlaA and mlaC share an operon with an encoded Resistance-Nodulation-cell Division antiporter potentially involved in anterograde phospholipid transport. Here, we describe the functional and mechanistical characterization of Cj MlaC. Complementation experiments in E. coli show that Cj MlaC functions independently of the native Mla system. Native mass spectrometry revealed that Cj MlaC uniquely exists as both monomer and dimer. Lipid binding stabilized the dimer and ion mobility mass spectrometry showed that conformational transitions precede phospholipid release, suggesting a cycle between a low-affinity monomer and a higher-lipid-affinity dimer. Cj MlaC binds phospholipid species distinct from Ec MlaC, showing an increased propensity for lysophospholipids, consistent with the unusually lysophospholipid-rich lipidome of C. jejuni, indicative of evolutionary adaptation to this unique lipid environment. Collectively, these findings uncover structural and mechanistic features of Cj MlaC and support divergent physiological roles for Cj and Ec MlaC in phospholipid trafficking.

BCR and CD40 signals are integrated to shape B cell transcriptional output, but how the two pathways are coordinated at the level of individual signaling nodes is incompletely understood. Using primary mouse B cells stimulated with anti-IgM, anti-CD40, or both (Both), we defined six DEG classes reflecting costimulation-dependence and cross-pathway antagonism. We also connected a CD40-driven, BCR-suppressed B-cell-identity module (Pax5, Aicda, Bcl6, Cd79a/b, Cd19) to an increase in Blimp1 protein at 24 h. Building on our previous work, which showed that cIAP prolongs IKK/ERK activity after single-pathway stimulation (Shinohara et al., 2016), we added an IAP inhibitor 10 min after Both stimulation and generated transcriptomic data. This profiling showed that cIAP is required not only for late canonical NF-{kappa}B-driven genes, as predicted, but also, unexpectedly, for the entire BCR-dominant, CD40-independent negative-feedback module (Cd5, Il10, Nfkbid, Spry1/2) identified in the first dataset. A separate module was instead amplified by IAP inhibition, with kinetics compatible with non-canonical NF-{kappa}B de-repression, directly addressing a question we left open in that study regarding the BCR-side partners of cIAP. Together, these correlative findings converge on a single model: cIAP is not a CD40-restricted adaptor but a hub shared by BCR and CD40, whose feedback simultaneously sustains canonical, fate-instructive signaling and restrains the non-canonical pathway, thereby coupling receptor-proximal signal dynamics to the B cell's downstream transcriptional and fate decisions.

Canonical McrBC enzymes are nucleotide-powered, motor-driven endonucleases that bind and cleave modified bacteriophage DNA. Non-canonical McrBC homologs like LlaI and BsuMI are distinguished by a unique three-gene organization and the ability to target DNA site-specifically. Here, we report the atomic-resolution crystal structures of the DNA-binding module LlaI.R1 and AAA+ motor LlaI.R2 from the Lactococcus lactis LlaI anti-phage defense system. The crystallized LlaI.R2 hexamer traps two distinct active site conformations that correlate to different states of the nucleotide hydrolysis cycle and reveal that the organization of the critical catalytic machinery present in canonical McrB homologs is also conserved in non-canonical R2 proteins. Although canonical McrB homologs are strictly GTP-specific, we find that the R2 proteins from LlaI and BsuMI do not discriminate between different nucleotides, even when in complex with their respective R1 partners. Using mutagenesis, we define surfaces on the LlaI.R1 structure that are critical for DNA-binding and interaction with LlaI.R2. These observations support computational modelling of the assembled LlaI restriction system bound to DNA. Together, our data provide new insights into the evolution of nucleotide specificity in McrBC restriction complexes and the molecular mechanisms governing McrBC-catalyzed DNA translocation and cleavage.

MYCN amplification drives replication stress in high-risk neuroblastoma, yet how MYCN-amplified tumour cells tolerate this stress to sustain proliferation remains poorly understood. Here we show that FBXL12, an SCF ubiquitin ligase substrate receptor that targets the Fanconi anaemia protein FANCD2 for degradation at replication forks, as well as the broader Fanconi anaemia and replication stress transcriptional program are elevated in high-risk and MYCN-amplified neuroblastoma. High FBXL12 expression independently predicts poor survival across neuroblastoma patient cohorts. FBXL12 loss stabilizes FANCD2 on chromatin, elevates ATR-dependent replication stress signalling and DNA damage during S phase, and impairs proliferation of MYCN-amplified neuroblastoma cells in vitro and in vivo. Mechanistically, MYCN directly engages the FBXL12-FANCD2 complex and antagonises FBXL12-mediated degradation of FANCD2 at replication forks, revealing that the oncogenic driver of replication stress also actively preserves the chromatin-bound FANCD2 pool required to tolerate it. Beyond S phase, FBXL12 loss disrupts FANCD2-dependent mitotic DNA synthesis and transmits unresolved replication intermediates into daughter cells. FBXL12-deficient cells consequently show transcriptional activation of MYC target gene, ATR, and mTOR signalling programs, and this pathway-concordant state confers differential sensitivity to ATR, and mTOR-targeting compounds, nominating candidate therapeutic strategies for this disease subset. Together, these findings define a MYCN-FBXL12-FANCD2 axis as a clinically relevant vulnerability in high-risk neuroblastoma.

Lineage-restricted transcriptional programs establish cell identity and can create selective dependencies in cancer. Here, we identify POU2AF2, encoding the transcriptional co-activator OCA-T1, as a critical lineage-specific dependency in a subset of diffuse large B-cell lymphoma (DLBCL). Pan-cancer dependency analyses and patient cohorts reveal elevated POU2AF2 expression in genetically aggressive DLBCL, where its depletion markedly suppresses tumor growth in vitro and in vivo. Mechanistically, POU2AF2 cooperates with the B-cell lineage-defining transcription factor POU2F2 (OCT2) to activate lymphocyte activation gene programs through direct chromatin engagement, thereby sustaining malignant transcriptional networks. We further identified a key epigenetic regulatory axis composed of the lineage-specific transcription factor TCF3 and the histone methyltransferase SET1A-COMPASS that drives POU2AF2 expression downstream of B-cell receptor signaling. Single-cell transcriptomic analysis reveals that POU2AF2 marks and sustains an innate-like B1 B-cell population in vivo, a candidate cell of origin for lymphoma. Together, these findings define a lineage-restricted POU2AF2/POU2F2 transcriptional module, controlled by a TCF3/SET1A epigenetic network, that sustains both innate-like B-cell identity and malignant fitness in DLBCL. Our study uncovers a previously unrecognized lineage-specific transcriptional dependency and highlights POU2AF2 and its associated regulatory circuitry as potential therapeutic targets in aggressive B-cell malignancies.

More than one thousand bifunctional terpene synthases combining prenyltransferase and terpene cyclase activities have been identified in bacteria and fungi, but only a handful of enzymes have been identified that combine terpene cyclase activity with a downstream processing activity. Drimenol synthase from the marine bacterium Aquimarina spongiae (AsDMS) consists of a class II terpene cyclase that converts farnesyl diphosphate into drimenyl diphosphate, and a haloacid dehalogenase-like phosphatase that hydrolyzes drimenyl diphosphate to generate the sesquiterpene alcohol drimenol. The first crystal structure of AsDMS to be reported revealed the architecture of domain assembly as well as dimeric quaternary structure, establishing a structural chemical foundation for cyclization and hydrolysis mechanisms [K. R. Osika, M. N. Gaynes, D. W. Christianson (2025) Proc. Natl. Acad. Sci. U.S.A. 122, e2506584122]. Here, we report crystal structures of the catalytically-inactive double mutant, D33A-D323A AsDMS, complexed with farnesyl diphosphate, geranyl diphosphate, and dimethylallyl diphosphate, which bind in the active sites of both the cyclase and phosphatase domains. Molecular recognition of the diphosphate group dominates binding interactions in both active sites. In the cyclase active site, only farnesyl diphosphate is sufficiently long for its terminal isoprenoid C=C bond to bind adjacent to the catalytic general acid that would initiate the cyclization cascade in the wild-type enzyme. In the phosphatase active site, all isoprenoid diphosphate groups bind similarly, but isoprenoid chain conformations vary. These structures provide a foundation for understanding substrate recognition and catalysis in both active sites. Finally, we present kinetic evidence suggesting that substrate channeling is operative in wild-type AsDMS.

Hematopoietic stem cell (HSC) aging is associated with epigenetic remodeling, yet the molecular mechanisms driving these changes, their overlap with stress-induced alterations, and whether this course can be durably reset remain incompletely understood. Here, we show that transient induction of the Yamanaka factors OCT4, SOX2, KLF4, and MYC in young mice durably delays and partially reverses physiological and LPS-driven HSC aging in mice. Transient reprogramming improved hematopoietic reconstitution, reduced myeloid bias, and limited DNA damage. Multi-omic analyses revealed reduced chromatin accessibility at AP-1-enriched regulatory regions, attenuated age-associated AP-1 transcriptional programs, and repression of transposable elements (TEs). Pharmacological AP-1 inhibition prevented LPS-induced TE activation and loss of HSC clonogenicity. Reverse transcriptase inhibition in aged mice reduced DNA damage and improved HSC function, demonstrating a functional contribution of TE activity to HSC decline. Together, these findings identify AP-1-associated chromatin remodeling as a candidate mechanism linking inflammatory stress, TE activation and HSC aging.