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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Widespread regulatory specificities between transcriptional corepressors and enhancers in Drosophila

Animal development and homeostasis critically depend on the accurate regulation of gene transcription, which includes the silencing of genes that should not be expressed. Repression is mediated by a specific class of transcription factors (TFs) termed repressors that, via the recruitment of co-repressors (CoRs), can dominantly prevent transcription, even in the presence of activating cues. However, the relationship between specific CoRs and enhancers has remained unclear. Here, we used functional genomics to uncover regulatory specificities between CoRs and enhancers. We show that enhancers can typically be repressed by only a subset of CoRs. Enhancers classified by CoR sensitivity also show distinct biological functions and endogenous chromatin features. Moreover, enhancers that are sensitive or resistant to silencing by specific CoRs differ in TF motif content, and their sensitivity to CoRs can be predicted based on TF motif content. Finally, we identified and validated specific TF motifs that have a direct impact on enhancers sensitivity or resistance towards specific CoRs, using large scale motif mutagenesis and addition experiments. This study reveals the existence of TF motif-based regulatory rules that coordinate CoRs-enhancer compatibilities. These specificities between repressors and activators not only suggest that repression occurs via distinct mechanisms, but also provide an additional layer in transcriptional regulation that allows for differential repression at close genomic distances and offers multiple ways for de-repression.

molecular biology↗

bioRxiv2022

Deep learning-enabled design of synthetic orthologs of a signaling protein

Evolution-based deep generative models represent an exciting direction in understanding and designing proteins. An open question is whether such models can represent the constraints underlying specialized functions that are necessary for organismal fitness in specific biological contexts. Here, we examine the ability of three different models to produce synthetic versions of SH3 domains that can support function in a yeast stress signaling pathway. Using a select-seq assay, we show that one form of a variational autoencoder (VAE) recapitulates the functional characteristics of natural SH3 domains and classifies fungal SH3 homologs hierarchically by function and phylogeny. Locality in the latent space of the model predicts and extends the function of natural orthologs and exposes amino acid constraints distributed near and far from the SH3 ligand-binding site. The ability of deep generative models to specify orthologous function in vivo opens new avenues for probing and engineering protein function in specific cellular environments.

molecular biology↗

bioRxiv2023

Foreign Ribosome Inactivating Proteins as immune effectors in insects

Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA. This ribosomal modification inhibits protein synthesis. During the last years, we have reported the existence of these toxins in insects, where their presence is restricted to mosquitoes from the Culicinae subfamily (e.g. Aedes aegypti) and whiteflies from Aleyrodidae family (e.g. Bemisia tabaci). Combination of phylogeny and synteny analyses showed that both groups of genes are derived from two independent horizontal gene transfer (HGT) events. Interestingly, we found that RIP encoding genes have been evolving under purifying selection, indicating that they have a positive impact on fitness of host organisms. We also demonstrated that A. aegypti RIP genes are transcribed and their transcripts are polyadenylated. Although the biological roles of these toxins remain open to speculation, defense activities have been postulated for plant and bacterial RIPs. Based on these pieces of evidence, we hypothesize that RIPs play a similar protective role in insects. In this work, we report the occurrence of a third HGT event in Sciaroidea superfamily, supporting that RIP genes fulfill an important functional niche in insects. Analysis on transcriptomic experiments from the three groups of insects indicate a convergence in expression profiles which are compatible with immune effectors. Finally, we show the induction in RIP expression after infection with pathogens. Moreover, we show transcriptomic evidence of parasite SRL depurination. Altogether, our results strongly support the role of these foreign genes as immune effectors that confer fitness advantage to host insects.

molecular biology↗

bioRxiv2023

The nucleolar aberrancies that drive ribosome impairment induced by RNA binding proteins are hallmarks of aging

The nucleolus is a dynamic structure where ribosome subunits are produced. Indeed, nucleoli respond to any change in cellular homeostasis by altering the rate of ribosome biogenesis, thus working as a stress sensor. Therefore, an imbalance in ribosome biogenesis promotes changes in morphology and function and can evoke a nucleolar stress response. Changes in the structure and composition of nucleoli impair ribosome biogenesis and have been described as nucleolar stress, a mechanism related to aging and cancer. Here, we show the role of the RNA binding protein Hnrnpk in nucleolar dynamics and ribosome function. Hnrnpk is a ribonucleoprotein in charge of escorting nascent transcripts to its processing and nuclear export to ribosomes. When Hnrnpk is overexpressed, the nucleolus is altered and shows stress-like phenotype, with accumulation and delocalization of components such as Ncl, driving ribosome biogenesis impairment and halting protein translation. Nucleolin haploinsufficiency is correlated with enlarged nucleoli, increased ribosome components and translation and induces a reduction in lifespan. Thus, gain of Ncl generated by Hnrnpk overexpression can cause ribosome biogenesis defects associated with ribosome impairment leading to ribosomopathies and bone marrow failure syndrome. Aging and bone marrow failure share common biological hallmarks. Indeed, Hnrnpk overexpression and nucleolar stress trigger cell cycle arrest and senescence of the cells, a feature of both processes. Together, these findings support the idea that nucleolar abnormalities contribute to ribosome impairment, thus triggering the onset of hematopoiesis and the aging process. Here, we decipher a novel master regulator of this mechanism: Hnrnpk.

molecular biology↗

bioRxiv2019

The unique topologies of N6-Adenosine methylation (m6A) in land-plant mitochondria and their putative effects on organellar gene-expression

Mitochondria are the main source of ATP production and also contribute to many other processes central to cellular function. Mitochondrial activities have been linked with growth, differentiation and aging. As relicts of bacterial endosymbionts, these organelles contain their own genetic system (i.e., mitogenome or mtDNA). The expression of the mtDNA in plants is complex, particularly at the posttranscriptional level. Following transcription, the polycistronic pre-RNAs in plant mitochondria are processed into individual RNAs, which then undergo extensive modifications, as trimming, splicing and C[->]U editing, before being translated by organellar ribosomes. Our study focuses on N6-methylation of Adenosine ribonucleotides (m6A-RNA) in plant mitochondria. m6A is the most common modification in eukaryotic mRNAs. The biological significance of this highly dynamic modification is under investigation, but its widely accepted that m6A mediates structural switches that affect RNA stability and activity. By performing m6A-pulldown/RNA-seq (m6A-RIP-seq) analyses of Arabidopsis and cauliflower mitochondrial transcripts (mtRNAs), we provide with detail information on the m6A landscapes in angiosperms mitochondria. The results indicate that m6A targets different types of mtRNAs, including coding sequences, UTRs, introns and non-coding RNA species. While introns and noncoding-RNAs undergo multiple m6A modifications along the transcript, in mRNAs m6A-modifications are preferably positioned near start-codons, and may modulate the translatability of the m6A-modified transcripts.

molecular biology↗

bioRxiv2025

Proteins are a source of glycans found in preparations of glycoRNA

Recent discoveries show that RNA can be modified with sialylated glycans (termed glycoRNA), thus broadening our understanding of cellular glycosylation beyond traditional proteins and lipids. However, the pathway of RNA-glycosylation and its biological function remain elusive. Following the original glycoRNA isolation protocol, we also detect labelled glycans in small RNA preparations. However, glycosylated molecules showed resistance to treatment with RNase A/T1 but were sensitive to proteinase K digestion under denaturing conditions. Using liquid chromatography-mass spectrometry (LC-MS) based proteomics, we detect various proteins that co-purify with small but not large RNA preparations isolated from human or murine cells, including the glycosylated membrane protein LAMP1. Importantly, we further demonstrate that recombinant soluble LAMP1 can be purified following the glycoRNA isolation method. These findings suggest that glycoproteins co-purify with RNA using current glycoRNA purification protocols, thus representing a considerable source of glycans in samples of glycoRNA.

molecular biology↗

bioRxiv2019

RNF219 regulates CCR4-NOT function in mRNA translation and deadenylation

Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the uncharacterized RING finger protein RNF219 co-purifies and strongly associates with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, that mediates translational repression in a deadenylase activity-dependent and -independent manner. Strikingly, although RNF219, inhibits the deadenylase activity of CCR4-NOT, it enhances its capacity to repress translation of a targeted mRNA, an effect of RNF219 that requires its interaction with CCR4-NOT. We propose that RNF219 is an interacting partner of the CCR4-NOT complex that switches the translational repressive activity of CCR4-NOT from a deadenylation-dependent to a deadenylation-independent mechanism.

molecular biology↗

bioRxiv2026

Splicing of ultraconserved poison exons controls mitotic fidelity and stem cell viability

SR proteins are essential splicing regulators whose expression is controlled in part through poison exons (PEs) -- ultraconserved non-coding exons that trigger nonsense-mediated decay -- yet the biological functions of these elements remain undefined. Here, we show that homozygous deletion of SRSF3-PE or TRA2{beta}-PE is selected against in mouse embryos and human induced pluripotent stem cells (iPSCs), and that conditional PE deletion causes apoptotic death in iPSCs but is tolerated in post-mitotic neurons, revealing a proliferative-state-specific requirement. Mechanistically, PE deletion elevates SR protein levels, triggers widespread splicing dysregulation, and disrupts the correct splicing of a mitotic gene network associated with spindle defects and mitotic errors. These findings establish ultraconserved poison exons as essential regulators of mitotic splicing fidelity and stem cell viability.

molecular biology↗

bioRxiv2019

Vulnerability correlates with life history traits and the load of deleterious mutations in fish.

Understanding why some species accumulate more deleterious substitutions than others is an important question relevant in evolutionary biology and conservation sciences. Previous studies conducted in terrestrial taxa suggest that life history traits correlate with the efficiency of purifying selection and accumulation of deleterious mutations. Using a large genome dataset of 76 species of fishes, we show that the rate of deleterious mutation accumulation (measured via dN/dS, i.e. non-synonymous over synonymous substitution rate) is associated to the vulnerability, the life-history strategies, and the latitude of species. Our results, focusing on a large clade of aquatic species, generalizes previous patterns found so far in few clades of terrestrial vertebrates. These results also suggest that vulnerable species accumulate more deleterious substitutions than non-threatened ones, which give insights in how life-history traits, populations sizes and genetic risk of extinction can be tightly interconnected.

molecular biology↗

bioRxiv2025

Data-driven strategies for drug repurposing

Drug discovery is a complex, time-intensive, and costly process, often requiring more than a decade and substantial financial investment to bring a single therapeutic to market. Drug repurposing, the systematic identification of new indications for existing approved drugs, offers a cost-effective and expedited alternative to traditional pipelines, with the potential to address unmet clinical needs. In this study, we present a comparative analysis of drug-target interaction data from three extensively curated resources: ChEMBL, BindingDB, and GtoPdb, evaluating their release histories, curation methodologies, and coverage of approved and investigational compounds and targets. To facilitate therapeutic interpretation, we manually classified ChEMBL targets into 12 high-level biological families and mapped 817 clinically approved drug indications into 28 broader therapeutic groups. This structured framework enabled a systematic profiling of physicochemical properties among approved drugs across therapeutic categories. Our analyses revealed associations between physicochemical characteristics and therapeutic groups, providing practical guidance for indication-specific compound prioritization and refining the repurposing studies. We also examined cross-indication drug approvals to identify areas with high repurposing potential. Finally, we implemented a pathway-based computational pipeline to predict repositioning opportunities for FDA-approved drugs across ten major cancer types, demonstrating its adaptability to other disease contexts. Overall, this work consolidates drug-target data and computational repurposing into a data-driven framework that advances drug discovery and translational applications.

molecular biology↗

bioRxiv2025

Comparative Analysis of Single-Nucleus and Single-Cell RNA Sequencing in Human Bone Marrow Mononuclear Cells: Methodological Insights and Trade-offs

Bone marrow mononuclear cells (BMMCs) are a heterogeneous pool of hematopoietic progenitors and mature immune cells that collectively sustain hematopoiesis and coordinate immune responses. The bone marrow serves not only as the primary site for blood cell production but also as a niche for various disorders, including blood cancers. Advances in single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) have significantly enhanced our understanding of the cellular biology and molecular dynamics within this complex microenvironment. The choice between these two approaches, however, is often shaped or constrained by the study design, such as research objectives, sample type, and preservation conditions. Consequently, methodological differences in library preparation and transcript capture efficiency can introduce systematic biases that complicate downstream analyses and interpretation, underscoring the need to identify and account for method-specific features. In this study, we conducted a comparative analysis of matched snRNA-seq and scRNA-seq datasets from 11 pairs of healthy donor bone marrow mononuclear cell samples, generated using the popular 10x Genomics platform. We evaluated method-specific biases using multiple quality metrics and compared cell type proportions and transcriptomic signatures captured by each approach. Integrative analysis of these datasets is feasible but not advisable due to systematic gene length biases that were observed between these approaches. Our results showed that despite inherent differences in library complexity, both protocols reliably captured all major cell types. This comparative analysis highlights intrinsic differences between snRNA-seq and scRNA-seq data, providing valuable insights into their respective advantages, limitations, and trade-offs. These findings can assist researchers in selecting the optimal method tailored to specific biological questions and sample characteristics, and also enable more method-aware data analysis and interpretation.

bioinformatics↗

bioRxiv2017

Post-Transcriptional Control Of EMT Is Coordinated Through Combinatorial Targeting By Multiple microRNAs

Epithelial-mesenchymal transition (EMT) is a process whereby cells undergo reversible phenotypic change, losing epithelial characteristics and acquiring mesenchymal attributes. While EMT underlies normal, physiological programs in embryonic tissue development and adult wound healing, it also contributes to cancer progression by facilitating metastasis and altering drug sensitivity. Using a cell model of EMT (human mammary epithelial (HMLE) cells), we show that miRNAs act as an additional regulatory layer over and above the activity of the transcription factors with which they are closely associated. In this context, miRNAs serve to both enhance expression changes for genes with EMT function, whilst simultaneously reducing transcriptional noise in non-EMT genes. We find that members of the polycistronic miR-200c~141 and miR-183~182 clusters (which are decreased during HMLE cell EMT and are associated with epithelial gene expression in breast cancer patients) co-regulate common targets and pathways to enforce an epithelial phenotype. We demonstrate their combinatorial effects are apparent much closer to endogenous expression levels (and orders of magnitude lower than used in most studies). Importantly, the low levels of combinatorial miRNAs that are required to exert biological function ameliorate the \"off-target\" effects on gene expression that are a characteristic of supra-physiologic miRNA manipulation. We argue that high levels of over-expression characteristic of many miRNA functional studies have led to an over-estimation of the effect of many miRNAs in EMT regulation, with over 130 individual miRNAs directly implicated as drivers of EMT. We propose that the functional effects of co-regulated miRNAs that we demonstrate here more-accurately reflects the endogenous post-transcriptional regulation of pathways, networks and processes, and illustrates that the post-transcriptional miRNA regulatory network is fundamentally cooperative.

molecular biology↗

bioRxiv2023

Condensate formation of the human RNA-binding protein SMAUG1 is controlled by its intrinsically disordered regions and interactions with 14-3-3 proteins

SMAUG1 is a human RNA-binding protein that is known to be dysregulated in a wide range of diseases. It is evolutionarily conserved and has been shown to form condensates containing translationally repressed RNAs. This indicates that condensation is central to proper SMAUG1 function; however, the factors governing condensation are largely unknown. In this work, we show that SMAUG1 drives the formation of liquid-like condensates in cells through its non-conventional C-terminal prion-like disordered region. We use biochemical assays to show that this liquid-liquid phase separation is independent of RNA binding and does not depend on other large, disordered regions that potentially harbor several binding sites for partner proteins. Using a combination of computational predictions, structural modeling, in vitro and in cell measurements, we also show that SMAUG1-driven condensation is negatively regulated by direct interactions with members of the 14-3-3 protein family. These interactions are mediated by four distinct phospho-regulated short linear motifs embedded in the disordered regions of SMAUG1, working synergistically. Interactions between SMAUG1 and 14-3-3 proteins drive the dissolution of condensates, alter the dynamics of the condensed state, and are likely to be intertwined with currently unknown regulatory mechanisms. Our results provide information on how SMAUG1 phase separation is regulated and the first known instance of 14-3-3 proteins being able to completely dissolve condensates by directly interacting with a phase separation driver, which might be a general mechanism in cells to regulate biological condensation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/527857v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@868746org.highwire.dtl.DTLVardef@1b3318org.highwire.dtl.DTLVardef@1b78868org.highwire.dtl.DTLVardef@5c7e5e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISMAUG1 is a human RNA-binding protein capable of condensation with unknown regulation C_LIO_LIA prion-like domain of SMAUG1 drives condensation via liquid-liquid phase separation C_LIO_LISMAUG1 interacts with 14-3-3 proteins via four phospho-regulated short linear motifs C_LIO_LI14-3-3 interactions change the dynamics of SMAUG1 condensates, promoting their dissolution C_LIO_LIThis is the first described regulatory mechanism for SMAUG1-driven condensation C_LI

molecular biology↗

bioRxiv2018

Histone deacetylase activity is required for Botrylloides leachii whole body regeneration.

The colonial tunicate Botrylloides leachii is exceptional at regenerating from a piece of vascular tunic after loss of all adults from the colony. Previous transcriptome analyses indicate a brief period of healing before regeneration of a new adult (zooid) in as little as 8-10 days. However, there is little understanding of how the resulting changes to gene expression, required to drive regeneration, are initiated and how the overall process is regulated. Rapid changes to gene expression often occur in response to chromatin changes, mediated by histone modifications such as histone acetylation. Here, we investigated a group of key epigenetic modifiers, histone deacetylases (HDAC) that are known to play an important role in many biological processes such as development, healing and regeneration.\n\nThrough our transcriptome data, we identified and quantified the expression levels of HDAC and histone acetyltransferase (HAT) enzymes during whole body regeneration (WBR). To determine if HDAC activity is required for WBR, we inhibited its action using valproic acid (VPA) and Trichostatin A (TSA). HDAC inhibition prevented the final morphological changes normally associated with WBR and resulted in aberrant gene expression. B. leachii genes including Slit2, TGF-{square}, Piwi and Fzd4 all showed altered gene expression upon HDAC inhibition in comparison to the control samples. Additionally, atypical expression of Bl_Piwi was found in immunocytes upon HDAC inhibition.\n\nTogether, these results show that HDAC function, specifically HDAC I/IIa class enzymes, are vital for B. leachii to undergo WBR successfully.

molecular biology↗

bioRxiv2016

Quantitative analysis of 3D alignment quality: itsimpact on soft-validation, particle pruning andhomogeneity analysis

Single Particle Analysis using cryo-electron microscopy is a structural biology technique to capture the three-dimensional conformation of biological macromolecules. The projection images used to construct the 3D density map are characterized by a very low signal-to-noise ratio to minimize radiation damage in the samples. As a consequence, the 3D alignment process is a challenging and error prone task and this job usually determines the success or failure of the macromolecule reconstruction. In this work, we present a soft-alignment validation approach, which can quantify the alignment precision and accuracy as well as the data homogeneity of the single particles when they are confronted with the resultant reconstructed 3DEM map. We have also applied this method to data homogeneity analysis and particle pruning, improving the data quality and as a consequence the final map resolution.

molecular biology↗

bioRxiv2020

Peptide location fingerprinting reveals modification-associated biomarkers of ageing in human tissue proteomes

Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age-related diseases, the untargeted identification of structural modifications in proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification-associated differences in mass spectrometry (MS) datasets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides (spectral counting) and map statistically significant differences to regions within protein structures. New photoageing biomarkers were identified in multiple proteins including matrix components (collagens and proteoglycans), oxidation and protease modulators (peroxiredoxins and SERPINs) and cytoskeletal proteins (keratins). Crucially, for many extracellular biomarkers, structural modification-associated differences were not correlated with relative abundance (by ion intensity). By applying peptide location fingerprinting to published MS datasets, (identifying biomarkers including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool to discover novel biomarkers.

molecular biology↗

bioRxiv2023

Analyzing the link between RNA secondary structures and R-loop formation with tree polynomials

R-loops are a class of non-canonical nucleic acid structures that typically form during transcription when the nascent RNA hybridizes the DNA template strand, leaving the DNA coding strand unpaired. Co-transcriptional R-loops are abundant in nature and biologically relevant. Recent research shows that DNA sequence and topology affect R-loops, yet it remains unclear how these and other factors drive R-loop formation. In this work, we investigate a link between the secondary structure of the nascent RNA and the probability of R-loop formation. We introduce tree-polynomial representations, a class of mathematical objects that enable accurate and efficient data analysis of RNA secondary structures. With tree-polynomials, we establish a strong correlation between the secondary structure of the RNA transcript and the probability of R-loop formation. We identify that branches with short stems separated by multiple bubbles in the RNA secondary structure are associated with the strong correlation and are predictive of R-loop formation.

molecular biology↗

bioRxiv2025

Lysine-R2HGylation identified as a post-translational modification in R2HG-elevated cancers

R-2-Hydroxyglutarate (R2HG), an oncometabolite predominantly produced by mutated isocitrate dehydrogenase 1/2 (IDH1/2) in various cancers, is known to drive cancer progression through noncovalent inhibition of -ketoglutarate (KG)-dependent enzymes. In this work, we propose an alternative mechanism wherein R2HG contributes to cancer development via covalent modification of biologically critical lysines, a process termed lysine-R2HGylation (KR2HG). We designed and synthesized R2HG-mimicking probes, demonstrating their effectiveness in facilitating KR2HG target profiling and site mapping. We identified KR2HG as a previously unrecognized post-translational modification, confirmed its C5-linkage on GSTP1(K209), and demonstrated that SIRT5 functions as a deacylase for GSTP1-KR2HG in vitro. Furthermore, we found that R2HG slightly but significantly inhibits the enzymatic activity of GSTP1 through KR2HG and dramatically suppresses monocyte differentiation via this catalytically important lysine modification. Our findings provide an alternative perspective on the role of R2HG in leukemia progression and offer a practical tool for the clinical investigation of R2HG-elevated cancers.

molecular biology↗