Biology subjects

Ascano, M.

Publications and source records attributed to Ascano, M..

7 recordsLinked to original sources

bioRxiv2026

Fatty Acid Synthase Binds Cellular mRNAs and its Depletion Reduces Translational Efficiency

Metabolic enzymes with non-canonical RNA binding activity have drawn a growing interest, due to their implications for basic biology and disease. Here we present the first endogenous RNA interactome of fatty acid synthase (FASN), an emerging oncological and virological target. Utilizing crosslinking-based approaches, we characterize the direct RNA association of FASN in two human cell lines, identifying over 6000 binding sites across over 3500 protein-coding genes, with a striking preference for the 5' UTR. We provide a functional cross-screen on the effects of FASN depletion on cellular translation. This integration with ribosome profiling indicated a preferential reduction in the translational efficiencies of FASN-associated mRNAs - an effect shown to be dependent on binding location and site number. Associated transcripts were enriched for morphogenic and cell division-related functions, as well as palmitoylated proteins. This correlative work primes further investigation of FASN as a regulator of translation of its target mRNAs.

molecular biology↗

bioRxiv2026

Extracellular vesicle cross-linking coupled with solid-phase purification facilitates discovery of ribonucleoprotein complexes assembled on EV RNAs.

Extracellular vesicles (EVs) are fundamental mediators of intercellular communication that transfer macromolecular cargo, including proteins and RNAs, to functionally reprogram recipient cells. However, the molecular processes by which transferred ribonucleoprotein (RNP) complexes engage recipient host machinery to influence cellular behavior are poorly understood, primarily due to a dearth of methods capable of tracking these post-transfer interactions. To address this gap, we developed two complementary chemical biology platforms: EV-CLASP and EV(r)-CLASP. While EV-CLASP maps the native, intra-vesicular RNP landscape packaged within the EV, EV(r)-CLASP captures post-transfer dynamics by identifying the host proteins that directly associate with EV-derived RNAs following cellular uptake. Both approaches leverage metabolic labeling and photo-crosslinking for high-specificity recovery. Donor cells are cultured with 4-thiouridine (4SU) to biosynthetically label newly synthesized RNAs during EV biogenesis. Purified EVs can be crosslinked using 365 nm UV light to capture intra-vesicular RNP composition. Alternatively, 4SU-labeled EVs can be delivered to recipient cells and subsequently crosslinked to identify the primary physical interactions formed within the recipient-cell environment. Crosslinked RNP complexes are isolated under stringent conditions using solid-phase reversible beads to minimize background, followed by quantitative mass spectrometry for protein identification. Together, EV-CLASP and EV(r)-CLASP provide a robust, versatile framework for defining the dynamic RNP assemblies that facilitate extracellular RNA communication, offering a powerful toolkit to uncover novel RNA-associated regulatory networks, therapeutic targets, and biomarkers across diverse biological and disease contexts.

cancer biology↗

bioRxiv2025

The Incoming Influenza Genome Assembles a Host RBP Network that Orchestrates Viral RNA Synthesis

Influenza A virus (IAV) replication initiates within minutes of entry, yet the earliest host determinants acting on the incoming viral genome remain undefined. Here, using VIR-CLASP to capture protein interactions with progenitor vRNA prior to any detectable viral transcription, we map the first host-viral RNA interface and identify [~]700 human RNA binding proteins (RBPs) proactively recruited by the negative-sense genome. These factors are enriched in pathways essential for RNA metabolism, chromatin remodeling, transcriptional regulation, and nuclear condensate organization, revealing that the vRNA engages a far broader host network than previously appreciated. Functional interrogation of top candidates demonstrates that GMPS, TOP2A, SRRM2, and SPEN serve as critical proviral factors operating at distinct stages of the IAV synthesis program: GMPS and SPEN are required for efficient production of vRNA, cRNA, and mRNA; TOP2A promotes mRNA capping and early transcript accumulation; and SRRM2, together with SR-proteins, coordinates splicing of viral M and NS transcripts. These findings support a model in which the incoming vRNA acts not merely as a transcriptional template but as a scaffold that initiates assembly of nuclear machinery required for productive viral replication. By defining the pre-replicative viral RNA interactome and its functional consequences, this work exposes an unrecognized layer of host control over IAV permissivity and establishes new points of vulnerability for antiviral intervention.

molecular biology↗

bioRxiv2025

Early Host-Virus RNA Interactions Reveal SPEN-Driven m6A Regulation as a Major Determinant of Henipavirus Infection

Early interactions between viral RNA and host-encoded RNA-binding proteins are pivotal in shaping the trajectory of RNA virus infection. Henipaviruses are emerging, highly lethal BSL-4 pathogens whose mechanisms of pathogenesis remain largely elusive. To illuminate the earliest moments of host-virus interplay, we employed Viral Cross-linking and Solid-phase Purification (VIR-CLASP) to capture host proteins bound to the incoming henipavirus genome within the first hour of infection. This approach establishes the first henipavirus RNA-host protein interactome, revealing 146 human proteins directly associated with the primary viral RNA. Among these, SPEN, RBM15, and RBM15B - canonical regulators of lncRNA Xist - emerged as key host factors that actively promote viral infection. Direct RNA sequencing further uncovered that SPEN depletion induces widespread hypomethylation, affecting ~98% of differentially modified m6A sites, ~87% of which localize to the L mRNA transcript encoding the viral RNA-dependent RNA polymerase. Collectively, these findings expose a critical layer of host dependency at the very onset of infection and reveal a previously unappreciated role for SPEN family proteins in facilitating henipavirus infection.

biochemistry↗

bioRxiv2020

ELAVL1 Exclusively Couples mRNA Stability with the 3'UTRs of Interferon Stimulated Genes

Upon detection of a pathogen, the innate immune system triggers signaling events leading to the transcription of mRNAs that encode for pro-inflammatory and anti-microbial effectors. RNA-binding proteins (RBPs) interact with these functionally critical mRNAs and temporally regulate their fates at the post-transcriptional level. One such RBP is ELAVL1, which is known to bind to introns and 3UTRs. While significant progress has been made in understanding how ELAVL1 regulates mRNAs, how its target repertoire and binding affinity changes within an immunological context remains poorly understood. Here, we overlap four distinct high-throughput approaches to define its cell-type and context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 overwhelmingly binds to intronic sites in a naive state, but during an innate immune response, ELAVL1 targets the 3UTR - binding both previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate the pathways involved in pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to best understand their post-transcriptional regulatory roles within specific biological circuitries.

molecular biology↗

bioRxiv2020

Viral cross-linking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

The initial interactions between incoming, pre-replicated RNA virus genomes and host protein factors are important in infection and immunity. Yet there are no current methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4SU-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The cross-linked RBPs are purified by solid-phase reversible immobilization (SPRI) beads with protein denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming viral genomes are labeled with 4SU, so cross-linking events specifically occur between proteins and pre-replicated viral genomic RNA. Since solid-phase purification under protein-denaturing conditions is used to pull-down total RNA and cross-linked RBPs, this facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes [~]7 days and VIR-CLASP takes 1 day.

immunology↗

bioRxiv2020

Small molecule inhibition of human cGAS reduces total cGAMP output and cytokine expression in cells

The cGAS-STING pathway is a major mechanism that mammalian cells utilize to detect cytoplasmic dsDNA from incoming viruses, bacteria, or self. CYCLIC GMP-AMP SYNTHASE (cGAS) is the sensor protein that directly binds dsDNAs. cGAS synthesizes cyclic GMP-AMP (cGAMP), which binds to the adaptor STIMULATOR OF INTERFERON GENES (STING), activating an INTERFERON REGULATORY FACTOR 3 (IRF3)-mediated immune response. Constitutive activation can result in interferonopathies such as Aicardi-Goutieres Syndrome (AGS) or other lupus-like autoimmune disorders. While inhibitors targeting mouse or human cGAS have been reported, the identification of a small molecule that targets both homologs of cGAS has been challenging. Here, we show that RU.521 is capable of potently and selectively inhibiting mouse and human cGAS in cell lines and human primary cells. This inhibitory activity requires the presence of cGAS, but it cannot suppress an immune response in cells activated by RNA, Toll-like receptor ligands, cGAMP, or recombinant interferon. Importantly, when RU.521 is applied to cells, the production of dsDNA-induced intracellular cGAMP is suppressed in a dose-dependent manner. Our work validates the use of RU.521 for probing DNA-induced innate immune responses and underscores its potential as an ideal scaffold towards pre-clinical development, given its potency against human and mouse cGAS.

immunology↗