Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor and one of the initiating enzymes essential for DNA repair. Understanding how to control PARP1 activity is critical for developing strategies to maintain geno...Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor and one of the initiating enzymes essential for DNA repair. Understanding how to control PARP1 activity is critical for developing strategies to maintain genome stability. Here, we report that the RNA-binding protein poly(rC)-binding protein 1 (PCBP1) interacts with and inhibits PARP1 activity under physiological conditions, and that this inhibition is relieved in the early phase of the DNA damage response. Mechanistically, histone deacetylase SIRT7 mediates the deacetylation of PCBP1 at K314 and K351, disrupting its interaction with the DNA-binding domain of PARP1 and thereby permitting PARP1 activation. Modulating PCBP1 expression or acetylation through gene editing or antagonistic peptides affects the efficiency of DNA damage repair in both cell and murine models. Reduced PCBP1 levels are associated with poor survival in cancer patients following radiotherapy. This regulatory role for PCBP1 in PARP1 activity may form the basis of a therapeutic strategy that combines the inhibition of PCBP1 deacetylation with DNA-damaging agents.
Sun W, Wu N, Xia M
… +22 more, Pan Y, Liu M, Fan S, Wang J, Zeng Y, Fan L, Chen J, Liu G, Yan H, Qiu Y, Xie Y, Jiang Z, Chen F, Yin Y, Zhang J, Li L, Xie W, Wang H, Zhang L, Yu M, Wang X, Yan J
CCCTC-binding factor (CTCF) is an evolutionarily conserved transcription factor with diverse regulatory roles. Its binding sites exhibit highly ordered nucleosomes and DNA hypomethylation, but how this epigenetic landsca...CCCTC-binding factor (CTCF) is an evolutionarily conserved transcription factor with diverse regulatory roles. Its binding sites exhibit highly ordered nucleosomes and DNA hypomethylation, but how this epigenetic landscape is established remains unclear. In this study, we develop a GpC methylation-assisted tracing (G-MAT) approach to investigate the interplay between DNA methylation and CTCF binding at a base-pair resolution, which reveals that CTCF-chromatin interaction frequently coincides with methylated DNA, which is likely mediated by the nucleosome remodeling and deacetylase (NuRD) complex. We show that NuRD is indispensable for CTCF's chromatin binding, emerging as a regulator of high-order genome architecture. Mechanistically, NuRD facilitates CTCF to interact with TET methylcytosine dioxygenase to maintain adjacent DNA hypomethylation, which is essential for activation of nearby genes. Notably, embryonic stem cells lacking NuRD exhibit impaired lineage commitment. Together, our study unravels a mechanism that elucidates the crosstalk between CTCF binding and the epigenome, with NuRD playing a crucial role as a mediator.
Bacteria have evolved a myriad of host-defense systems that protect against invasive mobile genetic elements. The DdmDE module, which consists of a DNA-guided, DNA-targeting prokaryotic Argonaute (pAgo) DdmE and a helica...Bacteria have evolved a myriad of host-defense systems that protect against invasive mobile genetic elements. The DdmDE module, which consists of a DNA-guided, DNA-targeting prokaryotic Argonaute (pAgo) DdmE and a helicase-nuclease enzyme DdmD, represents a model anti-plasmid system. Here, we demonstrate that a double-stranded DNA (dsDNA)-destabilizing force generates transient bubbles that provoke promiscuous binding by DNA-guided DdmE. The resulting nucleoprotein complex discriminates on-targets from off-targets primarily through differences in dissociation rates. Remarkably, DNA-bound DdmE recruits a DdmD dimer to drive dsDNA shortening against a resisting force. Further analysis attributes this shortening to target-centered, bidirectional dsDNA unwinding and single-stranded DNA (ssDNA) extrusion by DdmD, in which monomeric DdmD engages both unwound strands. During this process, free DdmD rapidly associates with the two extruded strands and catalyzes ssDNA digestion with a sequence preference for a 5' guanine. Our findings provide a dynamic perspective on how pAgos cooperate with accessory factors to achieve plasmid clearance.
Pancreatic ductal adenocarcinoma (PDAC) evades immune surveillance in part through autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I), though the basis for this vulnerability...Pancreatic ductal adenocarcinoma (PDAC) evades immune surveillance in part through autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I), though the basis for this vulnerability is unclear. Using synchronized endoplasmic reticulum (ER) exit assays, we show that PDAC cells retain MHC-I in the ER and inefficiently traffic it to the plasma membrane. We identify an autophagic capture complex composed of the ER-phagy receptor TEX264 and the cargo receptor NBR1 that targets MHC-I for degradation. Suppression of either receptor restores total and surface MHC-I levels. Capture is linked to antigen loading, as impaired peptide loading increases MHC-I binding to the TEX264-NBR1 complex, while high-affinity peptides reduce binding and promote increased surface localization. A genome-wide CRISPRi screen identified the ER-localized E3 ligase NFXL1 as a mediator of MHC-I ubiquitylation and capture. Elevated NFXL1 correlates with reduced MHC-I expression and poor prognosis, highlighting a targetable pathway regulating PDAC immunogenicity.
Sun J, Zhang Z, Yu Y
… +15 more, Cruz N, Zhang S, Xiang Y, Ito K, Lazarus C, Tang Z, Li B, Barrows D, Muir TW, Shi Y, Schneidman-Duhovny D, Wang SP, Urnavicius L, Patel DJ, Roeder RG
Mammalian H3K4 mono-methyltransferase MLL4 plays a critical role in enhancer-mediated gene activation. Here, we utilized a reconstituted MLL4 "fusion" complex (MLL4C) for structural and functional analyses and revealed t...Mammalian H3K4 mono-methyltransferase MLL4 plays a critical role in enhancer-mediated gene activation. Here, we utilized a reconstituted MLL4 "fusion" complex (MLL4C) for structural and functional analyses and revealed that MLL4 function is regulated by three structurally rigid modules, namely, SET-ASH2L-DPY30, RBBP5-WDR5, and PHD-FYR. Notably, the PHD-FYR module is evolutionarily conserved across MLL1-4 and is essential for MLL4- and p53-dependent transcription. Combining results from cryo-EM and crosslinking mass spectrometry, we obtained a complete model of MLL4C and elucidated its functional interactions with p53. Genomic analyses in HCT116 cells further showed that MLL4 colocalizes with p53 at p53 target genes and that MLL4 knockout results in a marked reduction in the expression of p53-regulated genes, accompanied by reduced p53 chromatin occupancy. These findings provide molecular insights into the catalytic and non-catalytic transcriptional functions of the MLL4 complex in establishing active enhancers and facilitating p53-dependent transcription activation.
Liu Q, Yoo S, Zhang ZA
… +12 more, Li L, Su H, Vannur L, Wooldredge AC, Hughes JB, Desprez PY, Hao N, Lithgow G, Andersen JK, Hansen M, Campisi J, Zhou C
Nearly all cellular processes are pH dependent. The acidic pH inside the lysosome (vacuole in yeast) is essential for cellular content degradation, signaling, and autophagy. Defects in lysosome/vacuole acidification are...Nearly all cellular processes are pH dependent. The acidic pH inside the lysosome (vacuole in yeast) is essential for cellular content degradation, signaling, and autophagy. Defects in lysosome/vacuole acidification are a conserved hallmark of aging and age-related diseases. Traditionally, the lysosome/vacuole is thought to import free protons (H⁺) from the surrounding neutral cytosol. Here, we uncovered a conserved lysosome/vacuole acidification mechanism from yeast to human involving lysosomal/vacuolar uptake of H pumped out by mitochondrial electron transport chain through mitochondria-lysosomes/vacuoles membrane contacts. Aging/senescence-associated disruption of mitochondria-lysosome/vacuole contacts causes lysosomal/vacuolar de-acidification, which can be reversed by either expressing an engineered linker to connect these two organelles or through an asymmetry-dependent rejuvenation process in daughter cells. Preserving lysosomal acidification in senescent human cells prevents the induction of major senescence-associated secretory phenotype factors and restores autophagic flux. These findings reshape our current understanding of the mechanisms underlying lysosomal/vacuolar (de-)acidification in both young and aged/senescent cells.
Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38- and JNK-activating MAP3 kinase ZAKα senses translational impairment. Combining AlphaFold3 prediction and RNA cro...Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38- and JNK-activating MAP3 kinase ZAKα senses translational impairment. Combining AlphaFold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAKα dynamically monitors the mRNA exit channel of elongating ribosomes. This is accomplished by ZAKα via direct interactions with the ribosomal proteins RACK1 and RPS27 as well as 18S rRNA helix-26. In this conformation, the RNA-binding S (sensing) and C-terminal domain of ZAKα span across the mRNA exit channel. Loss of ribosome processivity and mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAKα to stalled and collided ribosomes is associated with sequestration of the sterile alpha-motif (SAM) domain on RACK1, which allows for transient ZAKα dimerization, activation loop trans-autophosphorylation, and RSR activation. Our findings highlight how ZAKα senses both stalled and collided ribosomes in human cells through overlapping mechanisms.
HO functions as a signaling molecule regulating plant growth, development, and stress responses; however, its role in jasmonic acid (JA) signaling transduction and JA-regulated biological processes remains elusive. Here,...HO functions as a signaling molecule regulating plant growth, development, and stress responses; however, its role in jasmonic acid (JA) signaling transduction and JA-regulated biological processes remains elusive. Here, we report that General regulatory factor 8 (GRF8) is a critical factor inhibiting JA signaling, while HO removes this inhibition to facilitate the JA response during root growth and leaf senescence in Arabidopsis. GRF8 interacts with and inhibits MYC2, the key transcription factor in JA signaling. It recruits Jasmonate-ZIM domain (JAZ) repressors to enhance their interaction with and inhibition of MYC2. Additionally, GRF8 competes with Mediator 25 (MED25), a subunit of the Mediator coactivator complex, to interact with MYC2. Upon increased JA levels, HO sulfenylates GRF8 at cysteine-103, reducing its interaction with MYC2 and thereby releasing MYC2 activity to stimulate the JA response. Together, our findings suggest that HO-mediated sulfenylation of GRF8 provides a redox-based mechanism to modulate JA signaling in Arabidopsis.
Receptor-type E3 ubiquitin ligases enable extracellular signals to control ubiquitylation in the cytoplasm, playing widespread roles in development, metabolism, and immunity. Using cryoelectron microscopy, integrated wit...Receptor-type E3 ubiquitin ligases enable extracellular signals to control ubiquitylation in the cytoplasm, playing widespread roles in development, metabolism, and immunity. Using cryoelectron microscopy, integrated with biophysical and functional studies, we visualized a human E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex attenuates Hedgehog signaling by ubiquitylating Smoothened (SMO), a G-protein-coupled receptor (GPCR) that transduces morphogen signals. A long helix in the MMM complex engages SMO using an intramembrane degron and extends into the cytoplasm to suspend an activated and precisely oriented RING domain below the plasma membrane. This architecture enables ubiquitylation of the cytoplasmic surface of SMO, reducing SMO abundance at primary cilia. Our structure provides insights into MEGF8 mutations, which cause multi-organ birth defects, and defines a paradigm for how transmembrane E3 ligases control the cell surface abundance of GPCRs and other signaling receptors.
Argonaute proteins (AGOs) load small RNA duplexes and select one strand to form the RNA-induced silencing complex (RISC), but the mechanism of assembly remains unclear. We report four cryogenic-electron microscopy struct...Argonaute proteins (AGOs) load small RNA duplexes and select one strand to form the RNA-induced silencing complex (RISC), but the mechanism of assembly remains unclear. We report four cryogenic-electron microscopy structures of human AGO2 bound to a small interfering RNA (siRNA) duplex, which captures previously unanticipated intermediates. Unexpectedly, only the MID-PIWI lobe secures one duplex end, while α-helix 14 probes its stability. When the N domain reaches the opposite end, the L1 hairpin and Stalk wedge into the duplex and the PAZ domain engages the guide 3' end. This configuration peels the passenger from its 5' end while leaving it paired to the guide seed. AGO2 completely ejects the passenger by replacing it even with partially complementary target RNAs. This target-assisted passenger ejection (TAPE) also operates for the cleaved passenger strand. Our study uncovers that mRNAs are bifunctional molecules that not only serve as targets of RISCs but also help drive RISC assembly.
Taskin AA, Shankar S, Peselj C
… +12 more, Flotho A, Gomez-Fabra Gala M, Poveda-Huertes D, Myketin L, Mutlu D, Marada A, Schuck S, Jeske M, Büttner S, Luzarowski M, Meisinger C, Vögtle FN
The mitochondrial unfolded protein response (UPR) protects mitochondria from proteotoxic stress. Current models induce acute and severe mitochondrial disruption and propose cytosolic detection following the release of mi...The mitochondrial unfolded protein response (UPR) protects mitochondria from proteotoxic stress. Current models induce acute and severe mitochondrial disruption and propose cytosolic detection following the release of mitochondrial damage signals into the cytosol. However, this mode of toxicity contrasts sharply with physiological stress, such as the gradual accumulation of reactive oxygen species (ROS) during aging or chronic respiratory chain defects. Here, we employ a chemogenetic strategy in yeast to induce low levels of hydrogen peroxide (HO) in the mitochondrial matrix and show that mild oxidative stress activates the UPR independently of cytosolic damage. We identify the presequence proteases MPP and Oct1 as early ROS targets, thereby linking redox imbalance to UPR activation: oxidative stress induces glutathionylation of critical cysteines, impairing protease activity and causing the accumulation of unprocessed precursors in proteotoxic matrix aggregates. These aggregates are detected by intra-mitochondrial surveillance, activating UPR signaling. Thus, mitochondrial self-surveillance initiates rapid protective signaling as a primary response to mitochondrial dysfunction.
Endogenous double-stranded RNAs (dsRNAs) are immunogenic self-molecules that drive aberrant immune activation under pathological conditions. Here, we show that dsRNAs and their regulation by RNA-binding proteins are key...Endogenous double-stranded RNAs (dsRNAs) are immunogenic self-molecules that drive aberrant immune activation under pathological conditions. Here, we show that dsRNAs and their regulation by RNA-binding proteins are key determinants of the fine balance between aging and immunity in Caenorhabditis elegans and cultured human cells. We find elevated levels of dsRNAs with organismal aging and cellular senescence. We identify a moonlighting function for phenylalanyl-tRNA synthetase, FARS-1/FARSA, as a key factor necessary and sufficient for extending lifespan by downregulating dsRNAs, in particular, mitochondrial dsRNAs. FARS-1/FARSA possesses a previously unrecognized dsRNA-binding domain and mediates dsRNA downregulation with the RNA helicase, RHA-2/DHX37, independently of its canonical role in translation. Notably, increased dsRNA expression resulting from genetic inhibition of fars-1/FARSA upregulates immune response-related genes and enhances innate immunity against pathogens. Our study establishes that FARS-1/FARSA is an evolutionarily conserved dsRNA-binding protein that delays aging and promotes longevity by suppressing dsRNA accumulation.
Plasmids have fundamentally transformed how we resolve regulatory grammar across the tree of life. However, our understanding of how, or whether, chromatin structures form on plasmids transfected into mammalian cells rem...Plasmids have fundamentally transformed how we resolve regulatory grammar across the tree of life. However, our understanding of how, or whether, chromatin structures form on plasmids transfected into mammalian cells remains limited. We developed plasmid single-molecule chromatin fiber sequencing (plasmid Fiber-seq) to accurately map chromatin architectures along individual, full-length transfected plasmid molecules at near-single-nucleotide resolution. Application of this method to diverse plasmids and cell lines demonstrates that plasmids are chromatinized in an organized, sequence-dependent manner and adopt a heterogeneous and incomplete chromatin architecture relative to nuclear-encoded chromatin fibers. Focal occupancy of nucleosomes and transcription factors along transfected plasmids is central to their transcriptional activity, and plasmids can recapitulate nuclear genome-encoded chromatin architectures with varying fidelity. Combining plasmid Fiber-seq with high-throughput reporter assays reveals the molecular mechanisms underlying pathogenic non-coding variants, which disentangles the effects of transcriptional activators and repressors with near-single-nucleotide resolution. Our findings reveal principles for accurate, fine-scale mapping of chromatin-dependent regulatory grammar.
Duré C, Ghoshdastider U, Weber R
… +12 more, Khandekar A, Valdivia-Francia F, Renz PF, Sella F, Hyams K, Taborsky D, Yigit M, Ormiston M, Yamahachi H, Levesque M, Ellis SJ, Sendoel A
Somatic stem cells are characterized by their low overall protein-synthesis rates, a feature implicated in driving their stemness. However, how aging reshapes the translational landscape of stem cells remains poorly unde...Somatic stem cells are characterized by their low overall protein-synthesis rates, a feature implicated in driving their stemness. However, how aging reshapes the translational landscape of stem cells remains poorly understood. Here, we present an in vivo single-cell ribosome profiling strategy to monitor tissue-wide translational landscapes of the epidermis during aging. By implementing ribosomal elongation-inhibited cell isolation and switching to RNase I, we expand the applicability of single-cell ribosome profiling to in vivo systems and facilitate the evaluation of triplet periodicity, a hallmark of high-quality data. Leveraging this strategy, we document the in vivo translational landscapes of the major epidermal cell types, outline cell-type-specific translational efficiencies, and identify a pronounced translational reprogramming of AP-1 subunits specifically in aged epidermal stem cells. Our study illustrates the power of in vivo single-cell ribosome profiling to map cell-type-specific translational programs and offers a scalable strategy for tissue-wide interrogation of translational landscapes.
Targeting ferroptosis, a form of regulated cell death, holds potential for improving treatment efficacy in a range of cancers including hepatocellular carcinoma (HCC). The selenoprotein glutathione peroxidase 4 (GPX4) pl...Targeting ferroptosis, a form of regulated cell death, holds potential for improving treatment efficacy in a range of cancers including hepatocellular carcinoma (HCC). The selenoprotein glutathione peroxidase 4 (GPX4) plays a crucial role in suppressing ferroptosis by converting toxic phospholipid hydroperoxides into non-toxic lipid alcohols, yet the mechanisms regulating its synthesis remain poorly understood. This study identifies SHIELD (suppressor of hypoxia-induced lipid peroxidation and death), a long noncoding RNA (lncRNA) and direct HIF-1α target, as a regulator of ferroptosis in HCC. SHIELD inhibits ferroptosis by interacting with the RNA-binding protein GRSF1, which forms a ternary complex with GPX4 5'-UTR to enhance GPX4 mRNA translation and expression. Furthermore, targeting SHIELD with antisense oligonucleotides (ASOs) in combination with sorafenib significantly reduced tumor growth in patient-derived xenograft models. This discovery paves the way for improving the efficacy of tyrosine kinase inhibitors in HCC, which addresses the challenge of therapeutic resistance.
Redox signaling by nitric oxide (NO) is estimated to control a large part of the global proteome via S-nitrosylation (SNO-modification). Here, we report that RNA-binding proteins (RBPs) represent the most significantly e...Redox signaling by nitric oxide (NO) is estimated to control a large part of the global proteome via S-nitrosylation (SNO-modification). Here, we report that RNA-binding proteins (RBPs) represent the most significantly enriched class of S-nitrosylation targets, with broad coverage of spliceosomal factors. We demonstrate that NO regulates alternative splicing (AS) and that S-nitrosylation of PTBP1, a central regulator of AS, can massively shift and contextually alter gene expression while further enriching the transcriptome for SNO sites. PTBP1 S-nitrosylation changes RNA-binding domain conformation, RNA motif recognition, protein-RNA and protein-protein interactions, and intracellular trafficking to impact pathways for viral infection and neurodegeneration. Levels of SNO-PTBP1 are reduced in mouse and human Alzheimer's disease brains and correlate with adverse clinical outcomes. Overall, SNO-RBPs are characterized by conservation across diverse lineages and SNO sites and provide a blueprint for redox regulation of both transcriptome and proteome in physiology and disease.
Chen Y, Cheng CP, Cates K
… +15 more, Marinov GK, Lantz TC, Yang H, Liu I, Genuth NR, Andronescu C, Hung V, Bermudez A, Rothschild D, Georgeson J, Barocio SB, Kundaje A, Pitteri S, Ruggero D, Barna M
The ribosome is a ribozyme, but it also acts as a dynamic regulator of gene expression. Although ribosomal protein (RP) composition varies, dissecting the functional contributions of individual RPs beyond their housekeep...The ribosome is a ribozyme, but it also acts as a dynamic regulator of gene expression. Although ribosomal protein (RP) composition varies, dissecting the functional contributions of individual RPs beyond their housekeeping roles is challenging because of the lack of tools for manipulation in situ. Here, we developed Ribo-Tweezer, a degron-based system directly tethered to mature ribosomes that enables rapid, reversible, and selective depletion of specific RPs. Using Ribo-Tweezer in mouse embryonic stem cells (mESC), we find a previously uncharacterized role for RACK1 in stem cell fate control via translational regulation of zinc-finger transcriptional networks and long interspersed nuclear element-1 (LINE1) expression. This translation-transcription coupling provides a mechanism by which translation control is further amplified in gene regulation. Distinct translational programs induced by RPLP0 and RPLP1 depletion further demonstrate RP-specific regulatory functions in translation. Together, these findings establish Ribo-Tweezer as a powerful platform that has illuminated selective functions for RPs in gene regulation, which gives biological meaning to ribosome heterogeneity.
Liu Y, Yao Z, Zhang Y
… +16 more, Zhu Z, Wang Z, Chen Q, Yang Z, He Y, Chen X, Tian L, Du J, Wu J, Kim HJ, Huang J, Zhang Y, Ma W, Fan W, Taylor JP, Yang P
It has long been recognized that the intracellular replication of alphaviruses critically relies on several key host RNA-binding proteins (RBPs), including G3BP1/2 and FXR1/FXR2/FMR1. But how these RBPs modulate alphavir...It has long been recognized that the intracellular replication of alphaviruses critically relies on several key host RNA-binding proteins (RBPs), including G3BP1/2 and FXR1/FXR2/FMR1. But how these RBPs modulate alphaviral replication, and whether it would be possible to target them for antiviral treatment, is less explored. Here, using Semliki Forest virus (SFV) as a model, we report that SFV non-structural protein 3 (nsP3) exploits G3BP to drive its condensation and transforms antiviral stress granules into proviral nsP3-G3BP co-condensates. The gel-like co-condensates enrich and protect viral genomic RNAs from host RNase degradation and promote viral translation and replication. nsP3-RBP co-condensation is widespread among alphaviruses, and condensate disruption is a plausible antiviral approach. Thus, these findings uncover a general anti-alphavirus strategy based on the conserved reliance of virus-host protein co-condensation.
In a recent Nature article, Tou et al. introduce INSTALL, a strategy that couples immune-evasive circular single-stranded DNA (cssDNA) technology with recombinase-based genome editing to overcome innate immune barriers a...In a recent Nature article, Tou et al. introduce INSTALL, a strategy that couples immune-evasive circular single-stranded DNA (cssDNA) technology with recombinase-based genome editing to overcome innate immune barriers and enable the integration of large DNA cargos.
In a recent issue of Cell, Zuo et al. reveal a STAT1 modification by pyruvate, reducing type I interferon signaling. This study provides a possible explanation for why hyperglycemia in diabetes can impair anti-viral immu...In a recent issue of Cell, Zuo et al. reveal a STAT1 modification by pyruvate, reducing type I interferon signaling. This study provides a possible explanation for why hyperglycemia in diabetes can impair anti-viral immunity and indicates a new way to boost anti-viral innate immunity.