LGP2, a RIG-I-like receptor, plays a crucial role in antiviral immunity by enhancing MDA5 activity against specific viral infections. Here, using biochemical assays, cryo-electron microscopy (cryo-EM), and high-speed ato...LGP2, a RIG-I-like receptor, plays a crucial role in antiviral immunity by enhancing MDA5 activity against specific viral infections. Here, using biochemical assays, cryo-electron microscopy (cryo-EM), and high-speed atomic force microscopy, we reveal that LGP2 initially binds to the ends of double-stranded RNA (dsRNA) and subsequently translocates along the RNA via ATP hydrolysis. Our cryo-EM structure demonstrates that LGP2 forms filament-like assemblies with MDA5 along the internal region of dsRNA, promoting MDA5 filament nucleation. Additionally, LGP2 and MDA5 form short RNA filaments, which are further cross-bridged via caspase activation and recruitment domain (CARD)-CARD interactions, leading to the formation of filament microclusters. These microclusters, in turn, stimulate mitochondrial antiviral signaling (MAVS) filament formation. Our findings uncover a dynamic interplay between LGP2 and MDA5, revealing a previously unrecognized mechanism that enhances antiviral immune responses.
Natural proteins often form intricate multidomain, oligomeric architectures. This presents a prima facie challenge to cellular homeostasis, as topologically complex proteins seldom refold efficiently in vitro. Here, we s...Natural proteins often form intricate multidomain, oligomeric architectures. This presents a prima facie challenge to cellular homeostasis, as topologically complex proteins seldom refold efficiently in vitro. Here, we show that the efficient folding and assembly of the five-domain homotetramer β-galactosidase is obligatorily coupled to its synthesis on the ribosome, and we define the underlying mechanisms. During refolding from a denaturant, maturation of the catalytic domain is frustrated. Assembly outpaces monomer folding, and non-native oligomers accumulate. Efficient de novo folding is characterized by segmental domain folding, shaped by the binding of a nascent amphipathic helix to a cryptic pocket on uL23 on the ribosome surface. Homomer assembly also initiates cotranslationally via recruitment of a full-length subunit to the nascent polypeptide, and the failure to do so results in misassembly. Our results reveal how the ribosome can dictate the timing of folding and assembly to enable efficient biogenesis of a topologically complex protein.
Genome regulation is shaped not only by DNA sequence but also by epigenetic mechanisms that influence chromatin structure and gene expression. While epigenetics has classically focused on heritable DNA and histone modifi...Genome regulation is shaped not only by DNA sequence but also by epigenetic mechanisms that influence chromatin structure and gene expression. While epigenetics has classically focused on heritable DNA and histone modifications, growing evidence indicates that certain forms of DNA damage can also generate persistent changes in transcriptional states that are heritable in some scenarios. This review examines how diverse DNA damage-associated processes-including oxidative lesions, R-loops, telomeric damage, DNA double-strand breaks, and poly-ADP-ribosylation-intersect with the epigenome. We highlight the roles of oxidative DNA damage and repair in transcriptional regulation, the contribution of R-loops to gene expression and DNA methylation dynamics, and the impact of telomere-associated damage on chromatin organization and genome maintenance. DNA lesions, and in some cases DNA repair-associated proteins, can thus leave epigenetic "scars" that influence cellular identity, aging, and disease, expanding current views of epigenetic inheritance and genome stability.
Protein mutational landscapes are shaped by how amino acid substitutions affect stability and folding or aggregation kinetics. These properties are modulated by cellular proteostasis networks. Heat shock factor 1 (HSF1)...Protein mutational landscapes are shaped by how amino acid substitutions affect stability and folding or aggregation kinetics. These properties are modulated by cellular proteostasis networks. Heat shock factor 1 (HSF1) is the master regulator of cytosolic and nuclear proteostasis. Chronic HSF1 activity upregulation is a hallmark of cancer cells, potentially because upregulated proteostasis factors facilitate the acquisition and maintenance of oncogenic mutations. Here, we assess how HSF1 activation influences mutational trajectories by which p53 can escape cytotoxic pressure from nutlin-3, an inhibitor of the p53 regulator mouse double minute 2 homolog (MDM2). HSF1 activation broadly increases the fitness of dominant-negative p53 substitutions, particularly non-conservative, biophysically unfavorable amino acid changes within buried regions of the p53 DNA-binding domain. These findings demonstrate that HSF1 activation reshapes the oncogenic mutational landscape by preferentially supporting the emergence and persistence of biophysically disruptive, cancer-associated p53 substitutions, linking proteostasis network activity directly to oncogenic evolution.
Wang J, Minussi DC, Davis A
… +18 more, Wei R, Ye H, Sei E, Schalck A, Yan Y, Wu HJ, Bai S, Peng C, Hu M, Casasent A, Contreras A, Chen H, Hui D, Damodaran S, Edgerton ME, Kopetz S, Lim B, Navin N
Tumors are composed of a myriad of subclones that bulk DNA sequencing (DNA-seq) methods cannot accurately resolve. Single-cell DNA-seq methods were developed to address this issue, yet their data analysis remains challen...Tumors are composed of a myriad of subclones that bulk DNA sequencing (DNA-seq) methods cannot accurately resolve. Single-cell DNA-seq methods were developed to address this issue, yet their data analysis remains challenging. Here, we present CopyKit, a comprehensive tool for single-cell DNA copy-number analysis to resolve clonal substructure and reconstruct genetic lineages. Additionally, we introduce "scquantum" to estimate the integer copy-number states of single cells. We performed single-cell DNA-seq of 11,845 cells from one primary breast tumor, two liver metastases, and three primary tumors with matched metastatic tissues. These data identified the subclones from the primary tumors that seeded the metastatic lesions and their associated copy-number events. The data also provided evidence of both subclonal intermixing and spatial segregation in different regions of the liver metastasis. These applications show that CopyKit is a powerful approach for the analysis of high-throughput single-cell copy-number data.
Otobe Y, Deki-Arima N, Xinyan S
… +12 more, Itabashi K, Kurabayashi N, Nakamura U, Uchida A, Shimazaki R, Yamamoto K, Sakurai T, Fu YH, Ptáček LJ, Hirano A, Doi M, Yoshitane H
The circadian clock drives daily rhythms of gene expression and physiology. Advances in next-generation DNA sequencing have provided extensive insights into RNA expression, but more functional information at the protein...The circadian clock drives daily rhythms of gene expression and physiology. Advances in next-generation DNA sequencing have provided extensive insights into RNA expression, but more functional information at the protein level with sufficient depth has been limited by technical challenges. In this study, we generated a comprehensive mouse circadian proteome atlas (https://chronoproteinology.org/circadian_atlas) by analyzing 32 tissues, including the suprachiasmatic nucleus (SCN), using the next-generation mass spectrometer Orbitrap Astral. Data-independent acquisition of 584 samples, including developmental samples, revealed the spatiotemporal profiles of about 19,000 proteins. Proteome and phospho-proteome analyses of whole-cell and nuclear proteins in the liver revealed circadian changes in protein quantity and quality, as well as global changes in hPER2-S662G mutant mice, a genetic model of human familial advanced sleep phase (FASP). This multi-tissue circadian proteome atlas provides a fundamental resource for understanding when, where, and which proteins are expressed and function.
Gamble A, Ward TA, Wheeler OPG
… +18 more, Morris JP, Jones CM, Bennett LG, Vernon EG, Thanendran V, Ceppi I, Halder S, Borrello D, Walker TDJ, Rajan J, Suleiman H, Harrison D, Alshetiwi M, Dunphy G, Jackson-Jones LH, Cejka P, Unterholzner L, Staples CJ
Replication stress is a key driver of DNA damage and genome instability. Here, we report that replication stress induces an inflammatory response in the absence of DNA damage. The DNA-sensing factor interferon-γ-inducibl...Replication stress is a key driver of DNA damage and genome instability. Here, we report that replication stress induces an inflammatory response in the absence of DNA damage. The DNA-sensing factor interferon-γ-inducible factor 16 (IFI16) binds nascent DNA at stalled replication forks and signals via the adaptor stimulator of interferon genes (STING) to induce activation of nuclear factor κB (NF-κB) and the production of pro-inflammatory cytokines, independently of the cytosolic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS). Replication stress-induced fork remodeling generates a new DNA end that is vulnerable to degradation by nucleases and is protected by a range of factors, including the tumor suppressors BRCA1 and BRCA2. IFI16 acts directly at stalled replication forks to protect nascent DNA from degradation by the nucleases MRE11, EXO1, and DNA2. Furthermore, IFI16 is required for the interferon-mediated rescue of fork protection in BRCA-deficient cells, highlighting the critical role of IFI16 in the crosstalk between innate immunity and fork protection during replication stress.
Effective adaptive immunity requires generation of a diverse repertoire of antigen receptors via V(D)J recombination. To illuminate the underlying mechanisms, we combined biophysical simulations with experimental data to...Effective adaptive immunity requires generation of a diverse repertoire of antigen receptors via V(D)J recombination. To illuminate the underlying mechanisms, we combined biophysical simulations with experimental data to model chromatin folding and dynamics of the mouse immunoglobulin heavy chain gene (Igh) locus. Simulations that best recapitulated experimental data on locus structure and recombination of Igh alleles identified three novel chromatin folding principles. First, we found that prominent structural features of the Igh locus, such as the 3'-anchored stripe, required cohesin loading throughout the locus. Second, the Eμ enhancer was best modeled as a bi-directional loop extrusion blocker, though it does not bind CTCF. Third, we found that utilization of V genes to obtain maximum diversity required both widespread cohesin loading as well as long-range associations between H3K27ac-marked regions. Our findings provide a conceptual framework to understand chromatin folding principles that enable antibody diversity and reveal mechanisms of long-range genome communication.
Double-stranded (ds)RNA viruses replicate and transcribe their genome within a proteinaceous viral capsid to evade host cell defenses. While Reovirales members use conservative transcription, most dsRNA viruses, includin...Double-stranded (ds)RNA viruses replicate and transcribe their genome within a proteinaceous viral capsid to evade host cell defenses. While Reovirales members use conservative transcription, most dsRNA viruses, including cystoviruses, utilize semi-conservative transcription, in which a newly synthesized positive strand replaces the parental positive strand, which is released as mRNA. Here, we visualize semi-conservative transcription activation in cystovirus ɸ6 double-layered particles using cryogenic electron microscopy. We observe nucleotide-triggered disassembly of the domain-swapped outer capsid layer, subsequent expansion of the inner capsid layer, and stepwise assembly of transcription complexes at the opposing poles of the spooled dsRNA genome. These complexes consist of the viral polymerases embedded into a triskelion formed by the minor protein P7, which we show as essential for continuous transcription. The packaging hexamers proximal to the transcription sites channel the viral mRNA exit. Our results define the complex molecular pathway from the quiescent state to activated semi-conservative transcription.
The view of mRNA function as a translational template is being challenged beyond translation. However, how these non-canonical mRNAs function independently of their coding protein remains largely unexplored. Here, we fou...The view of mRNA function as a translational template is being challenged beyond translation. However, how these non-canonical mRNAs function independently of their coding protein remains largely unexplored. Here, we found that intercellular adhesion molecule 1 (ICAM1) depletion via CRISPR-Cas9 protein knockout and shRNA-mediated RNA knockdown produces opposite effects on cell proliferation in human cells, which is validated by overexpression of mutated coding ICAM1 mRNA and ICAM1 coding sequence (CDS). Mechanistically, cis-antisense transcripts of ICAM1/ICAM1-AS form a double-stranded RNA (dsRNA), which entraps the interleukin enhancer binding factor 2 (ILF2)/ILF3 complex to inhibit DNA binding in a length-dependent manner, thus suppressing EIF4E transcription and global protein synthesis. Clinical analysis highlights the coordinated downregulation of ICAM1/ICAM1-AS, independent of highly expressed ICAM1 protein in lung cancer. In conclusion, this study reveals a role for ICAM1 mRNA in regulating cellular transcription via the dsRNA-ILF2/3 axis. Our findings challenge the phenotype explanation of gene silencing between RNA knockdown and protein knockout and underscore independent mRNA functions.
In this issue of Molecular Cell, Zhu et al. show that mitochondria of cancer cells rely on the import of glutamine not only to fuel metabolite synthesis via the tricarboxylic acid cycle but also to charge mt-tRNA to allo...In this issue of Molecular Cell, Zhu et al. show that mitochondria of cancer cells rely on the import of glutamine not only to fuel metabolite synthesis via the tricarboxylic acid cycle but also to charge mt-tRNA to allow mitochondrial protein synthesis and respiration.
In this issue of Molecular Cell, Kral et al. identify a targetable, novel mechanism of pancreatic ductal adenocarcinoma (PDAC) tumorigenesis via SRSF1 splicing-mediated regulation of an Alu-derived exon in Aurora kinase...In this issue of Molecular Cell, Kral et al. identify a targetable, novel mechanism of pancreatic ductal adenocarcinoma (PDAC) tumorigenesis via SRSF1 splicing-mediated regulation of an Alu-derived exon in Aurora kinase A (AURKA).
Hoffmann C, Nagao T, Tsunoyama TA
… +9 more, Tromm JV, Logan C, Nakamura K, Wang H, Bianchi F, van den Bogaart G, Kusumi A, Hirabayashi Y, Milovanovic D
Mitochondria and the endoplasmic reticulum (ER) contain large areas that are in close proximity. Yet the mechanism of how these inter-organellar adhesions are formed remains elusive. Tight functional connections, termed...Mitochondria and the endoplasmic reticulum (ER) contain large areas that are in close proximity. Yet the mechanism of how these inter-organellar adhesions are formed remains elusive. Tight functional connections, termed "membrane contact sites," assemble at these areas and are essential for exchanging metabolites and lipids between the organelles. Recently, the ER-resident protein PDZ domain-containing protein 8 (PDZD8) was identified as a tether between the ER and mitochondria or late endosomes/lysosomes. Here, we show that PDZD8 can undergo phase separation via its intrinsically disordered region (IDR). Endogenously labeled PDZD8 forms condensates on membranes both in vitro and in mammalian cells. Electron microscopy analyses indicate that the expression of full-length PDZD8 rescues the decrease in inter-organelle contacts in PDZD8 knockout cells but not PDZD8 lacking its IDR. Together, this study identifies that PDZD8 condensates at the lipid interfaces act as an adhesive framework that stitches together the neighboring organelles and supports the structural and functional integrity of inter-organelle communication.
Guo L, Mann JR, Mauna JC
… +30 more, Copley KE, Wang H, Rubien JD, Bergmann CA, Carey JL, Merjane J, Ngo M, Xu J, Odeh HM, Lin J, Lee BL, Ganser L, Robinson E, Kim KM, Murthy AC, Paul T, Portz B, Gleixner AM, Diaz Z, Smirnov A, Padilla G, Lavorando E, Espy C, Shang Y, Huang EJ, Chesi A, Fawzi NL, Myong S, Donnelly CJ, Shorter J
RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, includ...RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs that prevent FUS fibrillization by promoting liquid phases and distinct short RNAs that prevent and reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. We define a short RNA that dissolves cytoplasmic FUS aggregates, restores nuclear FUS, and mitigates FUS toxicity in optogenetic models and ALS patient-derived motor neurons. Another short RNA dissolves cytoplasmic TDP-43 aggregates, restores nuclear TDP-43, and mitigates TDP-43 toxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have utility for ALS/FTD and related disorders.
In this issue of Molecular Cell, Ishiguro et al. describe new RNA modifications near the active site of the E. coli ribosome that appear only under anaerobic conditions. These modifications enhance ribosome activity and...In this issue of Molecular Cell, Ishiguro et al. describe new RNA modifications near the active site of the E. coli ribosome that appear only under anaerobic conditions. These modifications enhance ribosome activity and increase anaerobic growth rates.
Mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth, responding to amino acid availability. While mTORC1 is modulated by amino acid sensors like CASTOR1, the mechanisms driving its dy...Mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth, responding to amino acid availability. While mTORC1 is modulated by amino acid sensors like CASTOR1, the mechanisms driving its dynamic response to fluctuating amino acid levels remain unclear. Here, we investigate the role of CASTOR2, an understudied CASTOR1 homolog, in regulating mTORC1 activity. We show that CASTOR1 and CASTOR2 bind to arginine similarly but differ in their sensitivity: CASTOR1 responds to low arginine levels, whereas CASTOR2 responds to high arginine concentrations. Both proteins interact with the GATOR2 component Mios, inhibiting its binding to GATOR1. Arginine binding to CASTOR1/2 induces conformational changes at the aspartate kinase, chorismate mutase, and TyrA (ACT) domain (ACT2-ACT4) interface, leading to its dissociation from Mios. Functionally, we demonstrate that CASTOR proteins are highly expressed in muscle tissue and, in C2C12 cells, they regulate mTORC1 and myogenesis in response to different arginine availability. These findings highlight how CASTOR proteins function as dual arginine sensors to fine-tune mTORC1 activity.
Wei H, Song Q, Wang L
… +20 more, Deng Q, Wu B, Chen Y, Han T, Guo Y, Li Z, Dong F, Ma S, Zhao Q, Shi X, Pan C, Jiang W, Liu X, Chen Y, Jiao R, Yuan L, Liu C, Guo X, Cong Y, Li W
Approximately 10% of eukaryotic proteins are folded by the TRiC/CCT complex (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1), and only open-state TRiC can bind with programmed cell death 5 (P...Approximately 10% of eukaryotic proteins are folded by the TRiC/CCT complex (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1), and only open-state TRiC can bind with programmed cell death 5 (PDCD5). However, the physiological role of the PDCD5-TRiC interaction remains elusive. Here, we show that PDCD5 is required for flagellum biogenesis and ciliogenesis and present the PDCD5-TRiC structures in their open states at near-atomic resolution. Mechanically, we find that PDCD5 promotes substrates release by competing with PhLP2A to interact with TRiC, and the depletion of PDCD5 traps flagellum- and cilium-associated proteins within TRiC, finally leading to malformed flagella of spermatids and cilia in mouse ciliated cells. Moreover, we demonstrate that the function of PDCD5 in flagellum biogenesis and ciliogenesis depends on the interaction with TRiC by its C terminus. These findings identify PDCD5 as a TRiC regulator to promote a subset of proteins release.
Members of the bromodomain and extraterminal domain (BET) protein family play a central role in transcription by RNA polymerase II (RNA Pol II). Small-molecule inhibitors that block interaction between BET bromodomains a...Members of the bromodomain and extraterminal domain (BET) protein family play a central role in transcription by RNA polymerase II (RNA Pol II). Small-molecule inhibitors that block interaction between BET bromodomains and acetylated histones have been developed for disease therapeutics. However, the BET protein BRD4 does not require bromodomains to perform its major transcriptional elongation control, and mechanisms by which other BET proteins regulate RNA Pol II remain insufficiently understood. Addressing the disparity between pan-BET degraders and BRD4-specific depletion, we report that the BET protein BRD2 generally functions to promote transcriptional initiation in a bromodomain-dependent manner at both promoters and enhancers in human cell lines. We demonstrate that BRD2 bromodomains preferentially bind to histone H4 harboring MOF-mediated H4K16ac, while the BRD2 C-terminal domain facilitates recruitment of TFIID. Our studies provide mechanistic insight into distinct roles for BRD2 and BRD4 in transcriptional initiation and elongation control for proper regulation of gene expression.
RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-gu...RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.
The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acid tails (CAT tails) in an mRNA-free,...The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acid tails (CAT tails) in an mRNA-free, non-canonical elongation process. The relationship between CAT tail composition (alanine [Ala] and threonine [Thr] in yeast) and function has remained unknown. Using biochemical approaches in yeast, we discovered that mechanical forces on the NC regulate CAT tailing. We propose that CAT tailing initially operates in "extrusion mode," which increases NC lysine accessibility for on-ribosome ubiquitylation. Thr in CAT tails prevents the formation of polyalanine, which forms α-helices that lower extrusion efficiency and disrupt termination of CAT tailing. After NC ubiquitylation, pulling forces on the NC switch CAT tailing to an Ala-only "release mode," which facilitates NC release and degradation. Failure to switch from extrusion to release mode leads to the accumulation of NCs on large ribosomal subunits and proteotoxic aggregation of Thr-rich CAT tails.