Biology subjects

Tartaglia, G. G.

Publications and source records attributed to Tartaglia, G. G..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Amygdala TDP-43 pathology is associated with behavioural dysfunction and ferritin accumulation in amyotrophic lateral sclerosis.

BackgroundCognitive and behavioural symptoms associated with amyotrophic lateral sclerosis and frontotemporal spectrum disorders (ALSFTSD) are thought to be driven, at least in part, by the pathological accumulation of TDP-43. MethodsHere we examine post-mortem tissue from six brain regions associated with cognitive and behavioural symptoms in a cohort of 30 people with sporadic ALS (sALS), a proportion of which underwent standardized neuropsychological behavioural assessment as part of the Edinburgh Cognitive ALS Screen (ECAS). ResultsOverall, the behavioural screen performed as part of the ECAS predicted accumulation of pathological phosphorylated TDP-43 (pTDP-43) with 100% specificity and 86% sensitivity in behaviour-associated brain regions. Notably, of these regions, pathology in the amygdala was the most predictive correlate of behavioural dysfunction in sALS. In the amygdala of sALS patients, we show variation in morphology, cell type predominance, and severity of pTDP-43 pathology. Further, we demonstrate that the presence and severity of intra-neuronal pTDP-43 pathology, but not astroglial pathology, or phosphorylated Tau pathology, is associated with behavioural dysfunction. Cases were also evaluated using a TDP-43 aptamer (TDP-43APT), which revealed that pathology was not only associated with behavioural symptoms, but also with ferritin levels, a measure of brain iron. ConclusionsIntra-neuronal pTDP-43 and cytoplasmic TDP-43APT pathology in the amygdala is associated with behavioural symptoms in sALS. TDP-43APT staining intensity is also associated with increased ferritin, regardless of behavioural phenotype, suggesting that ferritin increases may occur upstream of clinical manifestation, in line with early TDP-43APT pathology, representing a potential region-specific imaging biomarker of early disease in ALS. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSThe amygdala is a key brain region in regulating behavior and emotional cognition and has been shown recently, through imaging studies, to be affected in ALS and FTD patients. What this study addsHere we examine the underlying pathology driving the association between the amygdala and behavioural symptoms in sporadic ALS demonstrating that region specific TDP-43 pathology and brain iron accumulation could represent potential early biomarkers of dysfunction. How this study might affect research, practice, or policyThe correlation between early TDP-43 pathology (detected by RNA aptamer) and increased ferritin (brain iron accumulation) occurring upstream of clinical manifestation represents a potential, region-specific (amygdala), early imaging biomarker in ALS. This means that people at risk could be identified early and stratified for clinical trials prior to substantial neuronal cell loss and symptom onset.

neuroscience↗

bioRxiv2024

Machine learning methods applied to classify complex diseases using genomic data

Complex diseases pose challenges in disease prediction due to their multifactorial and polygenic nature. In this work, we explored the prediction of two complex diseases, multiple sclerosis (MS) and Alzheimers disease (AD), using machine learning (ML) methods and genomic data from UK Biobank. Different ML methods were applied, including logistic regressions (LR), gradient boosting decision trees (GB), extremely randomized trees (ET), random forest (RF), feedforward networks (FFN), and convolutional neural networks (CNN). The primary goal of this research was to investigate the variability of ML models in classifying complex diseases based on genomic risk. LR was the most robust method across folds and diseases, whereas deep learning methods (FFN and CNN) exhibited high variability. When comparing the performance of polygenic risk scores (PRS) with ML methods, PRS consistently performed at an average level. However, PRS still offers several practical advantages over ML methods. Despite implementing feature selection techniques to exclude non-informative and correlated predictors, the performance of ML models did not improve significantly, underscoring the ability of ML methods to achieve optimal performance even in the presence of correlated features due to linkage disequilibrium. Upon applying explainability tools to extract information about the genomic features contributing most to the classification task, the results confirmed the polygenicity of MS. The prevalence of HLA gene annotations among the top genomic features on chromosome 6 aligns with their significance in the context of MS. Overall, the highest-prioritized genomic variants were identified as expression or splicing quantitative trait loci (eQTL or sQTL) located in non-coding regions within or near genes associated with the immune response and MS. In summary, this research offers deeper insights into how ML models discern genomic patterns related to complex diseases.

bioinformatics↗

bioRxiv2023

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS

TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, analysis of deeply-phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism, but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 aggregation and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply-phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic aggregation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic aggregation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS. Short AbstractRecent identification of cryptic-splicing events such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism in amyotrophic lateral sclerosis (ALS). However, the temporal nature of TDP-43 loss and its relation to clinical phenotype is not known. Here, we used a novel RNA aptamer to detect TDP-43 aggregation and used single molecule ISH to sensitively reveal TDP-43 loss-of-function, applying these methods in a deeply-phenotyped human post-mortem tissue cohort. We show that nuclear TDP-43 pathology is an early event, that coincides with STMN-2 cryptic splicing. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics and intervention prior to symptom onset in ALS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/563701v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1e49c3forg.highwire.dtl.DTLVardef@1ce05b0org.highwire.dtl.DTLVardef@d77205org.highwire.dtl.DTLVardef@7ed2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

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↗

bioRxiv2023

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimens fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a com-prehensive framework of live-cell spectroscopy methodologies - compatible with imaging - to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the pro-posed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation - inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes - not limited to liquid-liquid phase transition - and we anticipate their widespread application in future life-science research.

biophysics↗

bioRxiv2023

Internal Ribosome Entry Sites act as Effector Domain in linear and circular antisense long non-coding SINEUP RNAs

SINEUPs are antisense long non-coding RNAs that enhance translation of overlapping sense mRNAs through the activity of two domains: a SINEB2 sequence UP-regulating translation (Effector Domain, ED) and an antisense region providing target specificity (Binding Domain, BD). In this study, we demonstrate that the invSINEB2 sequence from the natural SINEUP AS Uchl1 RNA is an Internal Ribosomal Entry Site (IRES) when acting in cis and that known viral and cellular IRES sequences can act as Effector Domain in synthetic SINEUPs. To identify natural IRES-containing, non-coding RNAs with SINEUP-like activity, we focused on circular RNAs showing that the non-coding circ5533, transcribed from the c-myc locus, enhances endogenous protein expression of its target PX Domain Containing Serine/Threonine Kinase Like (Pxk) by increasing mRNA association to polysomes. In summary, this study shows that natural and synthetic SINEUPs include linear and circular transcripts with an embedded IRES sequence as ED.

molecular biology↗

bioRxiv2023

Identification and prediction of G-quadruplex RNA-binding proteins with roles in transcription and phase separation

RNA-binding proteins are central for many biological processes and their large-scale identification has demonstrated a broad range of functions. RNA G-quadruplexes are important regulatory elements occurring in both coding and non-coding transcripts, yet our knowledge of their structure-based interactions is limited. Here starting from theoretical prediictions, we show experimentally that a large number of chromatin-binding proteins bind to RNA G-quadruplexes and we classify these based on their RNA G-quadruplex-binding potential. Combining experimental identification of nuclear RNA G-quadruplex-binding proteins with computational analysis, we create a prediction tool that can assign probability score for a protein that it binds RNA G-quadruplexes. We show that predicted G-quadruplex RNA-binding proteins exhibit high degree of protein disorder and hydrophilicity, and suggest involvement in both transcription and phase-separation into membrane-less organelles, particularly the nucleolus. Finally, we present this tool as a web application for estimating RNA G4-binding propensity for proteins of interest at http://service.tartaglialab.com/new_submission/clever_G4_classifier.

molecular biology↗

bioRxiv2023

THE RIBOREGULATION MECHANISM OF HUMAN SERINE HYDROXYMETHYLTRANSFERASE IS ROOTED IN AN ALLOSTERIC SWITCH

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of these being characterized on structural grounds. Here we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzymes reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in the evolution of eukaryotic SHMT1 and the compartmentalization of one-carbon metabolism. The findings also provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

biochemistry↗

bioRxiv2023

ORC1 binds to cis-transcribed RNAs for efficient activation of replication origins

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. We propose that fluctuating concentrations of RNA during the cell cycle may play a sequential role in controlling origins through interaction with this flexible region of ORC1. Our results unveil a novel non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins. One sentence summaryThe human origin recognition complex subunit 1 ORC1, binds to RNAs transcribed from genes with origins of replication at the TSS, which is required for optimal origin activation.

molecular biology↗

bioRxiv2023

MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying mechanisms remain incompletely understood. In this work we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with unknown physiological substrate, displays an immune function by controlling inflammatory and type-I IFN responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as the first molecule regulated by MOK, by promoting Ser492-phospho-Brd4 levels. We further demonstrate that MOK regulates Brd4 functions by supporting its binding to cytokine gene promoters, therefore enabling innate immune responses. Remarkably, we show that MOK levels are increased in ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK-inhibitor to ALS model mice is able to modulate Ser492-phospho-Brd4 levels, suppress microglial activation and modify disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.

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

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a powerful method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can profoundly deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false-positive rates. Here, we present the R/Bioconductor package DEWSeq that makes full use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing (2.3-fold median). The improvement not only relates to the number of binding sites (e.g., 3.1-fold of known motifs for RBFOX2), but also their subcellular localisation (e.g., 1.9-fold of mitochondrial genes for FASTKD2) and structural targets (e.g., 2.2-fold increase of stem-loop regions for SLBP). DEWSeq therefore shows promise as an improved processing method for eCLIP protein-RNA interaction data.

molecular biology↗

bioRxiv2022

Reconstructing protein interactions at enhancer-promoter regions in prostate cancer

Here we introduce Promoter-ENhancer-GUided Interaction Networks (PENGUIN), a method to uncover protein-protein interaction (PPI) networks at enhancer-promoter contacts. By integrating H3K27ac-HiChIP data and tissue-specific PPI information, PENGUIN enables cluster enhancers-promoter PPI networks (EPINs) and pinpoint actionable factors. Validating PENGUIN in cancer (LNCaP) and benign (LHSAR) prostate cell lines, we observed distinct CTCF-enriched clusters, which identifies diverse chromatin conformations. In LNCaP, we found an EPIN cluster enriched with oncogenes and prostate cancer-associated SNPs. We uncovered a total of 208 SNPs in LNCaP EPINs and used CRISPR/Cas9 knockout and RNAi screens to confirm their relevance. PENGUINs application in prostate cancer demonstrates its potential for studying human diseases. The approach allows exploration in different cell types and combinations of GWAS data, offering promising avenues for future investigations. In conclusion, PENGUIN provides valuable insights into the interplay between enhancer-promoter interactions and PPI networks, facilitating the identification of relevant genes and potential intervention targets.

cancer biology↗

bioRxiv2022

A Theoretical Model reveals RNA sequestration in Alpha Synuclein Aggregates

Nucleic acids can act as potent modulators of protein aggregation, and RNA is able to either hinder or facilitate protein assembly depending on the molecular context. Here we used a computational approach to characterize the physico-chemical properties of regions involved in amyloid aggregation. In different experimental datasets we observed that, while the core is hydrophobic and highly ordered, external regions, more disordered, display a distinct tendency to interact with nucleic acids. To validate our predictions, we performed aggregation assays with -synuclein (aS140), a non-nucleic acid binding amyloidogenic protein, and a mutant truncated at the acidic C-terminus (aS103) that is predicted to sequester RNA. For both aS140 and aS103 we observed acceleration of the aggregation upon RNA addition with a significantly stronger effect for aS103. Due to the favorable electrostatics, we observed enhanced nucleic-acid sequestration ability for aS103 that entrapped a larger amount of RNA. Overall, our research suggests that RNA sequestration is a rather common phenomenon linked to protein aggregation and constitutes a gain-of-function mechanism to be further investigated. STATEMENT OF SIGNIFICANCEOur study indicates that aggregation confers RNA-binding ability to non-RNA-binding proteins such as alpha synuclein. The sequestration of RNA upon protein aggregation might alter RNA homeostasis and impact multiple biochemical cascades.

biochemistry↗

bioRxiv2022

Discovering host protein interactions specific for SARS-CoV-2 RNA genome

SARS-CoV-2, a positive single-stranded RNA virus, interacts with host cell proteins throughout its life cycle. These interactions are necessary for the host to recognize and hinder the replication of SARS-CoV-2. For the virus, to translate, transcribe and replicate its genetic material. However, many details of these interactions are still missing. We focused on the proteins binding to the highly structured 5 and 3 end regions of SARS-CoV-2 RNA that were predicted by the catRAPID algorithm to attract numerous proteins, exploiting RNA-Protein Interaction Detection coupled with Mass Spectrometry (RaPID-MS) technology. The validated interactors, which agreed with our predictions, include pseudouridine synthase PUS7 that binds to both ends of the viral RNA. Nanopore direct-RNA sequencing confirmed that the RNA virus is heavily modified, and PUS7 consensus regions were found in both SARS-CoV-2 RNA end regions. Notably, a modified site was detected in the viral Transcription Regulatory Sequence - Leader (TRS-L) and can influence the viral RNA structure and interaction propensity. Overall, our data map host protein interactions within SARS-CoV-2 UTR regions, pinpointing to a potential role of pseudouridine synthases and post-transcriptional modifications in the viral life cycle. These findings contribute to understanding virus-host dynamics and may guide the development of targeted therapies.

molecular biology↗

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↗

bioRxiv2022

A high-throughput approach to predict A-to-I effects on RNA structure indicates a change of double-stranded content in non-coding RNAs

RNA molecules undergo a number of chemical modifications whose effects can alter their structure and molecular interactions. Previous studies have shown that RNA editing can impact the formation of ribonucleoprotein complexes and influence the assembly of membrane-less organelles such as stress-granules. For instance, N6-methyladenosine (m6A) enhances SG formation and N1-methyladenosine (m1A) prevents their transition to solid-like aggregates. Yet, very little is known about adenosine to inosine (A-to-I) modification that is very abundant in human cells and not only impacts mRNAs but also non-coding RNAs. Here, we built the CROSSalive predictor of A-to-I effects on RNA structure based on high-throughput in-cell experiments. Our method shows an accuracy of 90% in predicting the single and double-stranded content of transcripts and identifies a general enrichment of double-stranded regions caused by A-to-I in long intergenic non-coding RNAs (lincRNAs). For the individual cases of NEAT1, NORAD and XIST, we investigated the relationship between A-to-I editing and interactions with RNA-binding proteins using available CLIP data. We found that A-to-I editing is linked to alteration of interaction sites with proteins involved in phase-separation, which suggests that RNP assembly can be influenced by A-to-I. CROSSalive is available at http://service.tartaglialab.com/new_submission/crossalive.

biochemistry↗