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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2022

Analytic representation of inhomogeneous-resolution maps of three-dimensional scalar fields

Refinement of macromolecular atomic models versus experimental maps in cryo-electron microscopy and crystallography is a critical step in structure solution. For an appropriate comparison, model maps should mimic imperfections of the experimental ones, mainly atomic disorder and its limited resolution, often inhomogeneous over the molecule. We construct these model maps as a sum of atomic contributions expressed through a specially designed function describing a solitary spherical wave. Thanks to this function, atomic contributions analytically depend on both an atomic disorder and the local resolution, a value associated now with each atom. Such fully analytic dependence of inhomogeneous-resolution map values on model parameters permits an efficient refinement of all these parameters together and, beyond structural biology, opens a way to solve similar problems in other research domains. One-Sentence SummaryAn analytic decomposition of 3D-oscillating functions results in efficient tools to calculate maps and refine atomic models.

molecular biology↗

bioRxiv2023

Ribosomal A-site interactions with near-cognate tRNAs drive stop codon readthrough

tRNAs serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. Besides this canonical role, tRNAs are involved in other processes like programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, we demonstrate that those that do, establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determines the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open a new direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

molecular biology↗

bioRxiv2025

A novel Notch and WNT signaling mechanism contribute to pediatric DCM: a pathway to new therapeutics.

BackgroundTherapies for pediatric idiopathic dilated cardiomyopathy (iDCM) are extrapolated from adult heart failure despite limited efficacy, suggesting fundamental biological differences. Our prior transcriptomic studies indicate activation of developmental signaling pathways, including Notch and WNT, in pediatric iDCM; however, their mechanistic contribution remains unknown. We tested whether reactivation of Notch and WNT/{beta}-catenin signaling drives pathological remodeling in postnatal hearts and whether pathway inhibition improves cardiac function. MethodsWe developed a juvenile rat model to reproduce age-dependent molecular features of pediatric iDCM using {beta}-adrenergic stimulation (isoproterenol, ISO) and secreted frizzled-related protein-1 (sFRP1), a circulating WNT modulator elevated in children with DCM. Cardiac function was assessed by echocardiography; pathway activation by immunoblotting and transcriptomics; myocardial stiffness by atomic force microscopy. Findings were compared with explanted pediatric and adult human myocardium. ResultsExplanted pediatric, but not adult iDCM hearts exhibited increased nuclear and cytoplasmic Notch intracellular domain (NICD) and {beta}-catenin. Combined ISO and sFRP1 treatment recapitulated key features of pediatric disease, including ventricular dilation, reduced ejection fraction, reactivation of the fetal gene program, and increased myocardial stiffness in the absence of fibrosis or hypertrophy. Bulk and single-nucleus RNA sequencing identified cardiomyocyte-specific activation of Notch and WNT pathways and reduced intercellular signaling diversity. Mechanistically, {beta}-catenin silencing attenuated Notch target gene activation and pathological remodeling in vitro. Pharmacologic Notch inhibition reduced NICD and {beta}-catenin accumulation, improved ventricular function, and normalized myocardial stiffness in vivo. ConclusionPediatric iDCM is characterized by pathological co-activation of developmental Notch-WNT signaling pathways that are not observed in adult disease. Reactivation of this axis promotes maladaptive remodeling and myocardial stiffening, and its inhibition improves cardiac function. These findings establish developmental signaling reactivation as a central mechanism of pediatric iDCM and support age-specific therapeutic strategies.

molecular biology↗

bioRxiv2019

A primer genetic toolkit for exploring mitochondrial biology and disease using zebrafish

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37 gene set and overall genomic structure ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also implicated to be a common component of a wide variety of other human illnesses ranging from neurodegenerative disorders like Huntingtons disease and Parkinsons disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system from genetics to imaging. This collection includes 22 mutant lines in 18 different genes created by locus-specific gene editing to induce frameshift or splice acceptor mutations leading to predicted protein truncation during translation. Also included are 6 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All of these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study of many different aspects of mitochondrial function with the goal of widening our understanding of their role in biology and human disease.

molecular biology↗

bioRxiv2026

DNA cut-ligation cyclization surpasses J-Factor limit by order of magnitude

We demonstrate DNA circularization efficiencies more than threefold greater than those predicted by classical Jacobson-Stockmayer theory for the ligation of linear double-stranded DNA. To quantify deviations from classical expectations, we experimentally calibrated the J-factor by ligating 452 bp DNA fragments bearing pre-cut, purified 4-nt overhangs at the optimal shortest minicircle length and at the highest DNA concentration; this reference value then enabled accurate calculation of expected cyclization efficiencies across the full range of DNA lengths (452-952 bp) and the three concentrations examined. These elevated cyclization efficiencies are enabled by simultaneous restriction cutting and ligation using the Type IIS enzyme BsaI-HFv2 in combination with T4 DNA ligase under finely optimized buffer conditions, yielding a 3.4-fold improvement over the classical estimate and achieving 75% cyclization at a high, practically relevant DNA concentration of 120 ng/{micro}l. We also identified a second enzyme, Esp3I, that exceeds the classical expectations by 2.3-fold (though less efficiently than BsaI-HFv2), while BbsI cut-ligation systematically underperformed expectations, providing insight into possible mechanisms underlying the outstanding performance of the first two enzymes. These results reveal a biologically mediated exception that overcomes the long-standing mechanistic expectations of Jacobson- Stockmayer theory and highlight the value of systematically screening enzyme combinations to discover additional systems capable of highly efficient intramolecular ligation.

molecular biology↗

bioRxiv2025

Internally twin-Strep tagged CD63 for rapid and efficient isolation of engineered extracellular vesicles encapsulating functional proteins

Extracellular vesicles (EVs) are important mediators of intercellular communication. Achieving high purity of intact engineered EVs through separation from the overwhelming background of unmodified vesicles and cellular or serum-derived contaminants remains a major challenge, yet is critical for fundamental studies of EV biology and EV-based therapeutics. To address this unmet need, we applied structure-guided engineering to canonical EV tetraspanins. Focusing on CD63 and CD9, we identified sites for internal insertion of the twin-Strep tag via flexible linkers into unstructured regions of their large extracellular loops. This design retains functional properties of the scaffolds while providing a robust molecular handle enabling rapid, efficient, and selective antibody-free affinity-based isolation of intact EVs using advanced Strep technology. Fusing proteins of interest (POIs) to the C-terminus of these scaffolds, either directly or via a photoactivatable protein, allowed POI loading into the lumen of twin-Strep tagged EVs with optional optogenetic control of POI release. Proteomic analysis confirmed the high purity of captured engineered EVs with >96% of contaminants removed, enabling sensitive detection of numerous EV-associated proteins. We also report the efficient removal of contaminating virus particles resembling EVs in size and density. Purified engineered EVs successfully delivered an encapsulated fluorescent reporter into target cells, bypassing dye labeling commonly used to track EVs. We demonstrate endosomal escape of the reporter, facilitated by EV decoration with vesicular stomatitis virus glycoprotein, and cytoplasmic release upon optogenetic activation. Our toolbox may serve as a broadly applicable strategy for the efficient production of intact and highly pure engineered EVs to support potential fundamental or translational EV studies.

molecular biology↗

bioRxiv2020

Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

The Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

molecular biology↗

bioRxiv2022

Telomerase-independent maintenance of telomere length in a vertebrate

Telomere shortening places a key limitation on cell proliferation1. In all vertebrates explored to date, this limitation is overcome by telomerase-dependent telomere extension. Failure to maintain telomere length results in premature ageing and functional impairments in highly replicative cell populations as telomeres erode2. Alternative lengthening of telomeres (ALT), a telomerase-independent mechanism, compensates for telomere loss in a subset of human cancer cell lines 2. Here, we demonstrate that the highly regenerative newt Pleurodeles waltl lacks telomerase activity, contains telomeres distinct from all known vertebrates in both sequence and structure, and deploys ALT for physiological telomere maintenance. This constitutes the first report of telomerase-independent resolution of the end-replication problem at the whole-organism level within Chordata. One-Sentence SummaryP. waltl telomere biology is distinct amongst vertebrates and uses ALT at the whole-organism level.

molecular biology↗

bioRxiv2023

Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

The most toxic forms of DNA damage are double-strand breaks (DSBs). We have previously shown that RNA polymerase II (RNAPII), phosphorylated at tyrosine 1 (Y1P) on the C- terminal domain, transcribes RNA at DSBs to promote efficient DNA repair. However, it is still unknown how transcription is regulated at DSBs. Here, we show that the trimeric SOSS1 complex (hSSB1, INTS3, and c9orf80) binds to Y1P RNAPII in response to DNA damage, hSSB1 binds to R-loops, and formation of the SOSS1 complex is required for the coexistence of replication protein A (RPA) and hSSB1 at DSBs. The damage-activated tyrosine kinase c- Abl phosphorylates hSSB1 to enable its binding to Y1P RNAPII and its recruitment to DSBs. Finally, we show both in vitro and in vivo that the SOSS1 complex and RNAPII form dynamic repair compartments at DSBs via liquid-liquid phase separation (LLPS). The loss of the trimeric SOSS1 leads to impaired DNA repair, highlighting its biological importance in the RNA-dependent DNA damage response. TeaserTrimeric SOSS1 complex and transcription contribute to phase separation at double-strand DNA breaks.

molecular biology↗

bioRxiv2024

G-quadruplexes in Haloferax volcanii

Archaea, a domain of microorganisms found in diverse environments including the human microbiome, represent the closest known prokaryotic relatives of eukaryotes. This phylogenetic proximity positions them as a relevant model for investigating the evolutionary origins of nucleic acid secondary structures such as G-quadruplexes (G4s), which play regulatory roles in transcription and replication. Although G4s have been extensively studied in eukaryotes, their presence and function in archaea remain poorly characterized. In this study, a genome-wide analysis of the halophilic archaeon Haloferax volcanii identified over 5, 800 potential G4-forming sequences. Biophysical validation confirmed that many of these sequences adopt stable G4 conformations in vitro. Using G4-specific detection tools and super-resolution microscopy, G4 structures were visualized in vivo in both DNA and RNA across multiple growth phases. Comparable findings were observed in the thermophilic archaeon Thermococcus barophilus. Functional analysis using helicase-deficient H. volcanii strains further identified candidate enzymes involved in G4 resolution. These results establish H. volcanii as a tractable archaeal model for G4 biology.

molecular biology↗

bioRxiv2025

Hypothetical LOC Genes as Biomarkers of Spaceflight Adaptation: A Comparative Study from ISS, Suborbital, and Earth-Based Experiments

Microgravity constitutes one of the most profound environmental stressors encountered by humans during spaceflight, capable of altering fundamental cellular processes and gene regulatory networks. While the effects of spaceflight on well-characterized protein-coding genes have been widely documented, little is known about the behavior of uncharacterized or poorly annotated genomic regions under these conditions. LOC (Locus) genes, often classified as long non-coding RNAs and excluded from conventional analyses, represent a largely unexplored component of the human transcriptome. In this study, we systematically investigated the transcriptional responses of LOC genes as part of the MESSAGE (Microgravity Associated Genetics) Science Mission, Turkiyes first human space biology initiative. Peripheral blood samples were collected from astronauts across five mission phases: pre-launch baseline, post-suborbital flight ([~]100 km), and on International Space Station (ISS) Days 4, 7, and 10 ([~]400 km). RNA-Seq analyses revealed six LOC genes with statistically significant expression changes (p < 0.05, Kruskal-Wallis test), alongside additional transcripts that, while not statistically significant, exhibited biologically meaningful temporal fluctuations. These dynamic profiles included continuous upregulation, transient activation with subsequent return to baseline, and delayed induction at later ISS stages, highlighting the functional diversity of LOC responses. To assess their translational potential, Open Reading Frame (ORF) analyses were performed on significant transcripts, revealing conserved ORF structures--most notably identical ORF33 sequences in LOC124905103 and LOC124900480-- suggesting coding capacity. Phylogenetic analyses further supported evolutionary clustering consistent with expression and ORF similarities. Collectively, these findings challenge the notion of LOC genes as transcriptional noise, instead positioning them as candidate biomarkers and functional elements of microgravity adaptation. By extending space biology research into the "dark genome," this study provides novel insights with potential implications for astronaut health monitoring and therapeutic development in long-duration missions.

molecular biology↗

bioRxiv2019

Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells and in vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

molecular biology↗

bioRxiv2026

A Transformer-based Multi-omics Model for Translation Efficiency in S. cerevisiae

Precise regulation of protein synthesis is fundamental to cellular homeostasis and remains a primary target for synthetic biology applications. However, the non-linear relationship between mRNA abundance and protein levels presents complexities that poses challenges for predictive engineering. Here, we present TRIM, a Transformer-based RNA Inference Model that leverages full-length mRNA sequences and multi-omics data to predict translation efficiency. By employing a Parallel Expert Mixer, TRIM achieves robust prediction accuracy (R2 [≥] 0.8,Pearson r [≥] 0.9). Trained on multimodal data from massive Saccharomyces cerevisiae isolates, TRIM demonstrates outstanding biological interpretability, helping to decipher complex translational patterns such as synergistic effects between bases, sequence-dependent codon preference in different stages, and distinct attention on key secondary structures. These results indicate that the integration of multi-omics data with holistic sequence modeling can effectively decode the cis-regulatory grammar of translation as well as providing a scalable and interpretable generative framework for future synthetic biology engineering. Availability and ImplementationThe source code and data used to produce the results and analyses presented in the manuscript are available from Github (https://github.com/ZeusLiu666/TRIM).

Molecular Biology↗

bioRxiv2021

Investigating densities of Symbiodiniaceae in two species of Antipatharians (black corals) from Madagascar

Here, we report the first methodological approach to investigate the presence and estimate the density of Symbiodiniaceae cells in corals of the order Antipatharia subclass Hexacorallia, known as black corals. Antipatharians are understudied ecosystem engineers of shallow (<30 m depth), mesophotic (30-150 m) and deep-sea (>200 m) reefs. They provide habitat to a vast number of marine fauna, enhancing and supporting coral reefs biodiversity globally. Nonetheless, little biological and ecological information exists on antipatharians, including the extent at which global change disturbances are threatening these corals. The assumption that they were exempted from threats related to climate change was challenged by findings of high density of dinoflagellates within three antipatharian colonies. Further methodical studies were necessary to investigate the regularity of these findings. An integrated design combining microscopy and molecular techniques was used to investigate the presence and estimate density of Symbiodiniaceae cells within two antipatharians species -Cupressopathes abies and Stichopathes maldivensis -from shallow and mesophotic reefs of SW Madagascar. Symbiodiniaceae-like cells were found within the two species from both shallow and mesophotic reefs, although the overall cell density was very low (0-4 cell mm-3). These findings suggest that high abundance of Symbiodiniaceae is not characteristic of antipatharians, which has relevant implications considering disruptions associated to climate change affecting other corals. However, the high densities of dinoflagellates found in antipatharian colonies exposed to higher light irradiance in other studies should be further examined.

molecular biology↗

bioRxiv2025

Small activating RNA AW1-51 (CEBPA-51) elicits targeted DNA demethylation to promote gene activation

Small activating RNAs are short double-stranded RNAs designed to upregulate transcription of target genes. By this virtue, they can be used to restore expression of genes frequently silenced in cancer. AW1-51 (also referred to as CEBPA-51), the first small activating RNA therapeutic to enter clinical evaluation, has demonstrated biological activity and safety in Phase II trials for hepatocellular carcinoma, both as monotherapy and in combination with sorafenib, and in Phase 1a/1b in combination with pembrolizumab for patients with advanced solid tumors. It targets the master regulator CCAAT enhancer-binding protein alpha, abnormally silenced by DNA methylation in a wide range of hematological and non-hematological malignancies. However, the molecular events enabling this mechanism are only partially elucidated. In this study, we uncovered the molecular basis for AW1-51-induced transcriptional reactivation of CCAAT enhancer-binding protein alpha demonstrating that by directly promoting DNA demethylation of its promoter restores its expression, protein synthesis, and consequently cell differentiation. These findings unveil AW1-51 as a prototype for RNA-based precision medicine enabling conditional expression of CCAAT enhancer-binding protein alpha in diseases characterized by aberrant gene silencing and extending its potential therapeutic impact beyond cancer.

molecular biology↗

bioRxiv2016

Gene regulation mechanism in drought-responsive grapevine leaves as revealed by transcriptomic analysis

Grapevine is economically important and widely cultivated fruit crop, which is seriously hampered by drought worldwide. It is necessary to understand the impact of glitches incurred by the drought on grapevine genetic resources. Therefore, in the present study RNA-sequencing analysis was performed using cDNA libraries constructed from both drought-stress and control plants. Results yielded, a total of 12,451 differentially expressed genes (DEGs) out of which 8,022 genes were up-regulated and 4,430 were down-regulated. Further physiological and biochemical analyses were carried out to validate the various biological processes involved in the development of grapevine in response to drought stress. Results also showed that decrease in rate of stomatal conductance in-turn decrease the photosynthetic activity and CO2 assimilation rate in the grapevine leaves and most ROS detoxification systems, including stress enzymes, stress related proteins and secondary metabolites were strongly induced. Moreover, various hormones were known to be induced in the present study in response to drought. Overall the present study concludes that these DEGs play both positive and negative role in drought tolerance by regulating different biological pathways of grapevine. However our findings have provided valuable gene information for future studies of abiotic stress in grapevine and other fruit crops.

Molecular Biology↗

bioRxiv2024

PCA-Plus: Enhanced principal component analysis with illustrative applications to batch effects and their quantitation

BackgroundPrincipal component analysis (PCA), a standard approach to analysis and visualization of large datasets, is commonly used in biomedical research for detecting similarities and differences among groups of samples. We initially used conventional PCA as a tool for critical quality control of batch and trend effects in multi-omic profiling data produced by The Cancer Genome Atlas (TCGA) project of the NCI. We found, however, that conventional PCA visualizations were often hard to interpret when inter-batch differences were moderate in comparison with intra-batch differences; it was also difficult to quantify batch effects objectively. We, therefore, sought enhancements to make the method more informative in those and analogous settings. ResultsWe have developed algorithms and a toolbox of enhancements to conventional PCA that improve the detection, diagnosis, and quantitation of differences between or among groups, e.g., groups of molecularly profiled biological samples. The enhancements include (i) computed group centroids; (ii) sample-dispersion rays; (iii) differential coloring of centroids, rays, and sample data points; (iii) trend trajectories; and (iv) a novel separation index (DSC) for quantitation of differences among groups. ConclusionsPCA-Plus has been our most useful single tool for analyzing, visualizing, and quantitating batch effects, trend effects, and class differences in molecular profiling data of many types: mRNA expression, microRNA expression, DNA methylation, and DNA copy number. An early version of PCA-Plus has been used as the central graphical visualization in our MBatch package for near-real-time surveillance of data for analysis working groups in more than 70 TCGA, PanCancer Atlas, PanCancer Analysis of Whole Genomes, and Genome Data Analysis Network projects of the NCI. The algorithms and software are generic, hence applicable more generally to other types of multivariate data as well. PCA-Plus is freely available in a down-loadable R package at our MBatch website.

bioinformatics↗

bioRxiv2024

Spatial mapping of dextran sodium sulphate-induced intestinal inflammation and its systemic effects

Inflammatory bowel disease (IBD) is a multifactorial disease and patients frequently experience extraintestinal manifestations affecting multiple sites. Causes of systemic inflammation remain poorly understood but molecules originating from the intestine likely play a role, with microbial and host small molecules polarizing host immune cells towards a pro- or anti-inflammatory phenotype. Using the dextran sodium sulphate (DSS) mouse model, which mimics the disrupted barrier function, microbial dysbiosis and immune cell dysregulation of IBD, we investigated metabolomic and phenotypic changes at intestinal and systemic sites. Using spatial biology approaches we mapped distribution and relative abundance of molecules and cell types across a range of tissues revealing significant changes in DSS-treated mice. Molecules identified as contributing to the statistical separation of treated from control mice were spatially localized within organs to determine their effects on cellular phenotypes through imaging mass cytometry. This spatial approach identified both intestinal and systemic molecular drivers of inflammation, including several not previously implicated in inflammation linked to IBD or the systemic effects of intestinal inflammation. Metabolic and inflammatory pathway interplay underpins systemic disease and determining drivers at the molecular level may aid the development of new targeted therapies.

molecular biology↗