Biology subjects

Colantoni, A.

Publications and source records attributed to Colantoni, A..

15 recordsLinked to original sources

bioRxiv2026

CHD8 orchestrates chromatin landscapes during early female neuronal differentiation

Background: Autism spectrum disorder (ASD) shows a strong male bias (~4:1). CHD8, a frequently mutated ASD gene, regulates neuronal development and Xist regulation in X-chromosome inactivation (XCI). However, its role in female neurodevelopment remains poorly understood due to the predominance of male-derived or sex-agnostic models. Methods: We performed integrated multi-omics analysis of female mouse embryonic stem cells (ES) differentiating to neuronal progenitor cells (NPCs) using wild-type, CHD8 knockdown (KD), knockout (KO), and domain-specific rescue lines (full-length, {Delta}Chromo, {Delta}Helicase). RNA-seq, CHD8 and H3K4me3 ChIP-seq, and ATAC-seq datasets were integrated to assess transcriptional, chromatin-binding and -accessibility changes during differentiation. Results: CHD8 occupancy was substantially remodelled during female neuronal differentiation, with 3,754 genes gaining NPCs-specific CHD8 binding predominantly at distal regulatory elements. CHD8 loss dysregulated 2,752 genes (1,134 upregulated and 1,618 downregulated), in NPCs. Differential accessibility analysis identified 4,486 chromatin regions with significant CHD8-dependent changes. Domain-specific rescue experiments showed that chromodomain and helicase activity makes distinct, non-redundant contributions to transcriptional recovery: {Delta}Chromo-rescued only ~ 1.0% of CHD8-dependent transcriptional changes, {Delta}Helicase rescued ~ 41.1%, and full-length CHD8 rescued ~ 70%. Integration of binding, expression, and accessibility data identified 8 high-confidence direct CHD8 target genes, and cross-referencing with the SFARI Autism Risk Gene database revealed that CHD8-dependent transcriptional changes converge on autism-related pathways. Conclusions: CHD8 acts as a key regulator of female-derived neuronal differentiation, recruited to H3K4me3-marked promoters in a chromodomain-dependent manner, with helicase activity providing non-redundant regulatory capacity at a subset of targets. These findings provide a molecular framework for CHD8-dependent transcriptional regulation in female NPCs and underscore the importance of including female-derived systems in neurodevelopmental disorder research.

neuroscience↗

bioRxiv2026

LBR nucleoplasmic domains regulate X-chromosome solubility and nuclear organization

The nuclear lamina plays a central role in genome organization, yet how specific lamina-associated proteins regulate chromosome architecture during development remains unclear. Here, we show that the nucleoplasmic domains of the Lamin B Receptor (LBR) are essential for X-chromosome localization at the nuclear periphery and chromatin architecture during neural differentiation. Using genetic dissection of LBR function, combined with genome-wide chromatin solubility profiling and transcriptional analyses, we demonstrate that loss of LBR N-terminal domains impairs proper cell differentiation and X chromosome inactivation (XCI), selectively disrupting chromatin structure in neural progenitors but not in pluripotent cells. Strikingly, these effects are disproportionately concentrated - but not limited to - on the inactive X chromosome, which undergoes a pronounced shift toward a more soluble chromatin state. Our findings establish the nucleoplasmic function of LBR as a key determinant of X-chromosome functionality and identify chromatin solubility and accessibility as a previously underappreciated layer of genome regulation by the nuclear lamina in XCI. Finally, our work provides definitive genetic evidence that LBRs nuclear architectural functions are molecularly separable from its metabolic sterol reductase activity, which is preserved in our model, and are critically necessary for XCI in differentiating mouse female XX ESCs models.

genetics↗

bioRxiv2025

RNA interactions drive structural and functional diversification of alpha-synuclein fibrils

Nucleic acids, particularly RNA, exert multiple roles on protein aggregation, yet their mechanistic involvement remains poorly understood. Building on prior work demonstrating RNA-driven acceleration of protein aggregation, we now demonstrate that RNA binding markedly reshapes the structural and functional properties of alpha-synuclein (aS) fibrils, a protein implicated in Parkinsons disease (PD). Although monomeric aS exhibits negligible RNA-binding properties, aggregation markedly increases its RNA-binding capacity. Our kinetic analyses reveal that RNA incorporation during initial stages of aggregation alters fibril elongation dynamics and modulates surface-mediated secondary nucleation in a seed concentration-dependent manner. Spectroscopic and microscopic characterization (FTIR, micro-FTIR) demonstrate that RNA promotes the formation of structurally distinct, more compact aS fibrils with heterogeneous morphology. Exploiting the recently discovered catalytic hydrolase activity of aS fibrils, we show that direct RNA binding inhibits enzymatic function, consistent with RNA-induced conformational remodeling that limits the exposure of critical residues. Additionally, RNA binding to fibril surfaces impairs proteolytic degradation by proteinase K, suggesting RNA stabilizes fibrils against clearance mechanisms. Moreover, RNA sequencing of fibril-bound nucleic acids uncovers a preference towards uridine-rich motifs that computational docking locates within hydrophilic cavities of disease-associated fibril structures. Collectively, we identify RNA as an active co-factor that remodels structure, propagation kinetics, stability, and function of aS fibrils. We offer a mechanistic link between RNA interaction and amyloid polymorphism relevant to synucleinopathies and, in broader contexts, to amyloid-associated diseases. Significance statementOur work represents the first systematic evidence that RNA directly interacts with protein fibrils and modulates key functional properties such as aggregation kinetics, proteolytic resistance and catalytic activity. We demonstrate that RNA co-aggregates with alpha-synuclein inducing structural conformations that depend on RNA for efficient assembly. Given the resemblance of these RNA-associated fibrils to those found in multiple system atrophy, our findings suggest that the final fibril structures in the patient brain are influenced by multiple cofactors and may differ substantially from recombinant fibrils formed in vitro, even when seeded with patient-derived material.

biochemistry↗

bioRxiv2025

RNase H1 counteracts DNA damage and ameliorates SMN-dependent phenotypes in a Drosophila model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is caused by a deficiency of the Survival Motor Neuron (SMN) protein. Mutations in SMN disrupt mRNA splicing and translation, leading to maladaptive changes in transcriptomes, proteomes, neuroinflammation, and metabolism, which drive motor neuron degeneration in SMA patients. Using a Drosophila SMA model, we found that systemic depletion of Smn leads to accumulation of RNA:DNA hybrids (R-loops), increased DNA damage, dysregulation of amino acids and sugar metabolism and activation of the innate immune response, recapitulating key pathological features reported in mammalian models and severe SMA patients. Persistent DNA damage in Smn-deficient flies alters cell proliferation rates in larval brains and induces extensive cell death in the developing eye. Importantly here, we show that stimulating the resolution of RNA:DNA hybrids with transgenic human RNAse H1 prevents the accumulation of DNA damage and attenuates the transcriptome and amino acid alterations induced by Smn depletion, mitigating the Smn-dependent cellular and developmental abnormalities, in Smn-deficient flies. Our data suggest that depletion of Smn causes an accumulation of aberrant transcripts and chronic DNA damage, which--along with the altered metabolomic profiles associated with Smn deficiency--trigger systemic inflammatory responses, ultimately affecting neuronal function and survival.

genetics↗

bioRxiv2025

scRAPID-web: a web server for predicting protein-RNA interactions from single-cell transcriptomics

SummarySingle-cell RNA sequencing (scRNA-seq) enables high-resolution studies of gene regulation, capturing gene expression at the individual cell level. We previously developed scRAPID, a computational pipeline for predicting protein-RNA interactions and identifying hub RNA-binding proteins (RBP) and RNAs through the integration of gene regulatory network (GRNs) inference from scRNA-seq data and catRAPID predictions. To make this tool accessible to a broader audience, we introduce scRAPID-web, a user-friendly web server supporting analysis of scRNA-seq data across eight model organisms. scRAPID-web offers customizable options to preprocess the input gene expression matrix, such as gene selection and cell type filtering. Users can choose from three GRN inference algorithms and decide whether to focus the analysis on specific gene types. Precompiled libraries allow fast filtering and motif-based validation of the inferred interactions. Results include detailed tables of predicted protein-RNA pairs and hubs, along with an interactive network visualization of potential RBP complexes built based on the inferred shared targets. scRAPID-web democratizes access to GRN-based analyses, providing insights into protein-RNA interactions and regulatory complexes in diverse cellular contexts. Availability and implementationscRAPID-web can be accessed at: https://tools.tartaglialab.com/scrapid.

bioinformatics↗

bioRxiv2025

Decoding RNA-RNA Interactions: The Role of Low-Complexity Repeats and a Deep Learning Framework for Sequence-Based Prediction

RNA-RNA interactions (RRIs) are fundamental to gene regulation and RNA processing, yet their molecular determinants remain unclear. In this work, we analyze several large-scale RRI datasets and identify low-complexity repeats (LCRs), including simple tandem repeats, as key drivers of RRIs. Our findings reveal that LCRs enable thermodynamically stable interactions with multiple partners, positioning them as key hubs in RNA-RNA interaction networks. These RRIs appear to be important for several aspects of RNA metabolism. Sequencing-based analysis of the lncRNA Lhx1os interactors validates the importance of LCRs in shaping contacts potentially involved in neuronal development. Recognizing the pivotal role of sequence determinants, we develop RIME, a deep learning model that predicts RRIs by leveraging embeddings from a nucleic acid language model. RIME outperforms traditional thermodynamics-based tools, successfully captures the role of LCRs and prioritizes high-confidence interactions, including those established by lncRNAs. RIME is freely available at https://tools.tartaglialab.com/rna_rna.

biochemistry↗

bioRxiv2024

Negative regulation of miRNAs sorting in EVs: the RNA-binding protein PCBP2 impairs SYNCRIP-mediated miRNAs EVs loading

While it is accepted that Extracellular Vesicles (EVs)-mediated transfer of microRNAs contributes to intercellular communication, the knowledge about molecular mechanisms controlling the selective and dynamic miRNA-loading in EVs is still limited to few specific RNA-binding proteins interacting with sequence determinants. Moreover, although mutagenesis analysis demonstrated the presence/function of specific intracellular retention motifs, the interacting protein/s remained unknown. Here, PCBP2 was identified as a direct interactor of an intracellular retention motif: CLIP coupled to RNA pull down and proteomic analysis demonstrated that it binds to miRNAs embedding this motif and mutagenesis proved the binding specificity. Notably, PCBP2 binding requires SYNCRIP, a previously characterized miRNA EV-loader as indicated by SYNCRIP knock-down. SYNCRIP and PCBP2 may contemporarily bind to miRNAs as demonstrated by EMSA assays and PCBP2 knock-down causes EV-loading of intracellular microRNAs. This evidence highlights that multiple proteins/miRNA interactions govern miRNA compartmentalization and identifies PCBP2 as a dominant inhibitor of SYNCRIP function.

molecular biology↗

bioRxiv2024

DROSHA, DICER and Damage-Induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break

Genome integrity is safeguarded by the DNA damage response (DDR). Controlled transcriptional dampening of genes surrounding DNA double-strand breaks (DSBs) has been shown to facilitate DNA repair. This phenomenon, defined as DSB-induced silencing in cis (DISC), involves the DDR apical kinase ATM and the Polycomb Repressive Complex 1 (PRC1). Conversely, DSBs have also been reported to induce de novo transcription of damaged-induced long non-coding RNAs (dilncRNAs) in a MRE11-RAD50-NBS1 (MNR) complex-dependent manner. MRN also controls the recruitment to DSB of the ribonuclease DROSHA, which together with DICER, stimulates DDR signaling and DNA repair. Here, we reconcile these apparently contrasting observations by showing that dilncRNA, together with DROSHA and DICER, but not GW182-like proteins required for miRNA-mediated gene silencing, controls DISC. Indeed, similarly to ATM, MRN inhibition abolishes DISC while pharmacological enhancement of DICER ribonuclease activity by Enoxacin improves DISC. Importantly, Enoxacin administration restores DISC upon ATM inhibition, demonstrating that DICER promotes DISC independently from ATM. Differently, Enoxacin does not restore DISC upon MRN inhibition, suggesting that DICER acts downstream to dilncRNA biogenesis and DROSHA recruitment. Mechanistically, we show that DROSHA and DICER control the recruitment of the PRC1 component BMI1 at DSBs and the consequent H2A-K119 ubiquitination. Upon DSBs formation, BMI1 and DROSHA interact in an RNA-dependent manner. Indeed, BMI1 associates to dilncRNA and do so in a DROSHA- and DICER-dependent manner. Importantly, inhibition of dilncRNA function by antisense oligonucleotides or Cas13-mediated targeting is sufficient to reduce BMI1 recruitment and DISC at individual loci. We propose that dilncRNAs together with DROSHA and DICER control DISC at genomic DSB by supporting PRC1 recruitment and chromatin ubiquitination.

molecular biology↗

bioRxiv2023

HuD (ELAVL4) gain-of-function impairs neuromuscular junctions and induces apoptosis in familial and sporadic amyotrophic lateral sclerosis models

Early defects at the neuromuscular junction (NMJ) are among the first hallmarks of the progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS). According to the "dying back" hypothesis, disruption of the NMJ not only precedes, but is also a trigger for the subsequent degeneration of the motoneuron in both sporadic and familial ALS, including ALS caused by the severe FUS pathogenic variant P525L. However, the mechanisms linking genetic and environmental factors to NMJ defects remain elusive. By taking advantage of co-cultures of motoneurons and skeletal muscle derived from human induced pluripotent stem cells (iPSCs), we show that the neural RNA binding protein HuD (ELAVL4) may underlie NMJ defects and apoptosis in FUS-ALS. HuD overexpression in motoneurons phenocopies the severe FUSP525L mutation, while HuD knockdown in FUSP525L co-cultures produces phenotypic rescue. We validated these findings in vivo in a Drosophila FUS-ALS model. Neuronal-restricted overexpression of the HuD-related gene, elav, produces per se a motor phenotype, while neuronal-restricted elav knockdown significantly rescues motor dysfunction caused by FUS. Finally, we show that HuD levels increase upon oxidative stress in human motoneurons and in sporadic ALS patients with an oxidative stress signature. On these bases, we propose HuD as an important player downstream of FUS mutation in familial ALS, with potential implications for sporadic ALS related to oxidative stress.

neuroscience↗

bioRxiv2023

Stand age diversity dampens forests sensitivity to climate change

Stand age significantly influences the functioning of forest ecosystems by shaping structural and physiological plant traits, affecting water and carbon budgets. Forest age distribution is determined by the interplay of tree mortality and regeneration, influenced by both natural and anthropogenic disturbances. Thus, human-driven alteration of tree age distribution presents an underexplored avenue for enhancing forest stability and resilience. In our study, we investigated how age distribution impacts the stability and resilience of the forest carbon budget under both current and future climate conditions. We employed a biogeochemical model on three historically managed forest stands, projecting their future as undisturbed systems, i.e., left at their natural evolution with no management interventions. The model, driven by climate data from five Earth System Models under four representative climate scenarios and one baseline scenario, spanned 11 age classes for each stand. Our findings indicate that Net Primary Production (NPP) peaks in the young and middle-aged classes (16- to 50-year-old), aligning with ecological theories, regardless of the climate scenario. Under climate change, the beech forest exhibited an increase in NPP and maintained stability across all age classes, while resilience remained constant with rising atmospheric CO2 and temperatures. However, NPP declined under climate change scenarios for the Norway spruce and Scots pine sites. In these coniferous forests, stability and resilience were more influenced. These results underscore the necessity of accounting for age classes and species-specific reactions in evaluating the impacts of climate change on forest stability and resilience. We, therefore, advocate for customized management strategies that enhance the adaptability of forests to changing climatic conditions, taking into account the diverse responses of different species and age groups to climate.

plant biology↗

bioRxiv2023

Prediction of protein-RNA interactions from single-cell transcriptomic data

RNA-binding proteins play a crucial role in regulating RNA processing, yet our understanding of their interactions with coding and non-coding RNAs remains limited. Experimental studies are typically restricted to a small number of cell lines and a limited set of RNA-binding proteins (RBPs). Although computational methods based on sequence and structure can predict protein-RNA interactions accurately, they often lack the ability to consider cell-type-specific gene expression and the broader context of gene regulatory networks (GRNs). In the present study, we assess the performance of several GRN inference algorithms in predicting protein-RNA interactions from single-cell transcriptomic data, and we propose a pipeline, called scRAPID (single-cell transcriptomic-based RnA Protein Interaction Detection), that integrates these methods with the catRAPID algorithm, which can identify direct physical interactions between RBPs and RNA molecules. Our approach demonstrates that RBP-RNA interactions can be inferred from single-cell transcriptomic data, with performances comparable or superior to those achieved for the well-established task of inferring transcription factor-target interactions. Notably, the incorporation of catRAPID significantly enhances the accuracy of identifying interactions, particularly with long non-coding RNAs, and enables the identification of hub RBPs and hub RNAs. Additionally, we show that interactions between RBPs can be detected based on their inferred RNA targets. We have made the software freely available at https://github.com/tartaglialabIIT/scRAPID.

bioinformatics↗

bioRxiv2023

Cortical neurons obtained from patient-derived GNAO1 iPSCs show altered differentiation and functional properties

Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Go, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca2+]i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.

neuroscience↗

bioRxiv2022

The PRALINE database: Protein and Rna humAn singLe nucleotIde variaNts in condEnsates

SummaryBiological condensates are membraneless organelles with different material properties. Proteins and RNAs are the main components, but most of their interactions are still unknown. Here we introduce PRALINE, a database for the interrogation of proteins and RNAs contained in stress-granules, processing bodies, and other assemblies including droplets and amyloids. PRALINE provides information about the predicted and experimentally validated protein-protein, protein-RNA and RNA-RNA interactions. For proteins, it reports the liquid-liquid phase separation and liquid-solid phase separation propensities. For RNAs, it provides information on predicted secondary structure content. PRALINE shows detailed information on human single-nucleotide variants, their clinical significance and presence in protein and RNA binding sites, and how they can affect condensates physical properties. AvailabilityPRALINE is freely accessible on the web at http://alvinlee.bio.uniroma1.it/praline. Supplementary informationGeneral information is at http://alvinlee.bio.uniroma1.it/praline/about, where we provide a detailed description of the datasets and the tools employed in the database. Data provided in PRALINE are available at http://alvinlee.bio.uniroma1.it/praline/downloads. The tutorial is at http://alvinlee.bio.uniroma1.it/praline/tutorial.

bioinformatics↗

bioRxiv2022

Multi-omics analysis reveals a crucial role for Retinoic Acid in promoting epigenetic and transcriptional competence of an in vitro model of human Pharyngeal Endoderm.

In vitro differentiation of human Pluripotent Stem Cells (hPSCs) into different cell types has enabled the study of developmental processes that are impossible to dissect in vivo. This innovation has allowed for the derivation of therapeutically relevant cell types that can be used for downstream applications and studies. The Pharyngeal Endoderm (PE) is considered an extremely relevant developmental tissue since it acts as a precursor to a plethora of organ systems such as Esophagus, Parathyroids, Thyroids, Lung, and Thymus. While several studies have highlighted the importance of these cells, an in vitro platform to generate human PE cells is still missing. Here we fill this knowledge gap, by providing a novel in vitro protocol for the derivation of bona fide PE cells from hPSCs. We demonstrated that our PE cells robustly express Pharyngeal Endoderm markers, they are transcriptionally similar to PE cells isolated from in vivo mouse development and represent a transcriptionally homogeneous population. Importantly, we elucidated the contribution of Retinoic Acid in promoting a transcriptional and epigenetic rewiring of PE cells. In addition, we defined the epigenetic landscape of PE cells by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of these data led to the identification of new putative regulatory regions and to the generation of a gene regulatory network orchestrating the development of PE cells. By combining hPSCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and modeling of their developmental defects in genetic syndromes.

developmental biology↗

bioRxiv2020

Epigenetic regulation of Wnt7b expression by the cis-acting long noncoding RNA lnc-Rewind in muscle stem cells

Skeletal muscle possesses an outstanding capacity to regenerate upon injury due to the adult muscle stem cells (MuSCs) activity. This ability requires the proper balance between MuSCs expansion and differentiation which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated lncRNA, lnc-Rewind, which is expressed in murine MuSCs and conserved in human. We find that, in mouse, lnc-Rewind acts as an epigenetic regulator of MuSCs proliferation and expansion by influencing the expression of skeletal muscle genes and several components of the WNT (Wingless-INT) signalling pathway. Among them, we identified the nearby Wnt7b gene as a direct lnc-Rewind target. We show that lnc-Rewind interacts with the G9a histone lysine methyltransferase and mediates the in cis repression of Wnt7b by H3K9me2 deposition. Overall, these findings provide novel insights into the epigenetic regulation of adult muscle stem cells fate by lncRNAs.

cell biology↗