Biology subjects

Viero, G.

Publications and source records attributed to Viero, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Ribosome Quality Control Mitigates Proteotoxic Stress in Aneuploid Cells

Aneuploidy is widespread in tumors, but how cancer cells adapt to aneuploidy-induced cellular stresses remains poorly understood. Here, we focus on the mechanisms employed to cope with proteostasis disruption, a major stress caused by aneuploidy. We show that aneuploid cells exhibit a significant accumulation of ribosomes enclosed within autophagosomes, ultimately degraded through lysosome-mediated processes. Our data also indicate that limited folding capacity of newly synthesized polypeptides leads to lysosome-mediated degradation of ribosomes. We also found that the E3 ligase ZNF598 marks these ribosomes for degradation, thus clearing translationally-impaired ribosomes. Importantly, highly aneuploid tumors display a positive correlation with ZNF598 expression, while being negatively associated with ribosomal signatures. This suggests that ribosome-associated quality control is crucial for cancer cell survival under proteotoxic stress. Our study uncovers molecular events in response to proteotoxic stress in aneuploid cells and suggests that components of ribosome-associated quality control, including ZNF598, could serve as promising targets in cancer therapy.

cell biology↗

bioRxiv2025

Spatiotemporal multi-omics atlas of the aging neuromuscular junction

The demographic shift toward a global aging population, coupled with rising prevalence of neurodegenerative diseases, poses major public health challenges. Aging is the primary risk factor for most neurodegenerative conditions, making the elucidation of its molecular mechanisms critical for developing effective interventions. Dysfunction of the neuromuscular junction (NMJ), the specialized synapse essential for motor function, is an early hallmark of aging and several neurodegenerative disorders, yet its molecular determinants remain incompletely understood. To shed light on potential common underlying mechanisms, we performed a spatiotemporal multi-omics analysis of the murine NMJ during aging, uncovering several genes showing decoupling between transcript and protein trajectories that may drive the related progressive motor decline. NMJs of soleus (SOL) and extensor digitorum longus (EDL) muscles, differing in fiber composition and vulnerability to aging and disease, displayed distinct spatiotemporal dynamics: fast-twitch EDL, more susceptible to degeneration, exhibited stronger mRNA-protein decoupling than aged slow-twitch SOL or younger SOL and EDL muscles. Elevated expression of miRNAs and RNA-binding proteins in aged EDL highlights the key role of post-transcriptional regulation in NMJ aging. This study provides new insights into the physiopathology of neuromuscular aging and offers a resource for investigating mechanisms shared between aging and neurodegenerative diseases. Additionally, it opens avenues for AI-driven discovery of drug targets and early biomarkers, potentially accelerating the development of therapeutic strategies.

neuroscience↗

bioRxiv2025

Empowering multiplexed ultra-throughout ribosome profiling with RiboWich

Ribosome profiling (RiboSeq) improved the understanding of mRNA translation, enabling the precise mapping of ribosome positioning along transcripts at single-nucleotide resolution. Although various library preparation protocols overcome the need for high input material, their technical complexity and limited efficiency hinder robust profiling, preventing them from keeping pace with other sequencing techniques and applications. To move towards high-throughput and single-cell RiboSeq technologies, we developed RiboWich (Ribosome sandWich). By directly ligating adaptors to ribosome-embedded RNA fragments, RiboWich eliminates the need for ribosome purification and size-selecting ribosome footprints and tackles two major bottlenecks, expanding RiboSeq for more advanced technologies. RiboWich offers robustness in profiling and excels in detecting upstream translons in immortalized and primary cells alike. By exploiting a dual-step multiplexing strategy, RiboWich enables the simultaneous profiling of at least 96 samples while retaining sensitivity and capturing condition-specific differences in translation. By enabling scalable, low-input translatome profiling, this advancement empowers proteogenomic approaches and AI/ML-driven data analysis to uncover regulatory dynamics, neoantigens, functional small translons, and drug-responsive signatures across diverse biological contexts and users. Altogether, RiboWich represents a straightforward, versatile, and scalable ribosome profiling conceptual platform that combines accessibility, sensitivity, and throughput potential, laying the foundation for advanced single-cell RiboSeq applications.

molecular biology↗

bioRxiv2025

Translation-specific disruption of Col1a1 expression in multiple models of Spinal Muscular Atrophy can be rescued by Risdiplam.

Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disorder caused by decreased levels of Survival of Motor Neuron (SMN) protein. If left untreated, SMA patients have a poor prognosis, marked by the degeneration of motor neurons, progressive muscle weakness and atrophy. The approval of SMN-restoring therapies that improve symptoms and lifespan in patients with SMA has created emerging, non-neuronal phenotypes and an urgent need for deepening our understanding of disease pathogenesis. Leveraging the knowledge that SMN loss drives alterations in translation, we used multiple tissues from a mouse model of SMA to uncover early translational alterations in key mRNAs and proteins, which act as contributors to pathogenesis and hallmarks of the disease. Among hundreds of differentially translated mRNAs, Col1a1 emerged as a translation-specific manifestation of early defects in the mouse model. These findings were confirmed in fibroblasts derived from patients with varying levels of disease severity. Notably, treatment with SMN-restoring therapies rescued COL1A1 protein levels, particularly in fibroblasts from patients with the most severe forms of the disease. Overall, our study identifies COL1A1 as an indicator of disease severity in SMA, which captures early molecular alterations and respond to SMN-modifying therapies.

molecular biology↗

bioRxiv2025

Model-independent reorganization of translation in TDP-43 Amyotrophic Lateral Sclerosis

The RNA-binding protein TDP-43 is a major contributor and a pathological hallmark of Amyotrophic Lateral Sclerosis (ALS), yet how TDP-43 dysregulation mechanistically alters protein synthesis across neuronal compartments and disease models remains unclear. Here, we dissected TDP-43-driven translational alterations in both in vitro and in vivo TDP-43 models of ALS. Through ribosome and polysome profiling, computational, and biochemical analyses, we observed robust TDP-43-associated translational remodelling at cellular and subcellular resolution. Our findings reveal a conserved mechanism across models, characterized by enhanced ribosome recruitment on polysomes, elongation impairment, axonal downregulation and instability of TDP-43 target mRNAs and redistribution toward non-target transcripts. Notably, TDP-43 dysregulation alters ribosome dynamics and selectively impairs translation of TDP-43 target mRNAs, whilst favouring the translation of other transcripts. This process reflects a compensatory but maladaptive response to TDP-43-induced mRNA destabilization. Together, these data demonstrate that alterations in TDP-43 disrupts neuronal proteostasis through ribosome reorganization and loss of mRNA homeostasis, providing a unifying mechanistic framework for translational imbalance in ALS and revealing early molecular events that may underlie motor neuron vulnerability.

molecular biology↗

bioRxiv2024

Patient-specific responses to SMN2 splice-modifying treatments in spinal muscular atrophy fibroblasts

The availability of three therapies for the neuromuscular disease spinal muscular atrophy (SMA) highlights the need to match patients to the optimal treatment. Two of these treatments (nusinersen and risdiplam) target splicing of SMN2, but treatment outcomes vary from patient to patient. An incomplete understanding of the complex interactions between SMA genetics, SMN protein and mRNA levels, and gene-targeting treatments, limits our ability to explain this variability and identify optimal treatment strategies for individual patients. To address this, we analyzed responses to nusinersen and risdiplam in 45 primary fibroblast cell lines. Pre-treatment SMN2-FL, SMN2{Delta}7 mRNA, and SMN protein levels were influenced by SMN2 copy number, age, and sex. After treatment, SMN and mRNA levels were more heterogeneous. In 43% of patients, response to both therapies was similar, but in 57% one treatment led to a significantly higher SMN increase than the other treatment. Younger age, higher SMN2 copy number, and higher SMN levels before treatment predicted better in vitro efficacy. These findings showcase patient-derived fibroblasts as a tool for identifying molecular predictors for personalized treatment.

molecular biology↗

bioRxiv2024

Interpreting single-cell messages in normal and aberrant hematopoiesis with the Cell Marker Accordion

Single-cell technologies offer a unique opportunity to explore cellular heterogeneity in health and disease. However, reliable identification of cell types and states represents a bottleneck. Available databases and analysis tools employ dissimilar markers, leading to inconsistent annotations and poor interpretability. Furthermore, current tools focus mostly on physiological cell types, limiting their applicability to disease. We developed the Cell Marker Accordion, a user-friendly platform providing automatic annotation and unmatched biological interpretation of single-cell populations, based on consistency weighted markers. We validated our approach on multiple single-cell and spatial datasets from different human and murine tissues, improving annotation accuracy in all cases. Moreover, we show that the Cell Marker Accordion can identify disease-critical cells and pathological processes, extracting potential biomarkers in a wide variety of disease contexts. The breadth of these applications elevates the Cell Marker Accordion as a fast, flexible, faithful and standardized tool to annotate and interpret single-cell and spatial populations in studying physiology and disease.

bioinformatics↗

bioRxiv2023

Two neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, a ubiquitously expressed RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, of both sporadic and familial origin, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt the subcellular transport and localization of mRNA. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins, both constitutively and in response to stimuli reaching the axon and presynaptic terminal. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two models of TDP-43 proteinopathy, consisting of virtually pure populations of mouse cortical neurons expressing a human TDP-43 fusion protein, wt or mutant, which accumulates as cytoplasmic aggregates. Neurons expressing human TDP-43 exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation in the axonal levels of polysome-engaged mRNAs playing relevant roles in those processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

neuroscience↗

bioRxiv2022

Filming space-time changes of gene expression with expressyouRcell

The last decade has witnessed massive advancements in high-throughput techniques capable of producing quantifications of transcript and protein levels across time and space, and at high resolution. Yet, the large volume of big data available and the complexity of experimental designs hamper an easy understanding and effective communication of the results. Here we present expressyouRcell, a unique and easy-to-use R package to map the multi-dimensional variations of transcript and protein levels in cell-pictographs. These variations are outcomes of differential and gene set enrichment analysis across space and time. Our tool directly associates these results with up to twenty specific cellular compartments, visualising them as pictographic representations of four different cellular thematic maps. expressyouRcell visually reduces the complexity of displaying gene expression and protein level changes across multiple time-points by generating dynamic representations of cellular pictographs. We applied expressyouRcell to six datasets, demonstrating its flexibility and usability in the visualization of simple and highly effective static and dynamic representations of time course variations in gene expression. Our approach complements classical plot-based methods for visualization and exploitation of biological data, improving the standard quantitative interpretation and communication of relevant results.

bioinformatics↗

bioRxiv2021

Nucleolar NOC1 controls protein synthesis and cell competition in Drosophila

NOC1 is a nucleolar protein necessary in yeast for both transport and maturation of ribosomal subunits. Here we show that in Drosophila NOC1 is essential for the correct animal development, and that its ubiquitous downregulation results in small larvae with reduced polysome abundance and decreased protein synthesis. NOC1 expression in multiple organs, such as the prothoracic gland and the fat body, is necessary for proper organ functioning. Reduction of NOC1 in clones from the imaginal discs results in small clones with cells that die by apoptosis, an event that is partially rescued using a M/+ background, suggesting that reduction of NOC1 causes the cells to acquire a loser state. This event was supported also by an increase in the transcription of Xrp1 and by activation of the pro-apoptotic eiger-JNK pathway, resulting in the upregulation of DILP8 as an indication of cellular damage. Here, we show that Drosophila NOC1 is important in the control of pre-rRNAs maturation and essential step in the regulation of ribosome biogenesis and its downregulation results in defects in growth and in cell competition, highlighting its novel function in this field. summary statementNOC1 is a nucleolar protein necessary for protein synthesis and ribosomal assembling. Its modulation induces cell competition and affects animal growth.

cell biology↗

bioRxiv2021

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3 splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

cancer biology↗

bioRxiv2020

Phospho-RNA sequencing with CircAID-p-seq

Accurate positional information concerning ribosomes and RNA binding proteins with respect to their transcripts is important to understand the global regulatory network underlying protein and RNA fate in living cells. Most footprinting approaches generate RNA fragments bearing a phosphate or cyclic phosphate groups at their 3' end. Unfortunately, all current protocols for library preparation rely only on the presence of a 3' hydroxyl group. Here, we developed circAID-p-seq, a PCR-free library preparation for 3' phospho-RNA sequencing. We applied circAID-p-seq to ribosome profiling, which produces fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast, highly efficient and low-cost sequencing of phospho-RNA fragments from eukaryotic cells and tissues. While assessing circAID-p-seq to portray ribosomes engaged with transcripts, we provide a versatile tool to unravel any 3'-phospho RNA molecules.

molecular biology↗

bioRxiv2020

FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

Mutations in the RNA binding protein (RBP) FUS cause amyotrophic lateral sclerosis (ALS) and result in its nuclear depletion and cytoplasmic mislocalisation, with cytoplasmic gain of function thought to be crucial in pathogenesis. Here, we show that expression of mutant FUS at physiological levels drives translation inhibition in both mouse and human motor neurons. Rather than acting directly on the translation machinery, we find that mutant FUS forms cytoplasmic condensates that promote the phase separation of FMRP, another RBP associated with neurodegeneration and robustly involved in translation regulation. FUS and FMRP co-partition and repress translation in vitro. In our in vivo model, FMRP RNA targets are depleted from ribosomes. Our results identify a novel paradigm by which FUS mutations favour the condensed state of other RBPs, impacting on crucial biological functions, such as protein translation.

neuroscience↗

bioRxiv2020

One-shot analysis of translated mammalian lncRNAs with AHARIBO

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode uncharacterized proteins, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated lncRNAs and peptide-producing lncRNAs. Here we present AHARIBO, a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs and corresponding de novo synthesized polypeptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds lncRNAs.

cell biology↗

bioRxiv2019

A long noncoding RNA acts as a post-transcriptional regulator of heat shock protein 70 kDa synthesis in the cold hardy Diamesa tonsa under heat shock

Cold stenothermal insects living in glacier-fed streams are stressed by temperature variations resulting from glacial retreat during global warming. The molecular aspects of insect response to environmental stresses remain largely unexplored. The aim of this study was to expand our knowledge of how a cold stenothermal organism controls gene expression at the transcriptional, translational, and protein level under warming conditions. Using the chironomid Diamesa tonsa as target species and a combination of RACE, qPCR, polysomal profiling, western blotting, and bioinformatics techniques, we discovered a new molecular pathway leading to previously overlooked adaptive strategies to stress. We obtained and characterized the complete cDNA sequences of three heat shock inducible 70 (hsp70) and two members of heat-shock cognate 70 (hsc70). Strikingly, we showed that a novel pseudo-hsp70 gene encoding a putative long noncoding RNA (lncRNA) which is transcribed during thermal stress, acting as a ribosome sponge to provide post-transcriptional control of HSP70 protein levels. The expression of the pseudo-hsp70 gene and its function suggest the existence of a new and unexpected mechanism to cope with thermal stress: lowering the pace of protein production to save energy and optimize resources for recovery.

zoology↗

bioRxiv2019

SMN-primed ribosomes modulate the translation of transcripts related to Spinal Muscular Atrophy

The contribution of ribosome heterogeneity and ribosome-associated factors to the molecular control of proteomes in health and disease remains enigmatic. We demonstrate that Survival Motor Neuron (SMN) protein, loss of which causes the neuromuscular disease spinal muscular atrophy (SMA), binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are positioned within the first five codons of a set of mRNAs which are enriched in IRES-like sequences in the 5UTR and rare codons at the beginning of their coding sequence. Loss of SMN at early-stages of SMA induces translational defects in vivo, characterized by ribosome depletion in rare codons at the third and fifth position of the coding sequence. These positional defects cause ribosome depletion from mRNAs bound by SMN-primed ribosomes and translational impairment of proteins involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs which encode proteins relevant to SMA pathogenesis.

neuroscience↗

bioRxiv2018

Hfq CLASH uncovers sRNA-target interaction networks enhancing adaptation to nutrient availability

By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to very efficiently adapt to constant changes in their environment. To better understand how Escherichia coli acclimatizes to changes in nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover sRNA-target interactions. Strikingly, we uncovered hundreds of novel Hfq-mediated sRNA-target interactions at specific growth stages, involving many novel 3UTR-derived sRNAs and a plethora of sRNA-sRNA interactions. We discovered sRNA-target interaction networks that play a role in adaptation to changes in nutrient availability. We characterized a novel 3UTR-derived sRNA (MdoR), which is part of a regulatory cascade that enhances maltose uptake by (a) inactivating repressive pathways that block the accumulation of maltose transporters and (b) by reducing the flux of general porins to the outer membrane. Our work provides striking examples of how bacteria utilize sRNAs to integrate multiple regulatory pathways to enhance nutrient stress adaptation.

systems biology↗

bioRxiv2017

Active ribosome profiling with RiboLace

Ribosome profiling, or Ribo-Seq, is based around large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information concerning ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-Seq approaches lack the ability to distinguish between fragments protected by ribosomes in active translation or by inactive ribosomes. To overcome these significant limitation, we developed RiboLace: a novel method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

molecular biology↗