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bioRxiv2024

PhyloForge: Unifying micro and macro evolution with comprehensive genomic signals

With the explosive growth of biological data, the dimensions of phylogenetic research have expanded to encompass various aspects, including the study of large-scale populations at the microevolutionary level and comparisons between different species or taxonomic units at the macroevolutionary level. Traditional phylogenetic tools often struggle to handle the diverse and complex data required for these different evolutionary scales. In response to this challenge, we introduce PhyloForge-a robust tool designed to seamlessly integrate the demands of both micro- and macro-evolution, comprehensively utilizing diverse phylogenomic signals, such as genes, SNPs, structural variations, as well as mitochondrial and chloroplast genomes. PhyloForges groundbreaking innovation lies in its capability to seamlessly integrate multiple phylogenomic signals, enabling unified analysis of multidimensional genomic data. This unique feature empowers researchers to gain a more comprehensive understanding of diverse aspects of biological evolution. PhyloForge not only provides highly customizable analysis tools for experienced researchers but also features an intuitively designed interface, facilitating effortless phylogenetic analysis for beginners. Extensive testing across various domains, including animals, plants, and fungi, attests to its broad applicability in the field of phylogenetics. In summary, the developmental background and innovative features of PhyloForge position it with significant potential in the era of large-scale genomics, offering a new perspective and toolset for a deeper understanding of the evolution of life.

molecular biology↗

bioRxiv2026

Longitudinal Plasma Proteome Changes Before and After Catheter Ablation in Atrial Fibrillation

BackgroundProteins in human plasma serve as critical markers for predicting disease risk and guiding therapeutic development. Current prediction models for atrial fibrillation (AF) largely rely on electronic health records; however, the plasma proteome of patients with AF reflects key biological processes, including inflammation, that are not captured by clinical variables alone. Circulating inflammatory mediators contribute to electrical and structural remodelling in the atria, thereby sustaining the AF phenotype. Identification of plasma proteins associated with AF may therefore improve understanding of the inflammatory and other biological processes underlying AF pathophysiology. ObjectiveIn this study, we profiled the plasma proteome of patients with paroxysmal AF (pxAF), persistent AF (persAF), and non-AF controls using Olink assay technology. MethodsPlasma samples from 30 individuals were analysed with the Olink Reveal panel. Differential expression analysis of normalised protein expression (NPX) values was performed between groups, with differentially expressed proteins (DEPs) defined by P < 0.05. ResultsWe identified 87 DEPs in pxAF and 107 DEPs in persAF compared with controls. From these, we shortlisted 11 candidate proteins that were upregulated in persAF at baseline and showed reduced expression 12 months after catheter ablation. This subset of proteins is implicated in the regulation of inflammation (CCL23, CXCL10, IL33), metabolism (ALDH3A1, NDUFS6), cell-matrix adhesion (AFAP1L1, LGALS7, SPOCK1), and physiological signalling (NOS1, PROK1, PTH). ConclusionCollectively, these plasma proteins highlight systemic molecular mechanisms contributing to AF pathogenesis and represent potential AF-specific biomarkers warranting further investigation in larger clinical cohorts and mechanistic studies.

molecular biology↗

bioRxiv2020

Direct RNA-RNA interaction between Neat1 and RNA targets, as a mechanism for RNAs paraspeckle retention

Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3UTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA-RNA interactions. Using a RNA pull-down procedure adapted to select only RNAs engaged in direct RNA-RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5 end of Neat1 demonstrating that they are located in the shell region of paraspeckles.

molecular biology↗

bioRxiv2022

An ultra high-throughput, massively multiplexable, single-cell RNA-seq platform in yeasts

Yeasts are naturally diverse, genetically tractable, and easy to grow in a myriad of experimental conditions such that researchers have the ability to investigate any number of genotypes, strains, environments, or the interaction thereof. However, studies of variation in the yeast transcriptome have been limited by the processing capabilities of available RNA sequencing techniques. Here we optimize a powerful, high-throughput single-cell RNA sequencing (scRNAseq) platform for yeasts. This platform utilizes a combinatorial barcoding strategy to enable massively parallel RNA sequencing of hundreds of yeast genotypes or growth conditions at once. This method can be applied to most species or strains of yeast for a fraction of the cost of traditional scRNAseq approaches. Thus, our technology permits researchers to leverage "the awesome power of yeast" by allowing us to survey the transcriptome of hundreds of strains and environments in a short period of time, and with no specialized equipment. The key to this method is that sequential barcodes are probabilistically appended to cDNA copies of RNA while the molecules remain trapped inside of each cell. Thus, the transcriptome of each cell is labeled with a unique combination of barcodes. Since we use the cell membrane as a container for this reaction, many cells can be processed together without the need to physically isolate them from one another in separate wells or droplets. Further, the first barcode in the sequence can be chosen intentionally to identify samples from different environments or genetic backgrounds, enabling multiplexing of hundreds of unique samples in a single experiment. In addition to greater multiplexing capabilities, our method also facilitates a deeper investigation of biological heterogeneity given its single-cell nature. For example, in the data presented here we report transcriptionally distinct cell states related to cell cycle, growth rate, metabolic strategies, stress responses, etc. all within clonal yeast populations grown in the same environment. Hence, our technology has two obvious and impactful applications for yeast research: the first is the general study of transcriptional phenotypes across many strains and environments, and the second is investigating cell-to-cell heterogeneity across the entire transcriptome.

molecular biology↗

bioRxiv2024

Assessment of salivary microRNA by RT-qPCR: Challenges in data interpretation for clinical diagnosis

Salivary microRNAs (miRNAs) have been recently revealed as the next generation of non-invasive biomarkers for the diagnostics of diverse diseases. However, their short and highly homologous sequences make their quantification by RT-qPCR technique highly heterogeneous and study dependent, thus limiting their implementation for clinical applications. In this study, we evaluated the use of a commercial RT-qPCR kit for quantification of salivary miRNAs for clinical diagnostics. MethodsSaliva was sampled from ten healthy volunteers for a time course analysis. A panel of six miRNA targets (with different sequence homologies) were analysed by one of the most commonly used commercially available RT-qPCR kit. Sensitivity and specificity of the tested miRNA assays were corroborated using synthetic miRNAs. The reliability of all tested assays to differentiate miRNA expression profiles were analysed, to statistically discriminate background noise from intrinsic individual signals. ResultsSignificant variabilities in expression profiles of six miRNAs from ten healthy participants were revealed, yet the poor specificity of the assays offered insufficient performance to associate these differences to biological context. Indeed, as the limit of quantification (LOQ) concentrations are from 2-4 logs higher than that of the limit of detection (LOD), the majority of the analysis for salivary miRNAs felt outside the quantification region. Most importantly, a remarkable number of crosstalk reactions exhibiting considerable OFF target signal intensities was detected, indicating their poor specificity and limited reliability. However, the spike-in of synthetic miRNA increased the capacity to discriminate endogenous salivary miRNA at the LOQ concentrations from those that were significantly lower. ConclusionsOur results demonstrate that comparative analyses for salivary miRNA expression profiles by this commercial RT-qPCR kit are most likely associated to technical limitations rather than to biological differences. In particular, assessment of fundamental parameters including LOD, LOQ and crosstalk of each assay is strictly necessary to interpret observed variations. The standardization of rigorous sample handling and experimental design according to technical parameters of each assay plays a crucial role in reducing data inconsistencies across studies. However, further technological breakthroughs are still required to overcome discrepancies in order to accelerate the translation of salivary miRNAs for clinical applications.

molecular biology↗

bioRxiv2025

Transmission potential of Culex and Aedes species for Madariaga virus, a member of the eastern equine encephalitis virus complex

Madariaga virus (MADV), widely distributed in Latin America, can cause severe disease in humans and equids, yet, key aspects of its transmission cycle remain unclear. To identify mosquitoes that could act as MADV vectors, we assessed the vector competence of Aedes aegypti, Ae. albopictus, Ae. taeniorhynchus, Culex tarsalis, Cx. coronator, and Cx. quinquefasciatus, following oral exposure to MADV isolated in Panama (all species) or Brazil (Ae. taeniorhynchus only). MADV RNA and infectious virus were quantified from mosquito bodies, legs, and saliva. At 14 days post-exposure, five species had virus in all biological sample types. Culex quinquefasciatus was susceptible to infection and dissemination but had no positive saliva samples. Aedes taeniorhynchus showed higher infection probabilities with MADV-BR. Time-course analysis revealed distinct dynamics in Ae. aegypti and Ae. albopictus. Our findings indicate MADV may be compatible with mosquito species present in endemic regions and areas at risk of virus introduction.

molecular biology↗

bioRxiv2018

miRNAs in platelet-poor blood plasma and purified RNA are highly stable: a confirmatory study

The relative stability of microRNAs (miRNAs) as compared with other RNA molecules has been confirmed in many contexts. When bound to Argonaute (AGO) proteins, miRNAs are protected from degradation, even when released into the extracellular space in ribonucleoprotein complexes, and with or without the protection of membranes in extracellular vesicles (EVs). Purified miRNAs also appear to present less of a target for degradation than other RNAs. Although miRNAs are by no means immune to degradation, biological samples subjected to prolonged incubation at room temperature, multiple freeze/thaws, or collection in the presence of inhibitors like heparin, can typically be remediated or used directly for miRNA measurements. Here, we provide additional confirmation of early, well validated findings on miRNA stability and detectability. Our data also suggest that inadequate depletion of platelets from plasma may explain the occasional report that freeze-thaw cycles can adversely affect plasma miRNA levels. Overall, the repeated observation of miRNA stability is again confirmed.

molecular biology↗

bioRxiv2026

Seminal extracellular vesicles from boar AI doses contain fertility-predictive protein and miRNA cargo and improve sperm physiology

Boar semen contains spermatozoa and seminal plasma (SP) that carries extracellular vesicles (EVs) among other components. However, artificial insemination (AI) doses produced by AI companies are highly diluted based solely on sperm concentration. The aim of this study was to evaluate the integrity of EVs isolated from AI doses, characterize the protein and miRNA content from high-fertility (HF) and reduced-fertility (RF) boars, and evaluate their functional impact on spermatozoa after dilution by a coincubation up to 24 hours at 38 {degrees}C. Proteomics identified 108 differentially expressed proteins between HF and RF EVs (97 upregulated in HF, 11 in RF), and transcriptomics revealed 80 differentially expressed miRNAs (DEMs) in EVs, 52 in SP, and 3 in spermatozoa, showing inverse expression in various shared DEMs between fertility rates, suggesting compartment-specific regulation. Functional coincubation demonstrated that EVs remain biologically active after dilution. HF EVs improved sperm quality parameters and reduced oxidative stress, while RF EVs increased total and progressive motility. Overall, our findings show that EVs from AI doses retain structural integrity, carry fertility-associated protein and miRNA signatures, and functionally modulate sperm quality in vitro. These features highlight porcine EVs as promising biomarkers and potential tools to optimize reproductive performance in swine production.

molecular biology↗

bioRxiv2026

The association of extracellular vesicle (EV)-cargo miR-330-3p with postoperative delirium and a potential mechanism of tau phosphorylation and neuron toxicity

BackgroundPostoperative delirium (POD) is a frequent and severe neurocognitive complication following cardiac surgery, associated with poor long-term outcomes. The underlying mechanisms are unclear, and objective biomarkers are urgently needed. MethodsWe used pre- and post-operative plasma samples from 59 patients undergoing cardiac surgery in three separate studies with rigorous delirium assessment using the Confusion Assessment Method in a case-control design. Small extracellular vesicles (sEVs) were isolated from plasma, and their miRNA cargo was profiled using RNA sequencing. Target miRNAs were validated by qRT-PCR, and digital PCR (dPCR). The functional impact of the lead candidate miRNA was investigated in vitro by assessing tau phosphorylation and cell viability in HT22 neuronal cell line. ResultsThere were no differences in sEV morphology or numbers between patients with and without POD. While three candidate miRNAs were initially validated by qRT-PCR, subsequent dPCR analysis confirmed that only the perioperative change in plasma sEV-cargo miR-330-3p expression was significantly greater in patients who developed POD (n = 20) compared with those who did not (n = 20) (5.22 copies/L plasma; 95% Confidence Interval (CI), 1.187 to 9.256; p = 0.0139). Receiver operating characteristic curve analysis for this change yielded an area under the curve of 0.745 (95% CI, 0.589 to 0.901). In vitro overexpression of miR-330-3p in a neuronal cell line significantly increased the phosphorylation of tau at Ser199 (p < 0.0001) and Ser396 (p < 0.001) and reduced cell viability (p < 0.001). ConclusionsOur findings suggest that sEV-bound miR-330-3p increases in patients with POD after cardiac surgery. In vitro results suggest a potential pathogenic role for miR-330-3p, linking a systemic signal to tau-related neuronal injury. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study identifies a specific perioperative increase in small extracellular vesicle (sEV)-cargo miR-330-3p in patients with postoperative delirium (POD) following cardiac surgery. C_LIO_LIWe provide the first evidence that miR-330-3p directly induces tau hyperphosphorylation and reduces neuronal viability in vitro, establishing a potential mechanistic link between systemic sEV signaling and neurodegeneration. C_LI What Are the Clinical Implications?O_LIThe measurement of perioperative change in miR-330-3p could serve as an objective biological marker to assist in the early identification and risk stratification of patients at high risk for POD. C_LIO_LIThe identified miR-330-3p/tau pathway represents a potential new therapeutic target; future interventions aimed at inhibiting this specific miRNA might help prevent or mitigate POD-related neuronal injury. C_LIO_LIThese findings emphasize the importance of monitoring dynamic sEV-cargo changes to better understand and manage perioperative neurocognitive disorders. C_LI

molecular biology↗

bioRxiv2023

S100A1's single cysteine is an indispensable redox-switch for the protection against diastolic calcium leakage in cardiomyocytes

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CM). Oxidative posttranslational modification (ox-PTM) of S100A1s conserved, single cysteine residue (C85) via reactive nitrogen species (i.e. S-nitrosylation or glutathionylation) was proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecules affinity towards Ca2+. In light of the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox-switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wildtype (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and {beta}-adrenergic receptor ({beta}AR) stimulation. However, in contrast ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ leak and ryanodine receptor (RyR2) hypernitrosylation during {beta}-AR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1s molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox-switch that is indispensable for S100A1s antiarrhythmic but not its inotropic potency in CM. We therefore propose a model where C85s ox-PTM determines S100A1s ability to beneficially control diastolic but not systolic RyR2 activity.

molecular biology↗

bioRxiv2021

A workflow for simultaneous detection of coding and non-coding transcripts by ribosomal RNA-depleted RNA-Seq.

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was highly efficient, resulting in less than 1.5% rRNA content in the final library, significantly better than other reported rRNA removal techniques. We identified >30,000 unique transcripts from all samples, including protein-coding genes and many unique species of non-coding RNA, in biologically-relevant proportions. Furthermore, normalized sequencing read count for select genes significantly negatively correlated with Ct values from RT-qPCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

molecular biology↗

bioRxiv2022

Evidence that Nek1 does not phosphorylate Rad54-S572 during recovery from IR

A main focus of the work in our lab is on the activity of Tousled Like Kinase 1 (TLK1) in the area of DNA Damage and Repair. As one of its key interactor, TLK1 phosphorylates NIMA related kinase 1 (Nek1), and Nek1 was reported by Spies et al. to phosphorylate and regulate the activity of the key HRR protein Rad541, which suggested an intriguing signal transduction pathway: TLK1>Nek1>Rad54. In an effort to confirm such relations, we now report that we have not been able to reproduce key findings from that study. Specifically, we found that Nek1 does not phosphorylate RAD54-S572 as was reported. We generated Nek1-KO mouse NT1 cells2 and Nek1-Knock-down in Hek293 (same cells as in Spies et al.), and the pRAD54-S572 signal does not change with our custom Ab, with or w/o IR. When we used an Ab from the Lobrich lab, it detected an immunoreactive band of wrong size for RAD54, which also did not change after IR even in synchronized G2 cells, contrary to their report. We also note that their P-assignment was based on guessing a weak consensus Nek1 sequence, and that site-directed mutagenesis of RAD54-S572 failed to yield biological effects in their in in vitro studies1. To conclusively establish that S572 is not a site of phosphorylation of Nek1, we carried out a IVK with purified Nek1 and RAD54 followed by MS analysis of the phosphatides, which revealed several but not S572. We also could not reproduce their copurification of Nek1-RAD54 by coIP, calling into question this interaction. Neither we could reproduce their results demonstrating the importance of Nek1 for HRR using the same SceI-mediated DR-GFP conversion assays.

molecular biology↗

bioRxiv2024

Chemical imaging unveils mitochondria as the major site of medicinal biguanide accumulation within cells

Metformin (MET), a commonly prescribed medication for managing type 2 diabetes, has demonstrated various beneficial effects beyond its primary anti-diabetic efficacy. However, the mechanism underlying MET activity and its distribution within organelles remain largely unknown. In this study, we integrate multiple technologies, including chemical labeling, immunostaining, and high-resolution microscopy imaging, to visualize the accumulation of MET in organelles of cultured cells. To achieve this objective, an alkynylated MET probe is developed that preserves biological activity similar to biguanide drugs. As determined by biorthogonal chemical labeling and imaging, the MET probe selectively localizes to substructures within cells, contrasting with its probe control. Furthermore, the MET probe can be competitively and efficiently washed out through biguanide administration, demonstrating the specific activity of this probe in monitoring the cellular dynamics of biguanide drugs. Our results indicate that the MET probe can reach near-saturated concentrations within two hours and is rapidly eliminated within an additional two hours once the exogenous source of the drug is removed. Furthermore, we reveal that the MET probe primarily accumulates in mitochondria, particularly within the mitochondrial matrix, and has a minor presence in other organelles, such as lysosomes and endosomes. Together, this study provides the first view of the MET subcellular localization and lays the foundation for future investigations on its molecular targets and mechanisms of action in promoting human health.

molecular biology↗

bioRxiv2025

Phenotypic and Functional Characterization of Oncohistone Mutations in Breast Cancers

Although mutations in genes encoding histones display a similar prevalence to that of some other somatic mutations in cancer, the underlying mechanisms by which histone mutations drive tumorigenesis have not been fully explored. Herein, we curated missense mutations occurring in core histone genes in breast cancers using data from MSK-IMPACT and cBioPortal to identify high frequency breast cancer-associated histone gene mutations. We characterized 17 high frequency oncohistone mutations in H2A H2B, and H3 that are enriched in breast cancer samples and occurred at glutamate (E), aspartate (D), serine (S), and arginine (R) residues. Many of these mutants co-occur with PIK3CA mutations in breast cancer samples. The oncohistone mutants were expressed in MCF-7 breast cancer cells or MCF-10A mammary epithelial cells and screened for effects on oncogenic phenotypes in a variety of cell- and tumor-based assays (i.e., proliferation, migration, invasion, competitive outgrowth, transformation). In addition, we examined the effects of selected mutants on DNA damage and gene expression. Our results indicate that the collection of oncohistone mutants that we screened have varying phenotypic and functional effects. Some can promote cancer-related phenotypes, with H2B-E76Q, H3-E97K, and H3-E105K eliciting strong oncogenic phenotypes and alterations in gene expression. All three of these mutants showed cooperativity with an activating mutation in PIK3CA (E545K), or a chemical activator of PI3K (UCL-TRO-1938), in assays of MCF-10A proliferation. H3-E105K also strongly promoted transformation of MCF-10A cells in an assay of growth on low attachment substrate independently of PIK3CA. Our results indicate that some high frequency oncohistone mutants can have oncogenic activity in breast cancers, and may act as potential cancer drivers. Collectively, these observations presented here can be used as a resource to connect biological and molecular outcomes to oncohistones in breast cancers. SignificanceIn this study, we identified and characterized 17 high frequency oncohistone mutations in H2A H2B, and H3 that are enriched in breast cancer samples and promote cancer-related phenotypes when expressed in cells. Many of these oncohistone mutations co-occur in breast cancer samples with mutations in PIK3CA, the most frequently mutated gene in breast cancer, and may act as potential cancer drivers.

cancer biology↗

bioRxiv2025

A novel approach for the extraction of nucleic acids using a hybrid paper-plastic device

Nucleic acid-based diagnostics play a crucial role in early and accurate disease detection. However, conventional extraction approaches are expensive, time-consuming, and require complex instrumentation and skilled personnel, which restricts their use in resource-limited settings. To develop and evaluate a simple, low-cost, and equipment-free paper-based microfluidic cassette device for rapid extraction of nucleic acids (DNA and RNA) from biological samples. A paper-plastic cassette was fabricated by laminating filter paper or glass fiber substrates between thermally bonded plastic sheets. The system was tested using Klebsiella pneumoniae (DNA) and HeLa cells (RNA) with different lysis buffer formulations. Extracted nucleic acids were quantified using a Qubit fluorimeter and assessed by gel electrophoresis and PCR amplification. Results were compared with standard commercial extraction kits. Among tested substrates, LF1 combined with lysis buffers 2, 4, and 5 yielded the highest quality DNA and RNA with 260/280 absorbance ratios of approximately 1.8 (DNA) and 2.0 (RNA). The nucleic acid yields were comparable to commercial kits, and the extracted material was suitable for direct downstream applications, including PCR. The developed paper-plastic cassette enables rapid, affordable, and equipment-free extraction of nucleic acids. This platform shows strong potential for point-of-care molecular diagnostics, particularly in low-resource and decentralized healthcare settings.

molecular biology↗

bioRxiv2022

Synergistic Combined-proteomics Guided Mapping strategy identifies mTOR mediated phosphorylation of LARP1 in nutrient responsiveness and dilated cardiomyopathy

Increased activity of the mammalian target of rapamycin (mTOR) signalling pathway, a crucial nutrient sensor, exacerbates ageing and ageing-related diseases, including cancer and heart failure. To further elucidate the physiological role of the serine/threonine kinase mTOR, we devised a novel tractable proteomics strategy that combines interaction proteomics, proximity-based proteomics and quantitative phosphoproteomics to identify interactors with and potential substrates of mTOR. We identified 58 candidate mTOR substrates, several of which were further validated. Interestingly, several of these candidate mTOR substrates are involved in various aspects of RNA biology, including regulating stability and processing. We characterized in-depth one of the validated mTOR substrates, LARP1, an RNA binding protein. mTOR-dependent phosphorylation of LARP1 is nutrient-sensitive and controls the RNA-binding ability of LARP1. We show that mTOR activity and LARP1 and LARP1 phosphorylation levels are increased in a congenital mouse model of dilated cardiomyopathy (DCM) caused by a mutation in the Lamin A gene. This implicates LARP1 in the development of DCM.

molecular biology↗

bioRxiv2023

Analysis of ageing-dependent thiol oxidation reveals early oxidation of proteins involved in core proteostasis functions

Oxidants have a profound impact on biological systems in physiology and under pathological conditions. Oxidative post-translational modifications of protein thiols are well-recognized as a readily occurring alteration of proteins. Changes in protein thiol redox state can modify the function of proteins and thus can control cellular processes. However, chronic oxidative stress causes oxidative damage to proteins with detrimental consequences for cellular function and organismal health. The development of techniques enabling the site-specific and quantitative assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early oxidation targets in key biological processes governing the de novo production of proteins, folding, and protein degradation. Comparison to existing datasets reveals evolutionary conservation of early oxidation targets. To facilitate accessibility and cross-species comparison of the experimental data obtained, we created the OxiAge Database, a free online tool for the research community that integrates current datasets on thiol redoxomes in aged yeast, nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster, and mouse Mus musculus. The database can be accessed through an interactive web application at http://oxiage.ibb.waw.pl.

molecular biology↗

bioRxiv2025

Genetic response to light and carbon source variations in Trichoderma harzianum: The key regulatory roles of env1, cre1, and blr2

In filamentous fungi, light plays a key role in regulating physiological processes such as growth, conidiation, secondary metabolism, and the expression of hydrolytic enzymes. The processes that depend on light are controlled by photoreceptors, including BLR1, BLR2, and ENV1, as well as by signaling pathways involving heterotrimeric G-proteins and cyclic adenosine monophosphate (cAMP). Trichoderma harzianum is a promising candidate for biotechnological use and is able to promote hydrolytic reactions under biomass degradation conditions. However, the genetic mechanisms underlying its response to light remain poorly understood, especially under degradative conditions. This study aimed to assess the expression of carbohydrate-active enzymes (CAZymes), transcription factors (TFs), and signaling pathway proteins under different light conditions and carbon sources. The results revealed distinct patterns of relative gene expression influenced by these environmental factors, highlighting the complex regulatory mechanisms at play in T. harzianum. Moreover, our results suggested that env1, cre1, and blr2 are critical for adjusting to different light conditions and carbon sources. This highlights the importance of both factors in regulating gene expression and supporting metabolic adaptation in T. harzianum. To our knowledge, such findings have not been previously reported in the context of cellulose degradation for this species. Overall, these results offer valuable insights into how T. harzianum responds to environmental changes, revealing a complex regulatory network that is not only crucial for optimizing fungal growth in industrial applications but also deepens our understanding of its biology and ecological interactions. Highlights{blacksquare} Trichoderma harzianum is a key candidate for biotechnology applications. {blacksquare}Light and carbon control CAZymes, photoreceptors, key regulators, and signaling. {blacksquare}T. harzianum has distinct gene expression patterns under different light conditions and carbon sources. {blacksquare}env1, cre1, and blr2 regulate metabolic adaptation in T. harzianum. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/657067v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@8167c7org.highwire.dtl.DTLVardef@1c46f6borg.highwire.dtl.DTLVardef@16eddc8org.highwire.dtl.DTLVardef@1a2afa6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗