Therapeutic mRNAs rely heavily on the incorporation of the RNA modification N1- methylpseudouridine for their efficacy. Schiffers et al. present N4-acetylcytidine as a robust alternative that further enhances protein production, broadening the chemical repertoire available for designing the next generation of mRNA medicines.1.
Adaptation to thermal stress requires cells to interpret temperature changes in molecular terms to initiate biochemical responses. In this issue of Molecular Cell, Ueno et al.1 uncover a protein phosphorylation circuit as a potential thermosensor, linking stress to changes in global mRNA splicing.
Nuclear pore complex assembly is an intricate process occurring in the double bilayer of the nuclear envelope. In this issue of Molecular Cell, Fischer et al.,1 Maslennikova et al.,2 and Wang et al.3 integrate genetic analysis across model organisms with functional studies and molecular simulations to better understand a key step-membrane fusion.
p53 plays a central role in the DNA damage response, inducing repair, cell-cycle arrest or apoptosis. Its loss is associated with replication stress and genomic instability. While several underlying mechanisms were suggested, the primary triggers of catastrophic genomic events like chromothripsis, a known driver of tumorigenesis linked with p53 loss, are still unclear. Using p53-depleted epithelial cells and fibroblasts, as well as patient-derived fibroblasts with germline p53 variants that spontaneously undergo chromothripsis, we found that p53 loss causes hypertranscription and increased nucleotide consumption. The resulting nucleotide shortage induces replication stress, causing telomere dysfunction, micronuclei formation, and chromothripsis. These effects were rescued by nucleoside supplementation or normalization of transcription levels, demonstrating a causal link between transcriptional activity, nucleotide availability, and genome stability. Emerging chromothriptic clones displayed restored DNA replication, telomere stabilization, and extrachromosomal DNA, suggesting key features that support clonal selection. We identify nucleotide pool homeostasis as a critical p53 function that suppresses replication stress, prevents chromothripsis, and protects against early tumorigenesis.
The cyclin E-cyclin-dependent kinase 2 (CDK2) complex is a component of mammalian cell-cycle machinery that drives cell division. Hyperactivation of cyclin E-CDK2 is frequent in human cancers. Small-molecule CDK2 inhibitors are tested in clinical trials for cancer patients. Here, we report that cyclin E-CDK2 has a cell-cycle-independent function in regulating the global transcriptional program of cancer cells. CDK2 phosphorylates bromodomain-containing protein-4 (BRD4) and regulates its chromatin association. Overexpression of cyclin E and the resulting activation of CDK2 in cancer cells alter the cancer cell transcriptome, repress the expression of interferon-stimulated genes, and confer resistance to immunotherapy. Conversely, CDK2 inhibition has the opposite effect and augments the efficacy of immune checkpoint blockade. CDK2 inhibition also increases tumor infiltration by dendritic cells (DCs) and enhances antigen cross-presentation to CD8 T cells. These studies reveal an additional function of cyclin E-CDK2 in tumorigenesis and identify inhibition of CDK2 with clinically available compounds as a strategy for enhancing immune checkpoint blockade.
Genomic integrity in transcriptionally active regions is pivotal for suppressing oncogenic mutations, yet the mechanisms that govern precise homologous recombination (HR) repair within these regions remain elusive. Here, we report that the IRAK1-spliceosome axis operates with small nuclear RNA (snRNA) as a central hub, potently promoting accurate repair at DNA double-strand break (DSB) sites within active chromatin in human cancer cells. Mechanistically, IRAK1 phosphorylates spliceosomal serine/arginine (SR)-rich proteins to recruit snRNA to DSBs, inducing robust condensation of the MRE11-RAD50-NBS1 (MRN) complex near transcriptionally active regions to create an ATM activation platform. Collectively, our findings define a prevalent mechanism governing region-specific precise repair in transcriptionally active domains, where snRNA acts as a "transcription repair bridge" to link transcriptional processes to HR repair and ultimately preserves genomic stability. Inhibiting IRAK1 axis impairs HR repair in transcriptionally active regions, causing a marked increase in mutation rates specific to these regions and cancer-cell chemosensitivity.
The human transcriptome contains millions of A-to-I editing sites arising from an unclear number of poorly characterized dsRNAs. Editing sites reveal the presence of dsRNA, but this method is limited by transcription levels, read depth, and ADAR expression and cannot identify unedited dsRNA. To address these limitations, we developed dsRNAscan. Applying dsRNAscan to the human genome predicted 5 million dsRNAs, mostly in repetitive and intergenic regions. Machine learning models trained on A-to-I editing and RNA structure-probing data identified ∼2.4 million high-confidence predictions, which were enriched at dsRNA-binding protein binding sites. Additionally, we predicted hundreds of dsRNAs conserved across vertebrates and observed thousands of editing-enriched regions suspected to arise from intermolecular dsRNAs formed with sense-antisense transcripts. Quantifying expression of intramolecular and intermolecular dsRNAs accessible to cytoplasmic immune sensors revealed that their ratio correlated with ADAR dependency across cancer cell lines. The human dsRNAome is available as a resource at https://dsrna.chpc.utah.edu/.
Single-molecule localization microscopy (SMLM) enables visualization of chromatin architecture at nanoscale resolution. However, high-performance DNA probes suitable for SMLM in both live cells and tissues remain limited. We developed Hoechst-6-Carboxytetramethylrhodamine (6-TAMRA) derivative (HoT) probes-rhodamine-based derivatives conjugated to a Hoechst moiety-through structural fine-tuning of rhodamine spirocyclization. HoTs are self-assembling, auto-blinking probes with excellent photostability and high temporal resolution. They permeate live cells, enabling long-term, real-time nanoscopic chromatin imaging in live and fixed cells and in tissue sections. In live cells, we identified nanoscale features in the 3D organization of chromatin and quantified DNA fiber kinetics at high resolution. We quantified DNA compaction in single cells within retinal and colon cancer sections. OligoSTORM (stochastic optical reconstruction microscopy)-labeled gene loci can be visualized and measured within their HoT-labeled chromatin footprints. Our work provides powerful tools for investigating chromatin structure and functions in living cells and tissues, with applications ranging from cancer diagnosis to retinal regeneration.
Intragenomic homologs are widespread, but their physiological roles are often masked by redundancy. Histone-like nucleoid structuring protein (H-NS), a nucleoid-associated protein in Gram-negative bacteria, typically coexists with homologs like StpA, whose functions are obscured by a lack of strong phenotypes. We demonstrate here that the interaction between H-NS and StpA fine-tunes the physico-chemical properties of nucleoid-associated compartments. Although H-NS forms dynamic condensates in vitro, StpA assembles into stable insoluble fibrils. However, together the two proteins form liquid-like droplets, whose fluidity and stability are tunable by their relative stoichiometry. By increasing the levels of StpA over H-NS, bacteria stabilize heterochromatin-associated compartments, thereby preserving gene repression and optimizing bacterial growth under stress. Structural differences at these proteins' dimerization sites help explain their distinct phase behaviors. Our findings reveal a paradigm in which intragenomic homologs that are positioned at the opposite ends of the phase spectrum can fine-tune subcellular organization to promote survival in fluctuating environments.
Cells undergoing division mount a unique response to DNA damage that ensures accurate chromosome segregation. The CIP2A-TOPBP1 complex has emerged as an important mitotic genome maintenance factor, but its function remains unclear. Here, we report that DDIAS is a DNA-binding effector of the CIP2A pathway in human cells. DDIAS physically interacts with TOPBP1, and its inactivation causes synthetic lethality with BRCA1 and BRCA2 deficiency. Homologous recombination (HR)-deficient tumors upregulate DDIAS to enable HR-deficient cells to tolerate their genomic instability. Mechanistically, DDIAS is a single-stranded DNA (ssDNA)-binding protein that promotes the repair of ssDNA carried from interphase into mitosis. Mitotic ssDNA in HR-deficient cells is exacerbated by poly(ADP-ribose) polymerase (PARP) inhibition, and DDIAS-dependent suppression of these lesions involves mitotic DNA synthesis, which promotes accurate chromosome segregation and survival. We propose that DDIAS defines a mitotic DNA repair system downstream of CIP2A that mitigates the threat of mitotic ssDNA for genome integrity.
Cancer cell proliferation requires a precise balance between biomass production and nutrient catabolism. The pyridine nucleotide cofactors nicotinamide adenine dinucleotide NAD(H) and NAD phosphate NADP(H) are central to this process, but their compartment-specific regulation is incompletely understood. Using in vivo isotope-labeled metabolite tracing in an orthotopic xenograft model, we find that human gliomas extensively synthesize proline, an amino acid previously associated with hypoxia tolerance. In glioma cells, we identify a hypoxia-enhanced proliferative sensitivity to environmental proline dependent on NADH to NADPH transhydrogenation from a spatially compartmentalized mitochondrial pool by the enzyme nicotinamide nucleotide transhydrogenase (NNT). We demonstrate NNT-dependent generation of mitochondrial NADPH is important for proline accumulation, maintenance of antioxidant systems, and reductive metabolism in hypoxic glioma cells in vitro and tumor progression in vivo. Collectively, these results highlight proline accumulation as a marker of mitochondrial NAD(P)(H) homeostasis and NNT as a specific metabolic dependency in human glioma.
RNA splicing has historically been thought to be highly efficient and accurate, with little opportunity for deviation from regulated alternative splicing. This dogma has been challenged by recent observations that biological noise may contribute substantially to transcriptome diversity. However, quantitative understanding of stochastic splicing variation is challenging because these transcripts are likely subject to rapid degradation. Here, we use deep sequencing across RNA compartments to track splicing intermediates in human cells and see abundant cryptic splicing associated with genomic features that promote splicing noise. We observe pervasive usage of low-fidelity splice sites, likely due to stochasticity in recruitment or binding of the spliceosome. These sites are turned over quickly and show evidence for nuclear and cytoplasmic degradation, suggesting widespread surveillance and rapid quality control of non-productive transcripts. Our findings provide insights into the propensity for error in RNA processing mechanisms and regulation of alternative splice sites across a gene.
Tumor cells rely on sustained protein synthesis despite fluctuating metabolic stress. To examine how metabolic state directly influences translational output, we investigated lactate utilization. Intracellular accumulation of lactate, a central glycolytic product, acutely represses mRNA translation. Mechanistically, alanyl-tRNA synthetase 1 (AARS1) charges tRNAs with lactate instead of amino acids. Unlike the rapid and selective transfer of alanine to cognate tRNAAla, slower lactyl transfer permits lactate modification of non-cognate tRNAs, broadly compromising elongation fidelity. Functionally, this direct metabolic control over a fundamental process of the central dogma reshapes the translatome, operating as an intrinsic metabolic brake that aligns biosynthetic capacity with energy state. Notably, aggressive tumors elevate lactate transporters, limiting intracellular lactate accumulation and evading translational repression. Pharmacological blockade of monocarboxylate transporters restores intracellular lactate accumulation, re-establishes translational repression, and impairs tumor progression in mice. These findings uncover a metabolite-tRNA charging event directly rewiring translational output and reveal a metabolic vulnerability with therapeutic potential.
