Biology subjects

Begley, T. J.

Publications and source records attributed to Begley, T. J..

9 recordsLinked to original sources

bioRxiv2026

Multi-dimensional DNA nanostructures isothermally assembled in hydrated ionic liquids

DNA nanostructures can be tailored to perform a wide variety of functions, with continued interest in biological applications. Some aspects of DNA nanostructure assembly can hinder the ability of nanostructures to be useful in physiological environments. Typical assembly methods employ magnesium ions to stabilize the structure, which leave the structure susceptible to damage by nucleases in body fluids. Further, DNA nanostructure assembly typically involves a thermal annealing protocol in which DNA strands are heated in a specific buffer to a high temperature and cooled slowly at specific rates, preventing convenient encapsulation of temperature-sensitive guest molecules. In this work, we demonstrate the assembly of a wide variety of DNA nanostructures and 3D crystals in a hydrated ionic liquid (choline dihydrogen phosphate, CDHP) instead of magnesium at constant moderate temperatures, thus avoiding thermal annealing. CDHP-assembled structures show enhanced biostability against a variety of nucleases. Molecular dynamics simulations show that choline ions stabilize DNA nanostructures by a direct and close-range interaction in contrast to the predominantly water-mediated interactions of Mg2+, leading to enhanced nuclease resistance in CDHP-containing environments. CDHP-assembled structures do not affect the viability of HepG2 cells and show higher cell internalization. Overall, this work develops a potential method to construct more biostable DNA nanostructures and 3D crystals in a simple one pot process. Assembly of DNA nanostructures under isothermal conditions is desirable for scaffolding biomolecules and to reduce the need for thermal annealing instruments, allowing nanostructure preparation in low-resource settings.

biophysics↗

bioRxiv2026

Oncogenes have the most distinct codon biases in the genome and codon signatures that oppose tumor suppressor genes

Oncogenes and tumor-suppressor genes play opposing roles in cancer biology to promote and restrict growth, respectively. Codon usage patterns interface with tRNA modifications to control translation, leading to gene-specific codon signatures with regulatory potential. As such, codon-biased translational regulation has been identified as a driver of proliferation and drug resistance in multiple cancers. We used advanced codon analytics methods to characterize and compare codon usage bias in oncogenes and tumor suppressor genes (TSGs) from humans and mice at group and gene-specific levels. We demonstrate that human oncogenes exhibit a distinct and opposing codon usage pattern to TSGs. This phenomenon is also present in mice but with less distinct oncogene bias relative to humans. Further comparison to 447 gene ontology groups demonstrated that human oncogenes have the most distinct codon usage patterns in the genome, while also highlighting that codon bias can separate functionally related genes and pathways from other biological processes. Using gene-specific codon analytics, we determined that human oncogenes have two types of extreme codon bias: a large group (N = 43) over-using G/C ending (GC3) codons and a smaller group (N = 12) over-using A/U (AU3) ending codons. While GC3 bias has been linked to increased translation in general, the AU3 finding suggests that genetic, environmental, or stress-related signals could drive the translation of this small group of oncogenes. The less extreme bias observed in mouse oncogenes and tumor suppressors likely underscores species-specific differences in oncogenic translation programs. Together, our findings highlight codon usage bias as a potential determinant of oncogene expression, provide a framework for ontology-based codon analysis, and uncover on species-specific differences in oncogene translation and codon usage biases.

cancer biology↗

bioRxiv2026

The Wobble Uridine tRNA Writer MnmA Shapes Codon-Dependent Stress Response Systems

Escherichia coli uses wobble uridine (U34) modifications to tune codon decoding, but how individual tRNA writer enzymes shape gene expression remains unclear. Here, we identify MnmA, the U34 thiolation enzyme for tRNALys, tRNAGln, and tRNAGlu, as a central regulator linking codon-directed translation to regulon control and stress response. Loss of MnmA depleted s2U-dependent wobble modifications, preventing geranyl-(ges2U) and seleno-(se2U)-based modifications, causing growth defects, reduced catalase activity, and multi-level gene expression dysregulation. The {Delta}mnmA cells showed broad adaptive transcriptional reprogramming associated with RpoS- and OxyR-regulated pathways, which was accompanied by compromised protein output. Endogenous and tagged-protein analyses revealed specific impairment of transcriptional regulators, adaptive and detoxification proteins, including RpoS, OxyR, FliA, KatE, and KatG. Polysome profiling and polysome-associated RNA sequencing showed that MnmA deficiency globally reduces translational capacity and uncouples mRNA abundance from translational efficiency, which is exacerbated during oxidative stress. We developed genome-wide codon-usage mapping analytics to identify five codon-defined gene clusters, with specific clusters enriched for Lys, Gln, and Glu codons disproportionately affected by MnmA loss. Together, these findings support that wobble uridine thiolation and downstream modifications pair with corresponding codon architecture to coordinate the translation of regulon controllers and stress-response networks linked to bacterial fitness. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC="FIGDIR/small/745044v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1a4058borg.highwire.dtl.DTLVardef@167f4cdorg.highwire.dtl.DTLVardef@1f8c7a8org.highwire.dtl.DTLVardef@1fc0767_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Growth-dependent tRNA Reprogramming and Codon Bias Link Translation to Metabolic State in Enterococcus faecalis

Enterococcus faecalis is a Gram-positive commensal bacterium of the human gut microbiome and an opportunistic pathogen responsible for many hospital-acquired infections. Despite the clinical importance of E. faecalis, how gene and protein expression are coordinated with growth remains poorly defined. Here, we profiled transcript, protein, and tRNA pool dynamics across distinct phases of E. faecalis growth. Differences in protein abundance and corresponding mRNA levels suggested growth phase-dependent posttranscriptional regulation. Growth-associated genes exhibited biased synonymous codon usage, with ribosomal and glycolytic proteins enriched in low-abundance codons read by queuosine-modifiable tRNAs. Analysis of tRNA modification and tRNA isoacceptor abundance revealed growth phase-dependent changes, particularly in anticodon stem loop modifications that influence synonymous codon translation. Changes in queuosine levels preceded shifts in ribosomal proteins, suggesting a contribution to codon-biased translation. Collectively, these findings reveal growth phase-associated remodeling of the E. faecalis tRNA pool and support a model in which queuosine-dependent translational reprogramming shapes protein expression during bacterial growth. IMPORTANCEEnterococcus faecalis is a common cause of hospital-acquired infections. Despite its clinical importance, a comprehensive understanding of the organisms physiology and adaptation to environmental changes remains incomplete. Here, we characterized protein, transcript, and tRNA dynamics across bacterial growth phases, uncovering a role for post-transcriptional regulation marked by tRNA reprogramming and biased synonymous codon usage. These findings enhance our understanding of E. faecalis growth and support a model of translational reprogramming therein.

microbiology↗

bioRxiv2025

Loss of s2U tRNA modification induces antibiotic tolerance in Yersinia pseudotuberculosis

Stress promotes phenotypic changes in bacteria that allow them to survive antibiotic treatment. This phenomenon, termed antibiotic tolerance, can cause treatment failure, highlighting a need to define bacterial pathways that promote survival. Previously, we found Yersinia pseudotuberculosis downregulates tusB, a gene involved in modifying tRNAs with s2U, in response to doxycycline. Here we find that deletion of tusB results in loss of s2U and induces antibiotic tolerance. Using a combination of sequencing-based approaches and analysis of gene codon usage, our data show that loss of s2U decreases translation of ribosomal proteins. Ribosomal proteins are highly enriched in codons that require s2U-modified tRNAs for efficient translation, and loss of s2U results in ribosome pausing at these codons. Our results highlight a previously unknown mechanism of antibiotic tolerance where reduction in ribosomal protein abundance can globally reduce translation, and describes a novel strategy bacteria use to slow growth by modulating s2U levels.

microbiology↗

bioRxiv2025

Genes and Pathways Comprising the Human and Mouse ORFeomes Display Distinct Codon Bias Signatures that Can Regulate Protein Levels

Arginine, glutamic acid and selenocysteine based codon bias has been shown to regulate the translation of specific mRNAs for proteins that participate in stress responses, cell cycle and transcriptional regulation. Defining codon-bias in gene networks has the potential to identify other pathways under translational control. Here we have used computational methods to analyze the ORFeome of all unique human (19,711) and mouse (22,138) open-reading frames (ORFs) to characterize codon-usage and codon-bias in genes and biological processes. We show that ORFeome-wide clustering of gene-specific codon frequency data can be used to identify ontology-enriched biological processes and gene networks, with developmental and immunological programs well represented for both humans and mice. We developed codon over-use ontology mapping and hierarchical clustering to identify multi-codon bias signatures in human and mouse genes linked to signaling, development, mitochondria and metabolism, among others. The most distinct multi-codon bias signatures were identified in human genes linked to skin development and RNA metabolism, and in mouse genes linked to olfactory transduction and ribosome, highlighting species-specific pathways potentially regulated by translation. Extreme codon bias was identified in genes that included transcription factors and histone variants. We show that re-engineering extreme usage of C- or U-ending codons for aspartic acid, asparagine, histidine and tyrosine in the transcription factors CEBPB and MIER1, respectively, significantly regulates protein levels. Our study highlights that multi-codon bias signatures can be linked to specific biological pathways and that extreme codon bias with regulatory potential exists in transcription factors for immune response and development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/636209v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@de9969org.highwire.dtl.DTLVardef@29e1dforg.highwire.dtl.DTLVardef@1abfebcorg.highwire.dtl.DTLVardef@e119b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

ROS-induced translational regulation--through spatiotemporal differences in codon recognition--is a key driver of brown adipogenesis

The role of translational regulation in brown adipogenesis is relatively unknown. Localized translation of mRNAs encoding mitochondrial components enables swift mitochondrial responses, but whether this occurs during brown adipogenesis, which involves massive mitochondrial biogenesis, has not been explored. Here, we used ribosome profiling and RNA-Seq, coupled with cellular fractionation, to obtain spatiotemporal insights into translational regulation. During brown adipogenesis, a translation bias towards G/C-ending codons is triggered first in the mitochondrial vicinity by reactive oxygen species (ROS), which later spreads to the rest of the cell. This translation bias is induced through ROS modulating the activity of the tRNA modification enzyme, ELP3. Intriguingly, functionally relevant mRNAs, including those encoding ROS scavengers, benefit from this bias; in so doing, ROS-induced translation bias both fuels differentiation and concurrently minimizes oxidative damage. These ROS-induced changes could enable sustained mitochondrial biogenesis during brown adipogenesis, and explain in part, the molecular basis for ROS hormesis.

molecular biology↗

bioRxiv2023

Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

The 40-50 RNA modifications of the epitranscriptome regulate posttranscriptional gene expression. Here we show that flaviviruses hijack the host tRNA epitranscriptome to promote expression of pro-viral proteins, with tRNA-modifying ALKBH1 acting as a host restriction factor in dengue virus infection. Early in the infection of human Huh-7 cells, ALKBH1 and its tRNA products 5-formylcytidine (f5C) and 2-O-methyl-5-formylcytidine (f5Cm) were reduced. ALKBH1 knockdown mimicked viral infection, but caused increased viral NS3 protein levels during infection, while ALKBH1 overexpression reduced NS3 levels and viral replication, and increased f5C and f5Cm. Viral NS5, but not host FTSJ1, increased f5Cm levels late in infection. Consistent with reports of impaired decoding of leucine UUA codon by f5Cm-modified tRNALeu(CAA), ALKBH1 knockdown induced translation of UUA-deficient transcripts, most having pro-viral functions. Our findings support a dynamic ALKBH1/f5Cm axis during dengue infection, with virally-induced remodeling of the proteome by tRNA reprogramming and codon-biased translation.

microbiology↗

bioRxiv2019

Epitranscriptomic regulation of the response to the air pollutant naphthalene in mouse lungs: from the perspectives of specialized translation and tolerance linked to the writer ALKBH8

BackgroundThe epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a tRNA methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation, via stop codon recoding, of proteins that contain selenocysteine. Corresponding selenoproteins play critical roles in protecting against reactive oxygen species and environmental stress. Using a novel animal model deficient in Alkbh8, we have investigated the importance of epitranscriptomic systems in the response to naphthalene (NA), an abundant polycyclic aromatic hydrocarbon, glutathione depleter and lung toxicant found in tobacco smoke, gasoline and mothballs.\n\nObjectivesOur goal was to define the molecular reprogramming of Alkbh8 deficient (Alkbh8def) mice and evaluate the roles that the epitranscriptomic writer ALKBH8 and selenoproteins play in mitigating NA-induced toxicity and lung dysfunction.\n\nMethodsWe performed basal lung analysis and NA exposure studies using WT, Alkbh8def and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for NA bioactivation. We characterized gene expression, molecular markers of damage, viability and tolerance to NA.\n\nResultsUnder basal conditions, lungs from Alkbh8def mice have increased oxidation-reduction potential (ORP) and 8-isoprostane levels, and have reprogrammed at the molecular level to display increased stress response transcripts. In addition, the ALKBH8 writer deficient lungs have a decreased GSH/GSSG ratio. Alkbh8def mice are more sensitive to NA than WT, showing higher susceptibility to lung damage both at the cellular and molecular levels. WT mice develop a tolerance to NA after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice, with writer deficient not surviving NA exposure.\n\nDiscussionWe conclude that the epitranscriptomic writer ALKBH8 plays a protective role against NA-induced lung dysfunction and promotes NA tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.

molecular biology↗