Biology subjects

Wu, V.

Publications and source records attributed to Wu, V..

14 recordsLinked to original sources

bioRxiv2026

Cryo-EM of a nucleotide-polymerizing ribozyme enables its predictive improvement

Ribozymes capable of self-replication from nucleotides would have been central to the hypothesized RNA World. The leading laboratory models for such molecules were converted from a class I ligase by in vitro evolution but then developed without 3D structures. Here, scaffolded cryo-EM of the substrate-free tC19Z RNA polymerase ribozyme at 3.1 [A] resolution shows how this conversion was achieved. An accessory domain evolved from random sequence grips the ancestral ligase through a loop-loop contact, a seam of magnesium ions, and a six-base stack, and rebuilds the ligases substrate binding site from different residues of its own. A previously unrecognized pairing, present before substrate binds, sequesters the 5' end that must otherwise pair with the template. Compensatory mutations to the ribozyme and template, designed to break this ectopic pairing, increase the extension rate. These results suggest that accelerating RNA structure determination may speed progress toward nucleotide-based self-replication.

biochemistry↗

bioRxiv2026

De novo design of RNA pseudoknots with deep learning

RNA design has been hindered by the limited accuracy of 3D structure prediction. Here, we show that intricate RNA structures can be generated with current deep learning tools through accurate de novo design of pseudoknot secondary structures. In an Eterna competition involving 57 pseudoknots, generative AI methods matched experienced human designers in solving most blind challenges, evaluated by single-nucleotide-resolution chemical mapping, compensatory mutagenesis, and cryogenic electron microscopy. AI-generated molecules with accurate secondary structures formed well-ordered 3D folds stabilized by noncanonical tertiary interactions not modeled during design. Success was guided by an RNet foundation model trained on prior chemical mapping data, suggesting that some difficult RNA design tasks may be tractable without first solving RNA 3D structure prediction.

biophysics↗

bioRxiv2026

Tau-induced ribosomal collisions impair memory through the activation of the integrated stress response

The formation of new long-term memories is reliant upon the spatial and temporal regulation of mRNA translation. Translational control has been demonstrated to be disrupted in neurodegenerative diseases, which exhibit impairments in both homeostatic translation and memory formation, such as Alzheimers disease (AD) and frontotemporal dementia (FTD). However, the precise mechanisms by which this dysregulation occurs, as well as the pathogenic consequences of this dysregulation have yet to be described. Here we establish that FTD-associated tau mutations impair protein synthesis prior to the onset of memory impairments by slowing ribosomal elongation speed, causing ribosomes to collide upon mRNAs. We reveal that this tau-induced ribosomal collision ultimately impairs memory-associated translation through activation of the integrated stress response (ISR) via GCN2. Pharmacological prevention of this ISR activation not only rescues memory formation in the PS19 mouse model of FTD, but also attenuates neuronal death, decreases tau phosphorylation and accumulation, and improves survival. Collectively, our data elucidates a novel mechanism by which mRNA translation is impaired early in neurodegeneration, identifies several pathological phenotypes which are traceable to impairments in mRNA translation, and highlights the therapeutic potential of rescuing these translational impairments.

neuroscience↗

bioRxiv2025

RNA switch model for localization and translation of the myelin basic protein mRNA

Oligodendrocytes myelinate the central nervous system by extending cellular projections that ensheath axons and elongate to form lipid-rich myelin. Classic studies visualizing RNA dynamics showed that myelin basic protein (MBP), one of the most abundant myelin proteins, is locally synthesized at the myelin sheath through the transport and local translation of Mbp mRNA. Mbp transport requires its 1.5-kb 3 untranslated region (3 UTR) and prior work identified candidate sub-sequences that may act as cis-acting transport stimulating RNA elements, including one with putative secondary structure. Here, a high-throughput reporter assay, dimethyl sulfate (DMS)-based RNA structure probing, and microscopy in primary rat oligodendrocytes identify a structured 127-nt region that we name the Mbp localization signal (MLS) as both necessary and sufficient for RNA enrichment to oligodendrocyte projections. Lysate pulldown experiments further identify hnRNP-F - a known constituent of the Mbp RNA granule that can suppress mRNA translation - as associated with the MLS; paradoxically, binding of this protein should compete with the ordered MLS RNA structure. These results suggest a model in which the MLS switches between two RNA conformations with distinct protein partners during the transition from Mbp mRNA transport to Mbp translation at the myelin sheath. Such regulation of RNA behavior by structure switching may generalize to other eukaryotic mRNAs whose behaviors shift across space and time. Significance StatementIn the brain, oligodendrocyte cells generate myelin, a type of insulation that wraps around neuronal axons in order to facilitate fast electrical signaling. A critical step in myelination is the local translation of MBP (myelin basic protein) in the myelin sheath. This requires the transport of Mbp mRNA, an incompletely understood phenomenon that we revisit using two recent approaches for mRNA structure and function. We refine a 127-nt region that is necessary and sufficient for mRNA transport to the myelin sheath. A proteomic screen reveals that this myelin localization signal (MLS) associates with a translation-suppressing protein called hnRNP-F, suggesting a model where Mbp mRNA switches between two states, one for transport and one for translation at the myelin sheath.

cell biology↗

bioRxiv2025

In vivo reprogramming of cytotoxic effector CD8+ T cells via fractalkine-conjugated mRNA-LNP

Selective in vivo reprogramming of cytotoxic effector CD8+ T (Teff) cells holds tremendous promise as a therapeutic tool but has not yet been accomplished. Here, we demonstrate that fractalkine-conjugated mRNA lipid nanoparticles (mRNA-LNP) can specifically target and deliver mRNA to CX3CR1+ Teff cells in vitro and in vivo. In mice, fractalkine-conjugated LNP target up to 90% of blood and splenic Teff cells, and delivery of IL-2-encoding mRNA to Teff cells enables robust exogenous IL-2 secretion. In rhesus macaques, fractalkine-conjugated mRNA-LNP target up to [~]100% of peripheral blood Teff cells and delivery of CD62L-mRNA enables transient CD62L expression. Collectively, these data demonstrate the potential of natural receptor ligand-based targeting of mRNA-LNP for effective and efficient transient in vivo modification of Teff cells.

immunology↗

bioRxiv2025

Multimodal imaging reveals a lysosomal drug reservoir that drives heterogeneous distribution of PARP inhibitors

For all drugs, effective target engagement requires sufficient intracellular concentrations of drug to be reached, but whether tumour heterogeneity impacts drug distribution and efficacy is poorly studied. PARP inhibitors have transformed treatment of high-grade serous ovarian carcinoma (HGSOC), but resistance remains a clinical hurdle in this highly heterogeneous tumour type. We developed a patient-derived explant multi-modal imaging pipeline, which demonstrated that cell-intrinsic PARP inhibitor accumulation is highly variable, both between patients and within tumours. Spatial transcriptomics revealed enrichment of apoptotic and lysosomal signatures in high-drug regions. Rucaparib, an intrinsically fluorescent PARP inhibitor, accumulates heterogeneously at the single-cell level, with rucaparib-high cells demonstrating increased drug response relative to rucaparib low. Mechanistically, lysosomal sequestration creates a rucaparib reservoir that determines drug levels in the nucleus. Perturbation of lysosomal content altered intracellular levels of weak base PARP inhibitors rucaparib and niraparib, but not olaparib. Together these data suggest that lysosomes act as a reservoir for a subset of PARP inhibitor drugs to improve drug response.

cancer biology↗

bioRxiv2025

Mapping the spatiotemporal dynamics of de novo protein synthesis during long-term memory formation

The formation of new associative long-term memory (LTM) following Pavlovian conditioning is dependent upon multiple, temporally distinct windows of mRNA translation. Current methods lack the temporal specificity to robustly characterize the dynamics of protein synthesis throughout the rodent brain following conditioning. Here we resolve these technological limitations and demonstrate that in awake mice, the retro-orbital (RO) injection of azidohomoalanine (AHA) enables the labelling and subsequent visualization of the brain de novo proteome, with labelling periods as short as 30 minutes. Combining this advancement in de novo proteomic labelling with tissue clearing, we identified brain region, cell-type, and neuronal sub-population specific changes in de novo protein synthesis in mice following an auditory threat conditioning paradigm. This approach also allowed us to track the changes in de novo protein synthesis over time, revealing that conditioning-induced changes in mRNA translation exhibit remarkable temporal specificity in brain regions such as the somatosensory cortex. Taken together, our findings highlight how this novel labelling technique can be used to map the highly intricate temporal and spatial dynamics of mRNA translation after behavioral conditioning.

neuroscience↗

bioRxiv2024

Select DYRK1A Inhibitors Enhance Both Proliferation and Differentiation in Human Pancreatic Beta Cells

The small molecule DYRK1A inhibitor, harmine, induces human beta cell proliferation, expands beta cell mass, enhances expression of beta cell phenotypic genes, and improves human beta cell function in vitro and in vivo. It is unknown whether the "pro-differentiation effect" is a DYRK1A inhibitor class-wide effect. Here we compare multiple commonly studied DYRK1A inhibitors. Harmine, 2-2c and 5-IT increase expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC2A2, SLC30A8, ENTPD3 in normal and T2D human islets. Unexpectedly, GNF4877, CC-401, INDY, CC-401 and Leucettine fail to induce expression of these essential beta cell molecules. Remarkably, the pro-differentiation effect is independent of DYRK1A inhibition: although silencing DYRK1A induces human beta cell proliferation, it has no effect on differentiation; conversely, harmine treatment enhances beta cell differentiation in DYRK1A-silenced islets. A careful screen of multiple DYRK1A inhibitor kinase candidate targets was unable to identify pro-differentiation pathways. Overall, harmine, 2-2c and 5-IT are unique among DYRK1A inhibitors in their ability to enhance both beta cell proliferation and differentiation. While beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor pharmaceutical development.

molecular biology↗

bioRxiv2024

Mitochondrial reverse electron transport in myeloid cells perpetuates neuroinflammation

Sustained smouldering, or low grade, activation of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis (MS)1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate neuroinflammation is currently unknown. Using a multiomics approach, we identified a new molecular signature that perpetuates the activation of myeloid cells through mitochondrial complex II (CII) and I (CI) activity driving reverse electron transport (RET) and the production of reactive oxygen species (ROS). Blocking RET in pro-inflammatory myeloid cells protected the central nervous system (CNS) against neurotoxic damage and improved functional outcomes in animal disease models in vivo. Our data show that RET in myeloid cells is a potential new therapeutic target to foster neuroprotection in smouldering inflammatory CNS disorders3.

neuroscience↗

bioRxiv2023

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target

With colorectal cancer (CRC) being the second most common cause of cancer-related deaths worldwide1, there is an urgent need for better diagnostic tools and new, more targeted therapies. Here we used genetically engineered mouse models (GEMMs), and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that unsupervised metabolic profiling can stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging (MSI) to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-mutant CRC, and propose one of its enzymes, i.e. adenosylhomocysteinase (AHCY), as a new therapeutic target. Collectively, we show that the profound genotype-dependent alterations in both lipid and small molecule metabolism in CRC may be exploited for tissue classification with no need for ion identification, and we applied further data analysis to expose a novel metabolic vulnerability of CRC.

cancer biology↗

bioRxiv2022

Comparative genomic insights into bacterial induction of larval settlement and metamorphosis in the upside-down jellyfish Cassiopea

Bacterial biofilm is crucial in inducing the larval transition from pelagic to benthic environments for marine organisms. Bacteria can therefore dictate species distribution and success of the individual. Despite the importance of marine bacteria to animal ecology, the identity of inductive microbes for many invertebrates are unknown. We isolated bacteria belonging to multiple phyla are capable of inducing settlement and metamorphosis in the upside-down jellyfish Cassiopea xamachana. The most inductive isolates belonged to the genus Pseudoalteromonas, a marine bacterium known to induce the pelago-benthic transition in other marine invertebrates. In sequencing the genome of the isolated Pseudoalteromonas and an inductive Vibrio, we found biosynthetic pathways previously implicated in larval settlement were absent in these Cassiopea inducing taxa. Comparative analysis of the Pseudoalteromonas and Vibrio revealed shared genes that could underlie the inductive capacity of these two bacteria. Thus, C. xamachana are capable of responding to multiple bacterial species, but they may be responding to a common cue produced by multiple taxa. These findings could provide hints to the ecological success of C. xamachana compared to sympatric congeneric species within mangrove environments and provide avenues to investigate the evolution of animal-microbe interactions.

microbiology↗

bioRxiv2021

Mass recalibration for desorption electrospray ionization mass spectrometry imaging using endogenous reference ions.

1BackgroundMass spectrometry imaging (MSI) data often consist of tens of thousands of mass spectra collected from a sample surface. During the time necessary to perform a single acquisition, it is likely that uncontrollable factors alter the validity of the initial mass calibration of the instrument, resulting in mass errors of magnitude significantly larger than their expected values. This phenomenon has a two-fold detrimental effect: a) it reduces the ability to interpret the results based on the observed signals, b) it can affect the quality of the observed signal spatial distributions. ResultsWe present a post-acquisition computational method capable of reducing the observed mass drift by up to 60 ppm in biological samples, exploiting the presence of typical molecules with a known mass-to-charge ratio. The procedure, tested on time-of-flight (TOF) and Orbitrap mass spectrometry analyzers interfaced to a desorption electrospray ionization (DESI) source, improves the molecular annotation quality and the spatial distributions of the detected ions. ConclusionThe presented method represents a robust and accurate tool for performing post-acquisition mass recalibration of DESI-MSI datasets and can help to increase the reliability of the molecular assignment and the data quality.

bioinformatics↗

bioRxiv2021

Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that "write" these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

genomics↗

bioRxiv2020

The identity of human tissue-emigrant CD8+ T cells

Lymphocyte migration is essential for human adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies to date have been restricted to immunological analyses of blood and various tissues. To address this issue, we used an integrated approach to characterize tissue-emigrant immune cells in thoracic duct lymph (TDL). In humans and non-human primates, lymphocytes were by far the most abundant immune lineage population in efferent lymph, and a vast majority of these lymphocytes were T cells. Cytolytic CD8+ T cell subsets were clonotypically discrete and selectively confined to the intravascular circulation, persisting for months after inhibition of S1P-dependent tissue egress by FTY-720. In contrast, non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Collectively, these data provide an atlas of the migratory immune system and define the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

immunology↗