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

Background: Cognitive 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. Methods: Here 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). Results: Overall, 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. Conclusions: Intra-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.

m6A modification inhibits miRNAs' intracellular function, favoring their extracellular export for intercellular communication.

Epitranscriptomics represents a further layer of gene expression regulation. Specifically, N6-methyladenosine (m6A) regulates RNA maturation, stability, degradation, and translation. Regarding microRNAs (miRNAs), while it has been reported that m6A impacts their biogenesis, the functional effects on mature miRNAs remain unclear. Here, we show that m6A modification on specific miRNAs weakens their coupling to AGO2, impairs their function on target mRNAs, determines their delivery into extracellular vesicles (EVs), and provides functional information to receiving cells. Mechanistically, the intracellular functional impairment is caused by m6A-mediated inhibition of AGO2/miRNA interaction, the EV loading is favored by m6A-mediated recognition by the RNA-binding protein (RBP) hnRNPA2B1, and the EV-miRNA function in the receiving cell requires their FTO-mediated demethylation. Consequently, cells express specific miRNAs that do not impact endogenous transcripts but provide regulatory information for cell-to-cell communication. This highlights that a further level of complexity should be considered when relating cellular dynamics to specific miRNAs.

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding.

This comprehensive Review delves into the chemical principles governing RNA-mediated crowding events, commonly referred to as granules or biological condensates. We explore the pivotal role played by RNA sequence, structure, and chemical modifications in these processes, uncovering their correlation with crowding phenomena under physiological conditions. Additionally, we investigate instances where crowding deviates from its intended function, leading to pathological consequences. By deepening our understanding of the delicate balance that governs molecular crowding driven by RNA and its implications for cellular homeostasis, we aim to shed light on this intriguing area of research. Our exploration extends to the methodologies employed to decipher the composition and structural intricacies of RNA granules, offering a comprehensive overview of the techniques used to characterize them, including relevant computational approaches. Through two detailed examples highlighting the significance of noncoding RNAs, NEAT1 and XIST, in the formation of phase-separated assemblies and their influence on the cellular landscape, we emphasize their crucial role in cellular organization and function. By elucidating the chemical underpinnings of RNA-mediated molecular crowding, investigating the role of modifications, structures, and composition of RNA granules, and exploring both physiological and aberrant phase separation phenomena, this Review provides a multifaceted understanding of the intriguing world of RNA-mediated biological condensates.

Cortical neurons obtained from patient-derived iPSCs with GNAO1 p.G203R variant 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 Gαo, 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.

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, the 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 pathology 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 accumulation 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 accumulation, 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.

The structural properties of full-length annexin A11.

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38-59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.

Predicting nuclear G-quadruplex RNA-binding proteins with roles in transcription and phase separation.

RNA-binding proteins are central for many biological processes and their characterization has demonstrated a broad range of functions as well as a wide spectrum of target structures. RNA G-quadruplexes are important regulatory elements occurring in both coding and non-coding transcripts, yet our knowledge of their structure-based interactions is at present limited. Here, using theoretical predictions and experimental approaches, we show that many chromatin-binding proteins bind to RNA G-quadruplexes, and we classify them based on their RNA G-quadruplex-binding potential. Combining experimental identification of nuclear RNA G-quadruplex-binding proteins with computational approaches, we build a prediction tool that assigns probability score for a nuclear protein to bind RNA G-quadruplexes. We show that predicted G-quadruplex RNA-binding proteins exhibit a high degree of protein disorder and hydrophilicity and suggest involvement in both transcription and phase-separation into membrane-less organelles. Finally, we present the G4-Folded/UNfolded Nuclear Interaction Explorer System (G4-FUNNIES) for estimating RNA G4-binding propensities at http://service.tartaglialab.com/new_submission/G4FUNNIES .

Computational Approaches to Predict Protein-Protein Interactions in Crowded Cellular Environments.

Investigating protein-protein interactions is crucial for understanding cellular biological processes because proteins often function within molecular complexes rather than in isolation. While experimental and computational methods have provided valuable insights into these interactions, they often overlook a critical factor: the crowded cellular environment. This environment significantly impacts protein behavior, including structural stability, diffusion, and ultimately the nature of binding. In this review, we discuss theoretical and computational approaches that allow the modeling of biological systems to guide and complement experiments and can thus significantly advance the investigation, and possibly the predictions, of protein-protein interactions in the crowded environment of cell cytoplasm. We explore topics such as statistical mechanics for lattice simulations, hydrodynamic interactions, diffusion processes in high-viscosity environments, and several methods based on molecular dynamics simulations. By synergistically leveraging methods from biophysics and computational biology, we review the state of the art of computational methods to study the impact of molecular crowding on protein-protein interactions and discuss its potential revolutionizing effects on the characterization of the human interactome.

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

Proteins are crucial in regulating every aspect of RNA life, yet understanding their interactions with coding and noncoding 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 physico-chemical principles 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). Here, we assess the performance of several GRN inference algorithms in predicting protein-RNA interactions from single-cell transcriptomic data, and 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 predicted from single-cell transcriptomic data, with performances comparable or superior to those achieved for the well-established task of inferring transcription factor-target interactions. The incorporation of catRAPID significantly enhances the accuracy of identifying interactions, particularly with long noncoding RNAs, and enables the identification of hub RBPs and RNAs. Additionally, we show that interactions between RBPs can be detected based on their inferred RNA targets. The software is freely available at https://github.com/tartaglialabIIT/scRAPID.

Two Receptor Binding Strategy of SARS-CoV-2 Is Mediated by Both the N-Terminal and Receptor-Binding Spike Domain.

It is not well understood why severe acute respiratory syndrome (SARS)-CoV-2 spreads much faster than other ß-coronaviruses such as SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. In a previous publication, we predicted the binding of the N-terminal domain (NTD) of SARS-CoV-2 spike to sialic acids (SAs). Here, we experimentally validate this interaction and present simulations that reveal a second possible interaction between SAs and the spike protein via a binding site located in the receptor-binding domain (RBD). The predictions from molecular-dynamics simulations and the previously-published 2D-Zernike binding-site recognition approach were validated through flow-induced dispersion analysis (FIDA)─which reveals the capability of the SARS-CoV-2 spike to bind to SA-containing (glyco)lipid vesicles, and flow-cytometry measurements─which show that spike binding is strongly decreased upon inhibition of SA expression on the membranes of angiotensin converting enzyme-2 (ACE2)-expressing HEK cells. Our analyses reveal that the SA binding of the NTD and RBD strongly enhances the infection-inducing ACE2 binding. Altogether, our work provides in silico, in vitro, and cellular evidence that the SARS-CoV-2 virus utilizes a two-receptor (SA and ACE2) strategy. This allows the SARS-CoV-2 spike to use SA moieties on the cell membrane as a binding anchor, which increases the residence time of the virus on the cell surface and aids in the binding of the main receptor, ACE2, via 2D diffusion.

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

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can 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 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. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.

Single-photon microscopy to study biomolecular condensates.

Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles.

The PENGUIN approach to reconstruct protein interactions at enhancer-promoter regions and its application to prostate cancer.

We introduce Promoter-Enhancer-Guided Interaction Networks (PENGUIN), a method for studying protein-protein interaction (PPI) networks within enhancer-promoter interactions. PENGUIN integrates H3K27ac-HiChIP data with tissue-specific PPIs to define enhancer-promoter PPI networks (EPINs). We validated PENGUIN using cancer (LNCaP) and benign (LHSAR) prostate cell lines. Our analysis detected EPIN clusters enriched with the architectural protein CTCF, a regulator of enhancer-promoter interactions. CTCF presence was coupled with the prevalence of prostate cancer (PrCa) single nucleotide polymorphisms (SNPs) within the same EPIN clusters, suggesting functional implications in PrCa. Within the EPINs displaying enrichments in both CTCF and PrCa SNPs, we also show enrichment in oncogenes. We substantiated our identified SNPs through CRISPR/Cas9 knockout and RNAi screens experiments. Here we show that PENGUIN provides insights into the intricate interplay between enhancer-promoter interactions and PPI networks, which are crucial for identifying key genes and potential intervention targets. A dedicated server is available at https://penguin.life.bsc.es/ .

Unveiling the role of PUS7-mediated pseudouridylation in host protein interactions specific for the SARS-CoV-2 RNA genome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the catRAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5' and 3' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2 UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies.

Transmembrane Helices 7 and 8 Confer Aggregation Sensitivity to the Cystic Fibrosis Transmembrane Conductance Regulator.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a large multi-spanning membrane protein that is susceptible to misfolding and aggregation. We have identified here the region responsible for this instability. Temperature-induced aggregation of C-terminally truncated versions of CFTR demonstrated that all truncations up to the second transmembrane domain (TMD2), including the R region, largely resisted aggregation. Limited proteolysis identified a folded structure that was prone to aggregation and consisted of TMD2 and at least part of the Regulatory Region R. Only when both TM7 (TransMembrane helix 7) and TM8 were present, TMD2 fragments became as aggregation-sensitive as wild-type CFTR, in line with increased thermo-instability of late CFTR nascent chains and in silico prediction of aggregation propensity. In accord, isolated TMD2 was degraded faster in cells than isolated TMD1. We conclude that TMD2 extended at its N-terminus with part of the R region forms a protease-resistant structure that induces heat instability in CFTR and may be responsible for its limited intracellular stability.

RNA sequestration driven by amyloid formation: the alpha synuclein case.

Nucleic acids can act as potent modulators of protein aggregation, and RNA has the ability to either hinder or facilitate protein assembly, depending on the molecular context. In this study, we utilized a computational approach to characterize the physico-chemical properties of regions involved in amyloid aggregation. In various experimental datasets, we observed that while the core is hydrophobic and highly ordered, external regions, which are more disordered, display a distinct tendency to interact with nucleic acids. To validate our predictions, we performed aggregation assays with alpha-synuclein (aS140), a non-nucleic acid-binding amyloidogenic protein, and a mutant truncated at the acidic C-terminus (aS103), which is predicted to have a higher tendency to interact with RNA. For both aS140 and aS103, we observed an acceleration of aggregation upon RNA addition, with a significantly stronger effect for aS103. Due to favorable electrostatics, we noted an enhanced nucleic acid sequestration ability for the aggregated aS103, allowing it to entrap a larger amount of RNA compared to the aggregated wild-type counterpart. Overall, our research suggests that RNA sequestration might be a common phenomenon linked to protein aggregation, constituting a gain-of-function mechanism that warrants further investigation.

LINE-1 regulates cortical development by acting as long non-coding RNAs.

Long Interspersed Nuclear Elements-1s (L1s) are transposable elements that constitute most of the genome's transcriptional output yet have still largely unknown functions. Here we show that L1s are required for proper mouse brain corticogenesis operating as regulatory long non-coding RNAs. They contribute to the regulation of the balance between neuronal progenitors and differentiation, the migration of post-mitotic neurons and the proportions of different cell types. In cortical cultured neurons, L1 RNAs are mainly associated to chromatin and interact with the Polycomb Repressive Complex 2 (PRC2) protein subunits enhancer of Zeste homolog 2 (Ezh2) and suppressor of zeste 12 (Suz12). L1 RNA silencing influences PRC2's ability to bind a portion of its targets and the deposition of tri-methylated histone H3 (H3K27me3) marks. Our results position L1 RNAs as crucial signalling hubs for genome-wide chromatin remodelling, enabling the fine-tuning of gene expression during brain development and evolution.

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. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.

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

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 an unknown physiological substrate, displays an immune function by controlling inflammatory and type-I interferon (IFN) responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as an effector protein 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 the ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK inhibitor to ALS model mice can modulate Ser492-phospho-Brd4 levels, suppress microglial activation, and modify the disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.