Biology subjects

Vandelli, A.

Publications and source records attributed to Vandelli, A..

11 recordsLinked to original sources

bioRxiv2026

A circRNA-derived IRES-like element enables programmable translational activation of the endogenous chaperone HSP90

Circular RNAs (circRNAs) are covalently closed transcripts that lack a 5' cap, consequently their translation depends on cap-independent initiation, in most cases through Internal Ribosome Entry Sites (IRESs). Whereas IRES-mediated translation has been studied almost exclusively in cis, the use of IRES-containing circRNAs to activate translation of an independent mRNA in trans has only recently begun to be explored. Here, we characterize SynFarsa, a synthetic circRNA-derived IRES-like element that promotes cap-lindependent translation in mouse and human cells. We engineered SynFarsa into a trans-acting regulator by fusing it to a short antisense sequence complementary to a selected region of a target mRNA. This reproduces the architecture of a circular SINEUP, in which IRES activity promotes translation in trans of a transcript recognized through antisense complementarity. Directing this construct to the hsp90 3'UTR increased HSP90 protein levels approximately 1.6-lfold without altering hsp90 mRNA abundance, whereas the hsp70 3'UTR and the 5'UTRs of both transcripts were not permissive, indicating that activity depends strongly on target site selection. In a FUSP525L neuroblastoma model of Amyotrophic Lateral Sclerosis, this increase in HSP90 was accompanied by a reduction in the number and size of cytoplasmic FUS aggregates. SynFarsa thus provides a circRNA-derived effector domain for targeted translational upregulation of an endogenous protein.

biochemistry↗

bioRxiv2026

The human RNA-DNA interactome is cell type-specific and dynamic

More than twenty years ago, the FANTOM consortium uncovered that mammalian genomes are pervasively transcribed, revealing multitudes of RNAs with unknown functions. A subset of these transcripts has since then been linked to transcriptional control and to chromatin organization via their ability to interact with DNA, suggesting that chromatin-associated RNAs could be key players in genome regulation. Although recent technological advances now enable the mapping of genome-wide RNA-DNA contacts, a lack of analyses integrating these methods with other genomic features and across multiple cellular contexts hinders our comprehensive understanding of the principles underlying RNA-DNA interactions and of their biological importance. As part of the FANTOM6 project, we thus generated RNA-DNA interaction maps in 16 different human cell types, then combined these contacts with multiple layers of other genomic data to investigate how patterns of interaction between RNA and DNA relate to chromatin organization and function. We show that the RNA-DNA interactome is highly dynamic yet reproducibly organized in cell-type specific networks, constituted of a great diversity of interactions that vary in function of their distance, the nature of their sources and the chromatin state of their targets. In particular, we detected numerous regulatory elements that exhibit marked changes in activity when differentially bound by transcripts, implying that thousands of RNA-DNA interactions can play a mechanistic role in gene expression. This regulatory function correlates with RNA-protein interactions and significantly associates with cell type-relevant and disorder-related traits. In addition to providing essential resources for future research in RNA-mediated chromatin regulation, cellular biology and human diseases, our study thus establishes the RNA-DNA interactome as a new genome regulatory layer that defines and maintains cellular identity and behavior.

genomics↗

bioRxiv2025

Integrating RNA Structure and Protein Interactions to Uncover the Mechanisms of Viral and Cellular IRES Function

BackgroundRNAs fold into complex structures that critically influence gene expression. A prominent class of regulatory elements resides in the 5' untranslated region (5' UTR), where internal ribosome entry sites (IRESs) promote cap-independent translation by directly engaging the ribosome. First discovered in viral genomes, IRESs have been classified into four types according to their structural compactness and factor requirements. While viral IRESs are well studied, cellular IRESs remain poorly understood: they display limited sequence conservation, reduced structural compactness, and variable dependence on auxiliary RNA-binding proteins known as IRES trans-acting factors (ITAFs). Whether their activity relies mainly on RNA structure or protein assistance remains unresolved. Here, we present a computational framework that combines in silico mutagenesis and RNA-protein interaction profiling to investigate IRES mechanisms and guide the design of synthetic elements. ResultsUsing the Hepatitis C Virus (HCV) IRES as a benchmark, we performed systematic single-nucleotide mutagenesis coupled with structural predictions. Mutations were classified as synonymous or non-synonymous based on their effect on the secondary structure. The HCV IRES showed overall robustness, but the domain interacting with eIF3 was particularly sensitive, consistent with its essential role in translation initiation. Extending this approach to other viral IRES families revealed distinct profiles of resilience: Aphthoviruses retained structural integrity despite extensive sequence variation, whereas Cripaviruses displayed higher variability. We then applied the same analysis to cellular IRESs, which proved more structurally sensitive, suggesting stronger reliance on cofactor support. To probe this connection, we used the catRAPID approach to predict interactions with translation-related proteins. The method distinguished IRESs with known ITAF binding, such as PTBP1, and highlighted stability-promoting mutations that increased the predicted affinity for translation factors. ConclusionsOur in silico analysis indicates that mutational tolerance mirrors IRES cofactor dependence: compact viral IRESs are structurally robust, whereas non-viral IRESs are more reliant on protein interactions. By linking structure prediction with interaction profiling, we identify variants that both stabilize IRESs and improve binding to ITAFs or translation factors. This framework provides mechanistic insight into sequence-structure-function relationships and supports the rational design of synthetic IRES elements for therapeutic and biotechnological applications.

biochemistry↗

bioRxiv2025

A spatial long-read approach at near-single-cell resolution reveals developmental regulation of splicing and polyadenylation sites in distinct cortical layers and cell types.

Genome-wide single-cell and spatial long-read approaches have gained traction, but mostly lack single-cell resolution - and yield limited read lengths. Here, we introduce spatial ISOform sequencing (Spl-ISO-Seq), which reveals exons and polyadenylation sites from long reads with near-single-cell resolution. Spl-ISO-Seq selects long cDNAs and doubles to triples read lengths compared to standard preparations. Adding a highly specific software tool (Spl-ISOquant) and comparing human post-mortem pre-puberty samples of the visual cortex (8-11 years) to post-puberty samples (16-19 years), we find that cortical layers harbor stronger splicing and poly(A)-site regulation than the adjacent white matter, with enrichment of multiple protein-domain types. For oligodendrocytes however, developmental splicing changes are stronger in white matter. Among cortical layers, layer 4 has the most developmental changes in alternative-exon inclusion in excitatory neurons and in poly(A) sites. We also find many repeat elements, especially ERV1 long terminal repeats downstream of developmentally-regulated layer 4 exons. Overall, alternative splicing changes are linked to synapses - specifically at the post-synapse. Age-linked splicing changes in layers 1-3 and 4 are associated with autism spectrum disorder but not with schizophrenia, amyotrophic lateral sclerosis and Alzheimers disease. These results root developmental splicing changes during puberty and the resulting protein changes in specific layers and cell types. More generally, our new technologies enable new observations for any complex tissue.

genomics↗

bioRxiv2024

Rationalizing the Effects of RNA Modifications on Protein Interactions

RNA modifications play a crucial role in regulating gene expression by altering RNA structure and modulating interactions with RNA-binding proteins (RBPs). In this study, we explore the impact of specific RNA chemical modifications--N6-methyladenosine (mA), A-to-I editing, and pseudouridine ({Psi})--on RNA secondary structure and protein-RNA interactions. Utilizing genome-wide data, including RNA secondary structure predictions and protein-RNA interaction datasets, we classify proteins into distinct categories based on their binding behaviors: modification-specific and structure-independent, or modification-promiscuous and structure-dependent. For instance, mA readers like YTHDF2 exhibit modification-specific and structure-independent binding, consistently attaching to mA regardless of structural changes. Conversely, proteins such as U2AF2 display modification-promiscuous and structure-dependent behavior, altering their binding preferences in response to structural changes induced by different modifications. A-to-I editing, which causes significant structural changes, typically reduces protein interactions, while {Psi} enhances RNA structural stability, albeit with variable effects on protein binding. To better predict these interactions, we developed the catRAPID 2.0 RNA modifications algorithm, which forecasts the effects of RNA modifications on protein-RNA binding propensities. This algorithm serves as a valuable tool for researchers, enabling the prediction and analysis of RNA modifications impact on protein interactions, thus offering new insights into RNA biology and engineering. The catRAPID 2.0 RNA modifications tool is available at http://service.tartaglialab.com/new_submission/catrapid_omicsv2_rna_mod. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/610603v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@136148corg.highwire.dtl.DTLVardef@17f2d65org.highwire.dtl.DTLVardef@7d4ce9org.highwire.dtl.DTLVardef@447e47_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Subgenomic flaviviral RNAs and human proteins: in silico exploration of anti-host defense mechanisms

BackgroundFlaviviruses pose significant global health threats, infecting over 300 million people annually. Among their evasion strategies, the production of subgenomic flaviviral RNAs (sfRNAs) from the 3 UTR of viral genomes is particularly notable. These sfRNAs interact with human proteins, disrupting key cellular processes such as RNA splicing and the interferon response. ResultsUtilizing a comprehensive in silico approach with the catRAPID algorithm, we analyzed over 300,000 interactions between sfRNAs and human proteins derived from more than 8,000 flavivirus genomes, including Dengue, Zika, Yellow Fever, West Nile, and Japanese Encephalitis viruses. Our study not only validated known interactions but also revealed novel human proteins that could be involved in sfRNA-mediated host defense evasion, including helicases, splicing factors, and chemokines. We propose that sfRNAs function as molecular sponges, sequestering specific proteins indicative of sfRNA-forming regions across flaviviruses. These findings represent a valuable resource for diagnostic and therapeutic developments. ConclusionsOur findings significantly expand the known interactome of sfRNAs with human proteins, underscoring their role in modulating host cellular pathways. By providing the first extensive atlas of sfRNA interactions, we offer new insights into how flaviviruses can manipulate host cellular machinery to facilitate viral survival and persistence. Intriguingly, we predict interaction with stress granules, a critical component of the cellular response to viral infection, suggesting a mechanism by which flaviviruses inhibit their formation to evade host defenses. This atlas not only serves as a resource for exploring therapeutic targets but also aids in the identification of sfRNA biomarkers for improved flavivirus diagnostics.

biochemistry↗

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

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

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↗

bioRxiv2021

A spoonful of disorder regulates interactions within Stress Granules and Processing Bodies

Condensation, or liquid-like phase separation, is a phenomenon indispensable for the spatiotemporal regulation of molecules within the cell. Recent studies indicate that the composition and molecular organization of phase-separated organelles such as Stress Granules (SGs) and Processing Bodies (PBs) are highly variable and dynamic. A dense contact network involving both RNAs and proteins controls the formation of SGs and PBs and an intricate molecular architecture, at present poorly understood, guarantees that these assemblies sense and adapt to different stresses and environmental changes. Here, we investigated the physico-chemical properties of SGs and PBs components and studied the architecture of their interaction networks. We found that proteins and RNAs establishing the largest amount of contacts in SGs and PBs have distinct structural properties and intrinsic disorder is enriched in all protein-RNA, protein-protein and RNA-RNA interaction networks. The increase of disorder in proteins is accompanied by an enrichment in single-stranded regions of RNA binding partners. Our results suggest that SGs and PBs quickly assemble and disassemble through fuzzy-like dynamic contacts modulated by unfolded domains of their components. Research HighlightsO_LIWe systematically studied RNA-RNA, protein-protein and RNA-protein interaction networks in stress granules and processing bodies; C_LIO_LIRNAs enriched in stress granules and processing bodies are more single-stranded and form a large number of contacts with both proteins and RNAs; C_LIO_LIProteins in stress granules and processing bodies are less structured and contact larger amounts of single-stranded RNAs. C_LI

molecular biology↗

bioRxiv2020

Structural analysis of SARS-CoV-2 andprediction of the human interactome

Specific elements of viral genomes regulate interactions within host cells. Here, we calculated the secondary structure content of >2000 coronaviruses and computed >100000 human protein interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic regions display different degrees of conservation. SARS-CoV-2 domain encompassing nucleotides 22500 - 23000 is conserved both at the sequence and structural level. The regions upstream and downstream, however, vary significantly. This part codes for the Spike S protein that interacts with the human receptor angiotensin-converting enzyme 2 (ACE2). Thus, variability of Spike S may be connected to different levels of viral entry in human cells within the population. Our predictions indicate that the 5 end of SARS-CoV-2 is highly structured and interacts with several human proteins. The binding proteins are involved in viral RNA processing such as double-stranded RNA specific editases and ATP-dependent RNA-helicases and have strong propensity to form stress granules and phase-separated complexes. We propose that these proteins, also implicated in viral infections such as HIV, are selectively recruited by SARS-CoV-2 genome to alter transcriptional and post-transcriptional regulation of host cells and to promote viral replication.

bioinformatics↗