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bioRxiv2026

Molecular basis of tRNA modification by the human m5C methyltransferase NSUN2

RNA 5-methylcytidine (m5C) is a prevalent modification that drives RNA stability and function. In humans, m5C is deposited on distinct RNA substrates by DNMT2/TRDMT1 and the NSUN family, to regulate diverse cellular processes, but how m5C writers recognise their substrates remains unclear. NSUN2 is a major m5C methyltransferase with broad roles in cell physiology and strong links to cancer and neurodevelopmental disorders 1. Here, we reconstitute an active human NSUN2-tRNA complex and capture its post-catalytic, tRNA-bound structure at 3.1 [A] resolution. Using an integrated approach combining biochemistry, cryo-electron microscopy, crosslinking mass spectrometry and molecular dynamics simulations, we show that NSUN2 remodels the tRNA to access the variable-loop target cytidine. Recognition is driven by RNA architecture, with NSUN2 exploiting the L-shaped tRNA scaffold to position the target base in the catalytic centre. We further show that Gly679 at the NSUN2-tRNA interface is important for the stability of the complex, providing a mechanistic basis for how the disease-associated Gly679Arg substitution can impair tRNA binding. Together, these findings establish an RNA-structure-guided mechanism for NSUN2 substrate recognition and methylation and provide general principles for m5C deposition on cellular RNAs and their fundamental role in disease.

molecular biology↗

bioRxiv2026

The broad-spectrum RumC1 bacteriocin targets a transient peptidoglycan intermediate of the nascent cell wall

RumC1 is a structurally unique bacteriocin with broad-spectrum efficacy, including against multidrug-resistant pathogens, yet acting by an undefined mechanism. By integrating genetics, biochemistry, computational modeling and single-cell fluorescence microscopy, we demonstrate that RumC1 is a distinct cell-wall-targeting toxin. First, all RumC1-resistant mutants isolated through a high-rate, genome-wide mutagenic screening exhibited specific impairments in peptidoglycan homeostasis regulation, pinpointing this pathway as critical for RumC1 activity. Second, RumC1 selectively accumulates within neosynthesized peptidoglycan, leading to cell growth arrest and death in a dose-dependent manner. Third, we characterize the RumIc1 immunity protein of the RumC1 biosynthetic cluster as a peptidase acting at the cell surface to protect the cells by trimming the stem peptide crucial for cell-wall assembly. As such, RumIc1 provides cross-protection against vancomycin, while RumC1 is demonstrated to act differently from this glycopeptide antibiotic. Collectively, these findings establish RumC1 as a toxin targeting a key peptidoglycan intermediate of cell wall maturation.

microbiology↗

bioRxiv2026

Capping protein regulates the balance of assembly among diverse actin networks in C. elegans zygotes

Actin cytoskeleton networks exhibit specialized architectural properties for specific cellular tasks, as determined by the actin-binding proteins (ABPs) associated with each network. Proper allocation of a limiting pool of actin monomers also helps shape the assembly of different F-actin networks. The ABP capping protein (CP) modulates F-actin network architecture through regulation of actin filament length by capping filament barbed ends. Using a combination of in vitro biochemistry and quantitative live-cell imaging, we characterize CP as a major regulator of inter-network competition between filopodia and mini-comets, two F-actin networks in the one-cell C. elegans embryo (zygote). We establish that this regulation is facilitated in part by competition for binding barbed ends between CP and the F-actin elongator formin CYK-1. Together, these results reveal a role for CP in determining F-actin network architecture and dynamics, regulating the coordination between actin assembly factors to assemble and maintain different dynamic F-actin networks, and allocation of G-actin between competing cortical F-actin networks. Summary for table of contentsCells assemble diverse actin cytoskeleton networks within a common cytoplasm for essential cellular processes. Yde et al. establish a role for Capping Protein, a regulator of actin filament length, in coordinating the balanced assembly of distinct actin networks in the C. elegans zygote.

cell biology↗

bioRxiv2026

A Type VII-secreted toxin enables inter-mycobacterial competition

Summary ParagraphMost bacteria live in complex environments where resources are scarce and competition is fierce. These organisms have evolved mechanisms to compete with other bacteria, often through the specialised secretion of proteinaceous toxins. Mycobacteria have not previously been reported to engage in this form of competition. The thick and unusual cell wall of mycobacteria, comprised of peptidoglycan, arabinogalactan and mycolic acids, is generally thought to be highly protective to these bacteria. Many enzymes have evolved to maintain this structure, including the GH183 family, which cleaves arabinogalactan. Here, we establish for the first time that some mycobacteria have weaponised a subset of these endo-D-arabinanases to enable inter-bacterial competition. We show that mycobacteria secrete an endo-D-arabinanase effector via the type VII secretion system that specifically targets the arabinogalactan layer of the Mycobacteriales cell envelope. Using structural biology and biochemistry, we identify the molecular basis for this activity and reveal a new protein family that protects the bacterium from the activity of this toxin. Finally, our data uncover widespread T7-secreted toxins in the Mycobacteriales, pointing to extensive inter-mycobacterial competition.

microbiology↗

bioRxiv2026

Stage-dependent tau post-translational modifications map the spatiotemporal progression of Alzheimer's disease

Alzheimers disease (AD) is defined by progressive tau aggregation, yet the molecular events driving this process remain poorly understood. Post-translational modifications (PTMs) are key regulators of tau biology and potential biomarkers of disease progression. Using immunoprecipitation-mass spectrometry and absolute quantification of tau isoforms, we profiled tau PTMs in soluble and insoluble brain fractions. We studied multiple brain regions (hippocampus, inferior temporal and frontal gyri), representing regions affected at different stages of pathology, from human donors spanning the AD spectrum and staged by ABC neuropathological scoring. We uncovered a stage-dependent PTM landscape across AD progression: early phosphorylation changes, including pT217 and pS262, precede later ubiquitination events, such as uK311, associated with tau aggregation. We also identified PTMs negatively correlated with aggregation, including mK258, suggesting potential protective roles. These findings refine our understanding of the spatiotemporal evolution of tau biochemistry and offer mechanistic and translational insights into AD tauopathy.

neuroscience↗

bioRxiv2026

Astrocytes modulate mitochondrial proteostasis and membrane potential in neurons through S100A6 - calcyclin-binding protein signaling

Calcyclin binding protein (CaCyBp) is an evolutionarily conserved protein that converts S100 protein family-derived signals to protein turnover through an ubiquitin ligase interaction domain within its C-terminus. Despite its predicted roles in cellular differentiation, CaCyBp expression alone and in relation to S100A6, its prototypic ligand, during fetal and postnatal organ development remains incompletely understood. Here, we combined cell-resolved neuroanatomy and biochemistry to reveal not only organ system-level CaCyBp expression but also its region-, circuit-, and neuron-specific distribution in the mouse brain from embryonic day 18.5. We extended these studies to human brains past gestational week 27, corroborating that neurons, but not astrocytes, microglia or oligodendrocytes, harbored CaCyBp. Conversely, S100A6 was detected only in astrocytes and ependymoglia in both species. Ultrastructural studies localized CaCyBp to mitochondria in neurons, with genetic loss-of-function and recombinant S100A6-induced signaling implicating CaCyBp in the regulation of the mitochondrial electrochemical gradient controlling ATP production. Overall, pleiotropic CaCyBp expression in neurons suggests a maintenance receptor-like role impacting cellular respiration, thus affecting proteostasis upon glia-derived S100A6 signals.

cell biology↗

bioRxiv2026

Serum albumin-fused interleukin-10 prevents neuroinflammation by promoting immunoregulation in the secondary lymphoid organs and limiting immune cell infiltration in the spinal cord

Interleukin-10 (IL-10) is a potent immunoregulatory cytokine that suppresses pro-inflammatory cytokine production, reduces antigen presentation by myeloid cells, promotes M2 macrophage polarization, and inhibits T cell activation. Despite these well-established immunoregulatory functions, efforts to harness recombinant IL-10 therapeutically have been limited by its short plasma half-life and poor retention in the secondary lymphoid organs (SLOs), key sites of autoreactive T cell priming in autoimmune disease. Previously, we engineered a fusion of serum albumin and IL-10 (SA-IL-10) with extended half-life and enhanced exposure in the SLOs following intravenous administration. Here, we integrate human transcriptomic analyses and a murine model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE), to investigate how sustained IL-10 exposure in the SLOs modulates immune responses under inflammatory conditions. Human single-cell RNA sequencing analyses revealed reduced IL-10 expression alongside increased IL-10 receptor expression across multiple immune cell populations in treatment-naive patients with multiple sclerosis (MS), motivating the investigation of IL-10-based immunomodulatory strategies. Prophylactic SA-IL-10 administration prevented the development and progression of EAE with superior efficacy to wild type IL-10 and comparable protection to fingolimod, an FDA-approved MS therapy. Immunophenotyping of the SLOs revealed that SA-IL-10 suppressed pathogenic, antigen-specific ROR{gamma}t+ Foxp3- TH17 T cells, CD86+ M1-like macrophages, CD86+ dendritic cells, and pro-inflammatory cytokine production, while expanding immunoregulatory CD206+ M2-like macrophages and increasing the frequency of multiple checkpoint markers (CTLA-4, PD-1, TIGIT, ICOS) on GATA3+ Foxp3- TH2 cells. Despite the absence of direct central nervous system targeting, SA-IL-10 treatment also reduced the infiltration of macrophages, dendritic cells, and CD4+ T cells into the spinal cord. Repeated SA-IL-10 administration was well tolerated, as treated EAE mice gained significantly more body weight over the course of treatment compared to PBS- and WT IL-10-treated controls, and exhibited plasma biochemistry parameters comparable to control animals at study endpoint. Together, these findings demonstrate that increasing IL-10 exposure in the SLOs suppresses neuroinflammation by promoting immunoregulation.

immunology↗

bioRxiv2026

yakRNA Design: A semantic multimodal RNA composer

Like proteins, RNAs have been a target for exploitation to generate synthetic molecules that can adopt enhanced and novel functions1. High-throughput assays2, combined with meticulous biochemistry, have led to the generation of some artificial RNAs but the ability to algorithmically program RNAs with an intended function remains difficult. While generative models have revolutionized protein design3,4, RNA design remains challenging due to the dearth of 3D RNA structures. Here we present You Always Know RNA (yakRNA) Design, a frontier language model that can simultaneously reason over sequence, biological function, consensus sequence, and secondary structure to generate functional RNAs. yakRNA Design is not trained on any 3D structural information but rather co-learned over a corpus of semantically labeled sequences that we designed. yakRNA Design, without any human intervention, fine-tuning, sequence optimization, scoring or selection, zero-shot designed 17 (out of 84 total designs) synthetic RNAs that were able to efficiently induce ribosomes to change their reading frames during elongation at rates comparable to or higher than most natural sequences. One of these efficient synthetic RNAs was found to have no identity to any sequence in the known universe, demonstrating how a model with semantic understanding of RNA has rich generative capabilities.

synthetic biology↗

bioRxiv2026

TFAM organizes DNA into compact higher order structures

TFAM (Transcription Factor A, Mitochondrial) is an essential human protein that plays two key roles in mitochondrial DNA (mtDNA) homeostasis. TFAM acts as a transcription factor that specifically binds to promoter regions, but it is also solely responsible for organizing mtDNA into nucleoids by nonspecifically covering the entire genome. Many studies have addressed TFAM in transcription regulation, but its role as a genome organizing entity is not well characterized. The current understanding of how TFAM compacts DNA into nucleoids is based on crystal structures of a TFAM monomer bound to short fragments of DNA (22-28 bp). However, this does not adequately reflect the biological role of TFAM in organizing the nucleoid where multiple TFAM molecules oligomerize on the 16.5 kb genome to form the nucleoid. Here, we present a biochemical and structural analysis of TFAM oligomerization on longer DNA. Our results show that TFAM compacts longer segments of DNA into higher order complexes that are homogenous yet exhibit continuous conformational dynamics. Significance statementMutations or damage to mitochondrial DNA (mtDNA) severely impairs cellular respiration and is implicated in many human diseases and aging. Transcription Factor A, Mitochondrial (TFAM) is an essential protein that is solely responsible for packaging mtDNA into nucleoids thereby shielding it from DNA damage. Despite its importance, the mechanism by which this is accomplished is poorly understood. Here, we use biochemistry to show that TFAM oligomerizes on DNA to form compact, homogenous higher order structures in solution. We also examined these complexes at a low resolution using cryo-EM, suggesting an organizational unit of mtDNA. This work reveals there may be a regular organization to mitochondrial nucleoids, providing the basis for further understanding mtDNA compaction by TFAM.

biophysics↗

bioRxiv2026

Culsma: A Formal Language for Laboratory Protocols

Laboratory protocols are commonly communicated in natural language, leaving room for variation in how experimental commitments are understood and carried out. We present Culsma, an executable specification language in which protocols are written as programs. The same program is legible to bench scientists and directly machine-readable by software that checks and runs it. It serves as the protocol's operational specification, making experimental commitments explicit for checking before execution and retaining them in results. Culsma addresses how experimental procedures are modeled: it defines operations by the experimental changes and relationships they establish, giving a fine-grained, compositional basis whose technique- and device-specific realizations remain extensible. We examine this boundary in workflows spanning molecular and cell biology, protein biochemistry, and immunoassays, which combine material handling, separation, and measurement differently, asking which distinctions the operation interface preserves. A maintained, source-linked benchmark broadens this evaluation with versioned checks of source-step correspondence and software execution.

bioinformatics↗

bioRxiv2026

Task-Specialized Protein Language Models Decode the Sequence Grammar of Post-Translational Modification Sites

Post-translational modifications (PTMs) regulate protein signaling, localization, degradation, and cellular decision-making, yet the sequence determinants that distinguish modified from chemically eligible but unmodified residues remain difficult to decode at proteome scale. Here, we examine whether adapting a general protein language model to PTM-site prediction can reveal the biochemical logic underlying residue-level modification. We fine-tune ESM2, a protein language model trained on tens of millions of evolutionarily diverse protein sequences, for phosphorylation, acetylation, and ubiquitination-site prediction. To address the pronounced class imbalance inherent in proteome-wide PTM annotation, we combine parameter-efficient fine-tuning with focal-loss training. The resulting task-specialized models show that PTM recognition depends on model capacity, annotation depth, and modification chemistry: phosphorylation benefits from larger models, whereas acetylation and ubiquitination peak at intermediate scale. More importantly, the fine-tuned phosphorylation model exposes three layers of biological organization: it recovers canonical kinase-recognition motifs without kinase-label supervision, resolves pathway-level functional relationships among proteins from sequence-derived embeddings, and preserves evolutionary signatures of homologous phosphorylation sites across 200 eukaryotic species. These results establish task-specialized protein language models as interpretable instruments for probing PTM-site biochemistry, kinase specificity, functional organization, and evolutionary conservation.

bioinformatics↗

bioRxiv2026

Protein solubility depends on centrifugation: Aiki-Sol, a per-regime predictor for E. coli

MotivationSequence-based predictors of recombinant protein solubility in Escherichia coli have plateaued (NESG independent-test AUC 0.760 [->]~ 0.80 over eight years of protein-language-model variants). The plateau hides a latent confound: the centrifugation regime used to separate the soluble from the insoluble fraction is a hidden variable collapsed into a single binary "soluble" label. The proteins biochemistry does not change between regimes; what changes is which fraction of the lysate is recovered as soluble. Existing predictors treat the regime as label noise rather than a feature, and sequence overlap between training and test partitions masks the resulting failure mode. ResultsWe release the Aiki-Sol Dataset, a tiered E. coli solubility corpus: a ~ 85K stringency-annotated benchmark, an Apache-licensed ~ 147K extension adding binary-only-labelled proteins, and a ~ 229K research-tier pool incorporating non-commercially-licensed sources. On the ~ 85K benchmark, scored on sequence-cluster-disjoint partitions, the strongest published binary comparator falls below chance on the 32,000 x g stratum (AUC 0.491 {+/-} 0.020); a fine-tuned ESM-2 650M backbone with five protocol-matched out-puts lifts pooled AUC by +0.108 (paired-bootstrap CI lower bound +0.090). The gain is curation, not architecture: structure-aware predictors given ESMFold structures do not outperform the sequence-only frame, and capacity scaled to 3B parameters does not exceed the conditioned 650M backbone. The released model, Aiki-Sol, jointly supervises five per-stringency outputs alongside a marginal output for stringency-unknown proteins; on five external cohorts it lifts cohort-mean AUC from 0.69-0.70 to 0.825, with a [≥] +0.10-0.16 lift on the three cohorts at measurably-zero training-pool overlap. Availability and implementationAiki-Sol model weights (Apache 2.0), the 147K-row license-clean training pool of the deployment checkpoint (CC BY 4.0), the cluster-disjoint per-stringency 5-fold partition assignments, per-cohort prediction CSVs, and source code for training, inference, and figure reproduction are available at https://github.com/aikium-public/aiki-sol and archived at Zenodo 10.5281/zenodo.20151817. The research-tier 229K checkpoint is released under CC-BY-NC-ND 4.0 (inheriting the most-restrictive upstream-source tier); its training CSV and the 84,809-protein stringency-annotated bench-mark of [§]2.1 mix non-commercial-tier upstream sources and are not redistributed verbatim. Upstream sources are documented in Data availability and SI [§]S1. The deployment artefact is distributed as a Python package (pip install aikisol) with a predict(seq) entry point. Contactvenkatesh@aikium.com. Supplementary informationSupplementary text, figures, and tables are available at Bioinformatics online.

bioinformatics↗

bioRxiv2026

Continuous negative autoregulation fine-tunes dosage-sensitive transcription factor expression to maintain post-mitotic neuron identity

How post-mitotic neurons maintain precise transcription factor (TF) levels throughout life remains a fundamental open question. Here, we challenge the prevailing model of positive autoregulation by demonstrating that UNC-3 (Collier/EBF1-4), a dosage-sensitive TF continuously required for cholinergic motor neuron identity in C. elegans, negatively regulates its own expression. Using genetics, biochemistry, and inducible protein depletion, we show this self-repression occurs directly at the transcriptional level and persists beyond development. CRISPR/Cas9 disruption of negative autoregulation causes motor neuron identity and locomotion defects, establishing its functional necessity. Mechanistically, the UNC-3 DNA-binding domain is required and sufficient for self-repression, with an AlphaFold2 screen implicating chromatin factors as interaction partners. Critically, UNC-3 self-repression is continuously counterbalanced by positive input from the HOX cofactor CEH-20/PBX, revealing a dynamic "balancing act" between opposing regulatory inputs that stabilize TF dosage over time. Mutations in the unc-3 ortholog EBF3 cause a neurodevelopmental syndrome, and disease-associated variants disrupt UNC-3 self-repression, revealing a key molecular mechanism underlying the disorder. We propose that negative autoregulation continuously counteracted by positive input represents a broadly applicable principle for maintaining dosage-sensitive TF expression to secure post-mitotic cell identity.

developmental biology↗

bioRxiv2026

A neonatal rat sepsis score captures the time course and severity of disease in a clinically relevant rat peritonitis model.

BackgroundNeonatal sepsis is a major cause of infant morbidity and mortality worldwide, particularly in preterm and very low birthweight babies. Fundamental differences between neonates and adults warrant clinically relevant models of neonatal sepsis. Here, we describe a preclinical fecal-slurry (FS)-induced peritonitis model of polymicrobial sepsis in neonatal rats, along with a novel neonatal rat sepsis score (nRSS) to monitor illness severity. MethodsPeritonitis was induced in 3-day-old Sprague Dawley rats by intraperitoneal injection of various doses (0.3-1.5mg/g body weight) of fecal slurry (FS); control pups received equivalent doses of vehicle. All pups received analgesics (buprenorphine), antibiotics (ampicillin and gentamicin), and fluids (saline) to model clinical standards of sepsis treatment. Time-dependent changes in circulating cytokines (IL-6, IL-1{beta}) and biomarkers of sepsis pathology (hemoglobin, glucose, alanine transaminase [ALT] levels) were assessed and correlated with nRSS scores. ResultsFS administration caused a dose-dependent increase in severity of sepsis over time, as indicated by increases in mortality rates (based on predefined criteria for euthanasia), nRSS scores, as well as time-dependent changes in circulating glucose, hemoglobin, IL-6, IL-1{beta}, and ALT activity levels. nRSS scores correlated with all quantitative measures of sepsis pathology. Notably, females showed higher mortality and higher early NRSS scores than males at moderate to high FS doses, yet biochemical markers and time of death did not differ between sexes, suggesting that the apparent female vulnerability may reflect more conspicuous behavioral manifestations of illness rather than greater underlying physiological severity. ConclusionInduction of peritonitis in rats at postnatal day 3 produced a consistent and reproducible model of polymicrobial neonatal sepsis. Illness severity was monitored using a newly developed nRSS. By minimizing distress and incorporating standards of care, this model and scoring system may serve as a platform for future investigations into the underlying mechanisms and potential therapeutic interventions for neonatal sepsis. ImpactO_LIA clinically relevant rat model of neonatal polymicrobial sepsis was developed, incorporating standards of care (analgesics, antibiotics, and fluid resuscitation) to better reflect the clinical context in which preclinical findings must ultimately translate. C_LIO_LIA novel neonatal rat sepsis scoring system (nRSS) was developed and validated, providing a sensitive, non-invasive measure of disease severity that correlates with biochemical markers and predicts mortality. C_LIO_LIFemale pups showed higher mortality and earlier behavioral signs of illness than males despite equivalent biochemistry, highlighting that clinical scores may capture sex-dependent vulnerability not apparent in standard biochemical measures. C_LIO_LITogether, this model and scoring system offer a refined platform for mechanistic and therapeutic studies of neonatal sepsis while advancing the welfare-conscious 3Rs principles essential to rigorous preclinical research C_LI

physiology↗

bioRxiv2026

Deletion of the MALAT1 RNA 3' end Promotes Transcript Decay, TALAM1 downregulation, and Inhibits Proliferation in Gastric and Breast Cancer Cells

The long non-coding RNA MALAT1 is a conserved oncogenic driver whose function relies on a 3 triple-helix motif. While its biochemistry is well-characterized in vitro, the endogenous requirement for this motif in regulating the stability of the transcript and other genes residing in its locus remains unclear. In this study, we employed a dual-sgRNA CRISPR-Cas9 approach to systematically excise triple-helix-forming sequences from the native MALAT1 locus in gastric (AGS) and breast (MCF7) cancer cells. Our findings demonstrate that the 3 end strongly contributes to MALAT1 stability. Perturbations ranging from genomic deletions to a single-base changes trigger transcript collapse and rapid exonucleolytic decay, while the biogenesis of the small RNA mascRNA (a byproduct of MALAT1, also involved in cancer) remains decoupled and unaffected. In cellulo, DMS probing reveals that edited transcripts retain structural complexity in the 3 region. Phenotypically, structural disruption of the 3 end significantly impairs proliferation of both cancer cellular models. These results identify the 3 triple-helix as a determinant of MALAT1 stability and provide endogenous validation for its role in the analyzed AGS and MCF7 cells.

molecular biology↗

bioRxiv2026

Differential and compensatory roles for type I phosphatidylinositol-4-phosphate-5-kinase isoforms in retinal function and health.

Phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) plays important roles in development, signaling, intracellular trafficking and regulation throughout the nervous system. Using selective and combined gene ablation strategies, we have determined the roles of this lipid and the kinase isoforms of the PIP5KI family primarily responsible for its synthesis in mouse retina. In rod cells, PI(4,5)P2 localizes predominantly to the plasma membrane of inner and outer segments and is enriched in membranes near the synaptic termini. Disruption of the gene encoding the {gamma} PIP5KI isoform, Pip5k1c, throughout the developing retina, using Cre expression driven by a Six3 transcription factor-dependent promoter, yields dramatic, but not complete, loss of the protein, with no apparent effects on morphology or function through the first 3-4 months after birth. Slowly progressing photoreceptor degeneration is observed at later ages. Complete loss of the {gamma} isoform in rods, driven by the rhodopsin promoter-based iCre75 transgene, leads to no obvious developmental defects, but results in an earlier-onset rod degeneration. Germ-line ablation of neither the Pip5k1a nor the Pip5k1b gene leads to any observable morphological defects. Homozygous Pip5k1a ablation leads to functional defects in photoreceptors as revealed by reduced a-wave and b-wave amplitudes in the electroretinograms. On the background of rod-specific Pip5k1c ablation, Pip5k1a deficiency greatly accelerates retinal degeneration. These results reveal a complex interplay among PIP5KI isoforms in ensuring proper photoreceptor function and health, with apparent partial redundancy in fulfilling their critical functions. They underscore the important role of PI(4,5)P2 in neuronal signaling and homeostasis. Significance StatementPhosphatidylinositol(4,5)P2, PI(4,5)P2, plays essential roles in nervous system development and function, but its roles in retina have been unknown. This study combines biochemistry, mouse genetics, light- and electron microscopy to reveal both specific and redundant functions for PIP2 formed by different kinase isoforms in the mammalian retina. It has implications for retinal function, disease and therapy, and for the broader field of phosphoinositide regulation.

neuroscience↗

bioRxiv2026

TrbA binds and locks a sliding clamp KorB to repress transcription on multi-drug resistance plasmids

Precise regulation of gene expression is essential for all living systems. Genes encoded on mobile genetic elements such as conjugative plasmids are also highly regulated to ensure stable inheritance, successful horizontal transfer, and adaptation to diverse bacterial hosts. The multi-drug resistance plasmid RK2 encodes an intricate regulatory network centred on the ParB-CTPase family protein KorB, which functions in both plasmid segregation and global gene regulation. We recently showed that KorB acts as a CTP-dependent DNA-sliding clamp that can be converted into a transcriptional repressor through direct interaction with the DNA-binding protein KorA. This clamp sliding-and-locking mechanism by KorAB enables highly effective transcriptional repression. Here, we investigate whether a third transcriptional regulator on the RK2 plasmid, TrbA, operates through a similar mechanism. Using a combination of structural prediction, biochemistry and in vivo transcriptional assays, we provide evidence that TrbA also directly interacts with KorB and functions as a clamp-locking factor. This KorB-TrbA interaction is mediated by a conserved aromatic interface that enables strong cooperative transcriptional repression. Disruption of this interface abolishes synergy, indicating that direct protein-protein interaction underpins KorB-TrbA cooperativity. Finally, bioinformatic analysis of a large plasmid database reveal that the tripartite KorB-KorA-TrbA system is present on numerous plasmids. Together, our findings establish TrbA as a bona fide KorB co-repressor and demonstrate how a single sliding clamp protein can integrate multiple partners to evolve a complex transcriptional regulatory network. IMPORTANCEPrecise regulation of gene expression ensures gene products are produced at the right time and in the right amounts. Recent works uncovered a new mechanism of bacterial gene regulation based on a clamp sliding and locking in a multi-drug resistance plasmid, RK2. KorB functions as a CTP-dependent DNA-sliding clamp capable of traveling over a long genomic distance. Sliding KorB is captured and locked in place by a partner protein, KorA, forming a stable complex at target promoters to repress transcription. Here, we show that another RK2 regulator, TrbA, also use this clamp sliding-locking mechanism, and identify an aromatic interface enabling TrbA-KorB-mediated transcriptional repression. Our findings show how a single sliding clamp integrates multiple partners to build a complex transcriptional regulatory network.

microbiology↗

bioRxiv2026

Diel metabolic plasticity of CAM photosynthesis in MD-2 pineapple (Ananas comosus) under contrasting tropical environments: biochemical patterns and agronomic implications.

The current understanding of Crassulacean Acid Metabolism (CAM), including semi-controlled studies in pineapple, does not fully explain outcomes observed under commercial field conditions. Although empirical agronomy confirms a strong climatic influence on growth and development, mechanistic explanations at the metabolic level--particularly for photosynthate allocation--remain scarce. This study evaluated how environmental variation affects diel CAM outputs and how such effects can be agronomically interpreted. MD-2 pineapple plants were cultivated in contrasting natural environments across Costa Rica. Leaf samples were collected at defined phenological stages and at the end of CAM Phases I and IV. Field data revealed distinct metabolic balances between soluble sugar accumulation and nocturnal malic acid content. Under high radiation and temperature, sucrose concentrations increased markedly, reflecting shifts toward leaf growth over stem reserve storage. These shifts were associated with differences in harvest index, highlighting the role of sucrose dynamics in phenotypic plasticity. From a seed selection perspective, integrating CAM diel profiling into research protocols--together with physiological age (thermal units)--could provide a stronger basis for classifying planting material beyond current fresh-weight standards. Such integration would improve the prediction of photosynthetic performance, early establishment success, and ultimately, crop uniformity at harvest. Approximately 38% of diel metabolic patterns deviated from the classical CAM model, indicating dynamic regulatory mechanisms under field conditions. Understanding these patterns could improve the interpretation of yield variability, natural flowering incidence, and harvest index across agroecosystems. Recognizing these CAM particularities offers a path to bridge the gap between fundamental CAM biochemistry and agronomic application, enabling the development of precise management strategies that respond to metabolic plasticity under real-world conditions. Closing this gap is essential to enhance productivity, uniformity, and sustainability in pineapple agroecosystems.

plant biology↗