In this issue of Molecular Cell, Tian et al. identify LENG8 as a conserved modular factor that enforces nuclear RNA quality control by coupling 5' splice site recognition to transcript retention and exosome-mediated degr...In this issue of Molecular Cell, Tian et al. identify LENG8 as a conserved modular factor that enforces nuclear RNA quality control by coupling 5' splice site recognition to transcript retention and exosome-mediated degradation.
Polycomb group (PcG) and Trithorax group (TrxG) proteins establish bivalent chromatin marked by H3K27me3, H2AK119ub1, and H3K4me3. However, how bivalent chromatin is formed in vivo in mammals is poorly understood. In mou...Polycomb group (PcG) and Trithorax group (TrxG) proteins establish bivalent chromatin marked by H3K27me3, H2AK119ub1, and H3K4me3. However, how bivalent chromatin is formed in vivo in mammals is poorly understood. In mouse oocytes, it arises at thousands of promoters, including noncanonical imprinted loci whose H3K27me3 is intergenerationally inherited by early embryos. Here, we show that H3K27me3 is deposited at H3K4me3-premarked promoters in an H2AK119ub1-dependent manner during oogenesis. We find that H2AK119ub1 deficiency causes transcriptional derepression and loss of H3K27me3 proportional to preexisting H3K4me3 levels in oocytes. Importantly, concomitant deficiency of H2AK119ub1 and MLL2-mediated H3K4me3 substantially restores transcriptional silencing and H3K27me3 deposition, leading to partial restoration of noncanonical imprinting in offspring. Taken together, we propose that H2AK119ub1 antagonizes MLL2 function to repress bivalent genes during oogenesis, thereby conferring heritable H3K27me3. This study reveals how PcG and TrxG counteraction shapes the maternal epigenome for the next generation's development.
Losa J, Simon F, Linnik D
… +16 more, Kaya SG, Stuart MCA, Stetsenko A, de Boer R, Ho FY, Incarnato D, Stevens J, van Eck J, Fraaije MW, Weiss LE, van Teeffelen S, Abeln S, Guskov A, Marrink SJ, Poolman B, Heinemann M
Molecular crowding in the bacterial cytoplasm restricts diffusion of large molecules, impacting cellular processes. To monitor cytoplasmic diffusion and rheology, we used single-particle tracking in Escherichia coli, fin...Molecular crowding in the bacterial cytoplasm restricts diffusion of large molecules, impacting cellular processes. To monitor cytoplasmic diffusion and rheology, we used single-particle tracking in Escherichia coli, finding a 3-fold variation in the diffusion of a 40-nm particle across exponential growth conditions. Known determinants of rheology did not account for this variation. Instead, we found a strong anticorrelation between the diffusion coefficient and the abundance of amino acid metabolism proteins (clusters of orthologous groups [COG] category "E"), persisting upon genetic perturbations, and that lower diffusion is associated with increased elasticity. Photoactivated light microscopy revealed that some amino acid metabolism proteins form clusters. Electron microscopy showed that these proteins can form amorphous agglomerates at physiological concentrations in vitro due to their high hydropathy, which also confers low disorder and compactness. These findings show that diffusion is controlled by the formation of protein agglomerates and thus reveal how condition-induced proteome changes affect cytoplasmic rheology.
Rapid cancer cell proliferation requires extensive macromolecular biosynthesis, yet how distinct anabolic pathways are coordinated remains incompletely understood. Here, we report that the trifunctional carbamoyl-phospha...Rapid cancer cell proliferation requires extensive macromolecular biosynthesis, yet how distinct anabolic pathways are coordinated remains incompletely understood. Here, we report that the trifunctional carbamoyl-phosphate synthase, aspartate transcarbamoylase, and dihydroorotase (CAD) activates key glycolytic enzymes to support biosynthesis and cancer cell proliferation. When cancer proteomics datasets were queried, a CAD activation signature was identified in diverse tumors. Metabolomics analysis revealed that CAD fuels central carbon metabolism, specifically the pentose phosphate pathway (PPP) and serine synthesis pathway (SSP). Mechanistically, CAD deamidates and activates glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate dehydrogenase (PHGDH), rate-limiting enzymes of the PPP and SSP, respectively, which are fully recapitulated by the glutaminase domain of CAD. Functional interrogation of cancer-associated CAD mutations and human hepatocellular carcinoma tumors predicts the metabolic signature endowed by G6PD and PHGDH deamidation. Simultaneous inhibition of G6PD and PHGDH effectively impeded tumor formation. This work identifies CAD as a central carbon metabolism signaling node and a potential therapeutic target.
Nössing C, Troitskaya O, Long JS
… +14 more, Craxton A, Brucoli M, Anderson E, Lilla S, McEwan DG, Minton AR, Barthet VJA, O'Prey J, Thomson G, Nixon C, Zanivan S, Strathdee D, MacFarlane M, Ryan KM
Inflammation can affect many diseases. We report here that inflammatory cytokines invoke caspase-8-mediated cleavage of the autophagy adaptor p62/SQSTM1 at aspartic acid 329 in human cells, producing a previously describ...Inflammation can affect many diseases. We report here that inflammatory cytokines invoke caspase-8-mediated cleavage of the autophagy adaptor p62/SQSTM1 at aspartic acid 329 in human cells, producing a previously described truncated form, which we term tr-p62. We show that TNF-driven cell death is tr-p62 dependent and that autophagy inhibition promotes death via tr-p62 accumulation. Mechanistically, p62 cleavage is receptor-interacting serine/threonine-protein kinase 1 (RIPK1) dependent, and tr-p62 stabilizes caspase-8 activating complex-IIb. tr-p62-driven cell death downstream of TNF is also RIPK1 and caspase dependent, promoting feedforward caspase-8 activation. p62 cleavage does not, however, affect necroptosis. Surprisingly, this caspase-8 cleavage site in p62 is absent in mice, and introduction of cleavable forms of p62 into mouse cells causes sensitization to TNF-induced death. Moreover, mice with CRISPR-Cas9-generated cleavable p62 exhibit TNF hypersensitivity and intestinal inflammation in vivo. These findings provide significant insights into TNF-induced cell death and introduce a mouse model that may provide better clarity for human-related studies of inflammatory disease.
Eukaryotic 28S rRNA contains expansion segments (ESs) that increase dramatically in length during evolution, yet their functions remain elusive. The nucleolus, where rRNA is transcribed, exhibits divergent architectures:...Eukaryotic 28S rRNA contains expansion segments (ESs) that increase dramatically in length during evolution, yet their functions remain elusive. The nucleolus, where rRNA is transcribed, exhibits divergent architectures: three-layered (tripartite) in amniotes but two-layered (bipartite) in other eukaryotes. Here, we identified distinct rRNA localization patterns within nucleolar compartments. Through in vitro reconstitution and simulations, we demonstrated that 28S rRNA induces layered nucleolar-like structures via multivalent RNA-RNA interactions. This structure-forming capacity correlates with the evolutionary expansion of ESs. 28S rRNAs from tripartite-nucleolus organisms possess longer ESs and exhibit enhanced multivalency compared with those from bipartite-nucleolus species. Deletion of specific ESs abolishes human 28S rRNA's ability to induce nucleolar-like structures, whereas transferring these segments to C. elegans 26S rRNA confers this capability in vitro. We conclude that rRNA ESs function as architectural elements that promote nucleolar complexity, providing mechanistic insight into how genomic expansion drives sophisticated cellular organization through enhanced intermolecular interactions.
A major source of poly(ADP-ribose) polymerase (PARP) activity in proliferating cells is unligated Okazaki fragments. Consequently, the anti-cancer PARP inhibitor olaparib impedes the maturation of nascent DNA strand frag...A major source of poly(ADP-ribose) polymerase (PARP) activity in proliferating cells is unligated Okazaki fragments. Consequently, the anti-cancer PARP inhibitor olaparib impedes the maturation of nascent DNA strand fragments during DNA replication. Here, we show that wild-type human cells overcome this impediment by triggering a process that facilitates nascent strand maturation in the presence of olaparib. We show that this process operates on very large nascent strand fragments and repairs thousands of olaparib-induced DNA single-strand breaks/gaps per genome. Critically, this process is dependent on the tumor suppressors BRCA1 and BRCA2 and is associated with the BRCA2-dependent accumulation of RAD51 recombinase in chromatin. Our data identify nascent strand gaps that are induced by olaparib independently of replication fork reversal and/or PRIMPOL-mediated repriming and that are repaired by a BRCA2-dependent process that we propose is daughter-strand gap protection and/or repair occurring hundreds of kilobases behind DNA replication forks.
The DDB1- and CUL4-associated factor (DCAF) family functions as substrate receptors within Cullin4-really interesting new gene (RING) ubiquitin ligases (CRL4s), facilitating proteasomal degradation of targeted substrates...The DDB1- and CUL4-associated factor (DCAF) family functions as substrate receptors within Cullin4-really interesting new gene (RING) ubiquitin ligases (CRL4s), facilitating proteasomal degradation of targeted substrates. Although CRL4-based targeted protein degradation (TPD) has emerged as a promising strategy to modulate undruggable proteins, the complex formation, substrates, and functional properties of many DCAFs remain poorly defined. In this study, using proximity biotinylation-based interactome analysis in human HEK293T cells, we systematically annotated interactors and functional associations of individual DCAFs. Furthermore, we identified substrates of the model DCAFs COP1 and DCAF3 using proximity biotinylation coupled with multi-omics approaches. By combining biochemical and cell-based analyses, we establish CRL4 complex formation and DCAF autodegradation as experimentally tractable proxy indicators to evaluate DCAF degradation activity and propose a set of high-activity DCAFs. These datasets establish a resource for functional characterization of DCAFs and provide a framework for their prioritization in TPD.
ER-phagy involves the selective autophagosomal engulfment of ER fragments, but the signaling events, selection mechanisms, and membrane source of ER-phagic autophagosomes remain elusive. Here, using state-of-the-art supe...ER-phagy involves the selective autophagosomal engulfment of ER fragments, but the signaling events, selection mechanisms, and membrane source of ER-phagic autophagosomes remain elusive. Here, using state-of-the-art super-resolution multi-SIM imaging, we reveal that stresses (prolonged starvation, cholesterol dyshomeostasis, and high-Ca insults) trigger the expansion of sheet ER subdomains containing high levels of luminal Ca in mammalian cells, which are subsequently degraded by ER-phagy. Autophagosome formation and sequestration of ER sheets require the concerted actions of FAM134B and lipidated LC3, whereas the autophagy proteins ATG14 and ATG9 are partially dispensable. Electron microscopy and cryo-electron tomography show that the membranes of autophagosomes enclosing high-Ca-containing ER sheets are directly remodeled from the ER. The ER-localized cation channels PIEZO1 and TRPV1 are enriched at and mediate Ca transients from high-Ca-containing ER sheets, triggering liquid-liquid phase separation of the autophagosome-initiating FIP200 complex to initiate ER-phagy. Thus, distinct mechanisms are employed for the formation of high-Ca-containing ER-enclosing autophagosomes and non-selective autophagosomes.
The formation of biomolecular condensates, such as Yb bodies in Drosophila ovarian somatic cells, is significantly contributed to by phase separation of scaffold proteins. Client proteins transiently accumulate via inter...The formation of biomolecular condensates, such as Yb bodies in Drosophila ovarian somatic cells, is significantly contributed to by phase separation of scaffold proteins. Client proteins transiently accumulate via interactions with these scaffolds; however, how client flux is regulated remains unclear. Here, we investigate Shutdown, a client protein of Yb bodies-the site of Piwi-piRNA-induced silencing complex (piRISC) precursor (pre-Piwi-piRISC) formation-and show that cytosolic Shutdown connects Armitage and Piwi to promote Piwi deposition into Yb bodies before quickly returning to the cytosol. This return allows Armitage to transfer pre-Piwi-piRISC to mitochondria for maturation. Shutdown's acidic N terminus mediates self-repulsion, conferring its transient residence within Yb bodies. A point mutation in armitage, analogous to a human armitage/MOV10L1 mutation associated with azoospermia, traps Shutdown in Yb bodies, blocking Piwi-piRISC generation. This study reveals the mechanism underlying Shutdown's transient localization in Yb bodies and its essential role in Piwi-piRISC biogenesis and fertility.
As animals evolved from external to internal fertilization, sperm flagella, once transiently propelling sperm in water to reach nearby eggs, developed to beat for days in the viscous female reproductive tract. How flagel...As animals evolved from external to internal fertilization, sperm flagella, once transiently propelling sperm in water to reach nearby eggs, developed to beat for days in the viscous female reproductive tract. How flagella are remodeled accordingly remains unclear. Unlike externally fertilizing zebrafish and sea urchins, mammalian flagella feature a barrel between radial spokes (RSs) RS1 and RS2. Here, we show that this RS1-RS2 barrel (RRB) is a unique T-complex protein-1 ring complex (TRiC) that folds locally translated polypeptides to sustain flagellar motility. Cryo-electron microscopy (cryo-EM) reveals a flagellum-specific TRiC structure. An in situ cryo-electron tomography (cryo-ET) map of flagellar axonemes captures the RRB TRiC in an active, substrate-receptive state, with additional densities suggestive of folding substrates and cofactors. Mammalian flagella contain components of translation machineries and locally synthesize proteins. Cross-linking mass spectrometry identifies candidate locally translated axonemal proteins and folding substrates. Furthermore, a TRiC ATPase inhibitor markedly represses mouse sperm motility. Our findings provide insights into flagellar remodeling in internally fertilizing species.
Garcia GR, Palangat M, Moraly J
… +13 more, Donovan BT, Wang B, Phadke I, Sturgill D, Chen J, Zhang G, Holewinski RJ, Short B, Rivero GA, Swoboda DM, Taylor N, McGraw K, Larson DR
The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA-binding proteins U2AF1 and U2AF2 form a heterodimer (U2AF) that shuttles b...The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA-binding proteins U2AF1 and U2AF2 form a heterodimer (U2AF) that shuttles between the nucleus and cytoplasm, regulating splicing in the nucleus and translation in the cytoplasm. Our study in human bronchial epithelial cells (HBECs) identifies an unexpected role for U2AF in mitochondrial function. We demonstrate that U2AF interacts with nuclear-encoded mitochondrial (NE-mt) mRNAs and proteins, inhibits translation, localizes to the mitochondria, and regulates mRNA localization to mitochondria. Moreover, an oncogenic point mutation in U2AF1(S34F) disrupts this regulation, leading to altered mitochondrial structure, increased translation, large changes in the mitochondria proteome, and oxidative phosphorylation (OXPHOS)-dependent metabolic rewiring, recapitulating changes observed in bone marrow progenitors from patients with myelodysplastic syndromes. These findings reveal a non-canonical role for U2AF, where it modulates multiple aspects of mitochondrial function by regulating the translation and mitochondrial localization of nuclear-encoded mRNAs.
Ferroptosis is an iron-dependent form of cell death converging on lipid peroxidation first identified by examining compounds with enhanced lethality to KRAS mutant cells. Despite over 90% of pancreatic ductal adenocarcin...Ferroptosis is an iron-dependent form of cell death converging on lipid peroxidation first identified by examining compounds with enhanced lethality to KRAS mutant cells. Despite over 90% of pancreatic ductal adenocarcinoma (PDAC) tumors harboring KRAS mutations, PDAC exhibits relative resistance to ferroptosis compared with other tumor types, and the mechanisms behind this resistance remain unclear. Here, we report that exposure to pancreatic tumor interstitial fluid in synergy with hypoxia induced robust protection against ferroptosis in a manner dependent on the hypoxia-inducible transcription factor 2 (HIF-2). HIF-2 upregulates the expression of both components of the system Xc cystine transporter and transsulfuration pathway enzymes CBS and CTH to increase intracellular cysteine levels, enabling anti-ferroptotic glutathione production. HIF-2 also induces the Parkin mitophagy factor and suppresses mitochondrial function and reactive oxygen species (ROS) generation. Altogether, our findings uncover an unforeseen role of the HIF-2 transcription factor as a coordinator of anti-ferroptotic mechanisms in pancreatic cancer.
In a recent article in Nature, Zabala-Letona et al. propose that polyamines-small, positively charged metabolites-regulate RNA splicing by electrostatically shielding acidic sites on spliceosome proteins from kinase-medi...In a recent article in Nature, Zabala-Letona et al. propose that polyamines-small, positively charged metabolites-regulate RNA splicing by electrostatically shielding acidic sites on spliceosome proteins from kinase-mediated phosphorylation. This work adds to a growing body of evidence suggesting that metabolites can regulate cell biology through non-specific macromolecular interactions.
In this issue of Molecular Cell, Garcia et al. reveal an unexpected role for the splicing factor U2AF in repressing translation and influencing the localization of nuclear-encoded mitochondrial mRNAs to the outer mitocho...In this issue of Molecular Cell, Garcia et al. reveal an unexpected role for the splicing factor U2AF in repressing translation and influencing the localization of nuclear-encoded mitochondrial mRNAs to the outer mitochondrial membrane.
During hCMV infection, the highly abundant viral lncRNA RNA2.7 reshapes host gene expression by sequestering RNA-binding proteins, stabilizing host mRNAs, and enforcing G1 arrest. In this issue, Fisher et al. explore how...During hCMV infection, the highly abundant viral lncRNA RNA2.7 reshapes host gene expression by sequestering RNA-binding proteins, stabilizing host mRNAs, and enforcing G1 arrest. In this issue, Fisher et al. explore how this RNA-driven strategy optimizes the cellular environment for viral replication.
In eukaryotic ribosomes, ∼2% of RNA nucleotides undergo 2'-O-methylation, a conserved cellular mechanism thought to be critical for maintaining and regulating translation. Here, we use ribosome profiling, translation ass...In eukaryotic ribosomes, ∼2% of RNA nucleotides undergo 2'-O-methylation, a conserved cellular mechanism thought to be critical for maintaining and regulating translation. Here, we use ribosome profiling, translation assays, proteomics, and high-resolution structural analyses to show that loss of native 2'-O-methylation in yeast ribosomes disproportionally affects the translation of ribosomal protein transcripts, driven by changes in codon usage and recognition of structured RNA. Translation reprogramming by the hypomethylated ribosomes is supported by reduced thermostability and high-resolution evidence of altered ribosomal structures and conformations. Consistent with the roles of the affected proteins in sustaining cellular fitness, hypomethylated ribosomes under stress exhibit a selective loss of downregulated proteins and misassembly associated with methylation loss. Our data provide structural and mechanistic insights into how 2'-O-methylation supports ribosome integrity and mediates cellular stress responses.
The SPFH (stomatin, prohibitin, flotillin, and HflK/C) family proteins are proposed scaffolds for organizing functional membrane microdomains (FMMs) on various cellular membranes. Erlin1 and Erlin2, two endoplasmic retic...The SPFH (stomatin, prohibitin, flotillin, and HflK/C) family proteins are proposed scaffolds for organizing functional membrane microdomains (FMMs) on various cellular membranes. Erlin1 and Erlin2, two endoplasmic reticulum (ER)-residing SPFH members, as heteromeric complexes, participate in ER-associated protein degradation (ERAD). However, the mechanisms underlying Erlin-mediated FMM organization and ERAD regulation remain poorly understood. Here, through cryoelectron microscopy (cryo-EM), we find that the human Erlin1/2 complex forms a 26-mer cage assembly, defining a nanometer-sized microdomain on the luminal leaflet. The intramembrane region of each subunit constitutes a specific phosphatidylinositol-binding pocket. ER proteins can be recruited to both the interior and exterior of these cages. By caging cargoes, the Erlin1/2 complex physically secludes them from their substrates or binding partners, conferring another layer of regulation on their functions. Moreover, individual cages can cluster to organize FMMs of different sizes. These dynamic properties underscore a general regulatory role of Erlin1/2 in various ER-related biological processes, including coronaviral replication.
During infection with human cytomegalovirus (HCMV), the viral long non-coding RNA RNA2.7 becomes the most abundant polyadenylated transcript in the cell, yet its function has remained enigmatic. By combining RNA sequenci...During infection with human cytomegalovirus (HCMV), the viral long non-coding RNA RNA2.7 becomes the most abundant polyadenylated transcript in the cell, yet its function has remained enigmatic. By combining RNA sequencing, metabolic labeling of newly synthesized RNA, and ribosome profiling, we define how RNA2.7 modulates host gene expression and promotes viral propagation. We show that RNA2.7 stabilizes numerous host mRNAs by sequestering a broad array of RNA-binding proteins, reshaping the cellular transcriptome. Accordingly, RNA2.7 is essential for HCMV-induced cell-cycle arrest at the G1-S transition specifically when infection occurs in G1, thereby enhancing viral replication in actively cycling cells. Notably, RNA2.7 expression alone is sufficient to block cell-cycle progression, and screening RNA2.7 fragments identifies a region containing an extended polyadenosine stretch that is required for this activity. Our findings reveal how RNA2.7 promotes viral replication by modulating host mRNA stability and enforcing cell-cycle arrest, creating favorable conditions for infection.
Excessive RNA damage activates cellular stress responses, triggering cell death. However, pathways that negatively regulate RNA damage responses are largely uncharacterized. Using genetic screens, we find that the ubiqui...Excessive RNA damage activates cellular stress responses, triggering cell death. However, pathways that negatively regulate RNA damage responses are largely uncharacterized. Using genetic screens, we find that the ubiquitin ligase RNF25 provides tolerance to RNA damage caused by the nucleoside analogue azacytidine, a chemotherapeutic agent used to treat acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Mechanistically, we show that azacytidine is incorporated into mRNA, where it causes lesions that stall elongating ribosomes, leading to cytotoxic activation of the GCN2-dependent integrated stress response (ISR). Furthermore, we establish that RNF25 prevents ISR hyperactivation by ubiquitylation of ribosomal protein eS31, thereby suppressing cell death upon azacytidine treatment. Our study reveals an mRNA damage tolerance mechanism that determines cellular survival in response to azacytidine, highlighting RNA damage-induced stress response as a potentially critical component of chemosensitivity in AML and MDS.