Daniela Ribeiro

During translation, transfer RNAs (tRNAs) deliver specific amino acids to the ribosome in a coordinated manner with the sequence encoded by the messenger RNA. Despite their central role in protein synthesis, their precise contribution to the modulation of translation remains poorly understood, primarily due to lack of methods to characterize tRNA abundances and their modifications from actively translating ribosomes. Here we develop tRIBO-seq, a simple and robust nanopore-based method to selectively capture ribosome-associated native tRNA populations (ribo-tRNAs) from actively translating ribosomes, providing tRNA abundance, modification and fragmentation information from a single experiment. Using tRIBO-seq, we find that ribo-tRNAs, but often not total tRNAs, are significantly altered on stress. Notably, we find that tRNAome alterations strongly vary depending on the stress type; while viral infection and leucine deprivation leads to changes in ribo-tRNA abundances, methionine starvation causes a dramatic loss of methyl-based tRNA modifications. By contrast, we find that arsenite exposure does not alter tRNA abundances nor modification patterns, but rather causes major fragmentation events in selected subsets of ribo-tRNAs. Altogether, tRIBO-seq offers a robust and reproducible approach to map the full tRNA landscape—capturing tRNA abundance, modification and fragmentation patterns—across both total and actively translating tRNA populations, revealing the dynamics of the tRNAome with unprecedented resolution, in a single experiment.

Influenza A virus (IAV), as all other viruses, is completely dependent on the host translation machinery components, including host transfer RNAs (tRNAs), to effectively decode its genome. However, while the human genome is biased towards cytosine (C) and guanosine (G)-ending codons, the IAV genome is skewed towards adenine (A) and uridine (U)-ending codons. Nevertheless, translation of IAV’s RNA genome is highly efficient. Here we show that host tRNA and tRNA epitranscriptome dynamics are important regulators of IAV RNA translation and host antiviral responses. We show that the levels of several tRNA modifications, including 5-methylcarboxymethyluridine (mcm ⁵ U ₃₄ ) and 5-methoxycarbonylmethyl-2-thiouridine (mcm ⁵ s ² U ₃₄ ), and their cognate writers, vary over the course of IAV infection. Additionally, we demonstrate that a set of tRNAs are preferentially recruited to ribosomes upon IAV infection, in line with IAV codon usage requirements. We further show that loss of ELP3, the catalytic subunit of the elongator complex, which is involved in the catalysis of mcm ⁵ U ₃₄ and of mcm ⁵ s ² U ₃₄ , induces tRNA hypomodifications, impairs translation of codon biased IAV genes and triggers the integrated stress response (ISR), interfering with IAV propagation. Taken together, our results uncover the relevance of host tRNAs and their modifications for optimal expression of viral genomes and host antiviral responses, setting the tRNA epitranscriptome as a promising target for the development of host-based antiviral therapies.

Peroxisomes and mitochondria are important platforms for antiviral signal transduction, as detection of cytosolic viral RNA by retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs) induces the activation of the mitochondrial antiviral signaling adaptor (MAVS) at both organelles. To decisively elucidate the mechanistic differences and similarities in kinetics and end products between these two pathways, we developed a doxycycline‐inducible system that allows for precise control of MAVS expression and activation at either peroxisomes or mitochondria, in a timely manner, and across various cell types. Our findings demonstrate that both peroxisomal and mitochondrial MAVS induce type I and III interferon‐dependent antiviral signaling and that the peroxisomal signaling occurs significantly faster than its mitochondrial counterpart. Importantly, using not only the doxycycline‐inducible system but also the conventional activation of the MAVS pathway through direct stimulation of RIG‐I‐like receptors, we demonstrate that the rapid and robust antiviral response resulting from peroxisomal MAVS activation is mechanistically due to the faster oligomerization of MAVS at the membranes of this organelle, when compared to mitochondria. These data emphasize the versatility and speed of the peroxisome‐dependent antiviral response and may lead to the identification of specific targets to develop novel host‐directed antiviral strategies.

Peroxisomes are dynamic organelles that play important roles in cellular metabolic and signaling pathways and are implicated in several disease conditions. Although a detailed comprehension of peroxisome dynamics is crucial in various contexts of health and disease, its accurate assessment still presents significant challenges, mainly due to their morphological heterogeneity. Here, we discuss different strategies to study peroxisome dynamics and present a semi-automated macro, PeroxiDynA, that enables the analysis of peroxisome dynamics from single-plane confocal images of mammalian cells in ImageJ. Our findings confirm that PeroxiDynA can be successfully employed to determine relative peroxisome number and spatial distribution. A comparison between PeroxiDynA and other image analysis strategies reveals that it results in increased reliability in the analysis of peroxisome abundance and morphology. Although future improvements are still required to minimize the time and limitations imposed during analysis of peroxisomal features, we propose that PeroxiDynA is a useful tool for biomolecular studies, not only to analyze peroxisomes but also the dynamics of small biological structures in cells from mammals and other organisms.

Alzheimer's disease (AD) remains a complex and unmet medical challenge, requiring innovative approaches to address its multifaceted pathology. In this study, we explored chromeno[3,4-b]xanthones as a novel multifunctional scaffold, synthesized via the straightforward cyclization of their precursor, (E)-2-styrylchromones. Compounds 10 and 11q-s exhibited potent and selective cholinesterase inhibition (IC₅₀ 1.7-9.0 μM for AChE and BChE), along with significant antiamyloid activity (inhibition exceeding 50% at 50 μM). Among them, compound 11r demonstrated the most well-balanced multifunctional profile against all four AD-relevant targets. Molecular docking studies revealed key π-stacking, hydrogen bonding, and halogen interactions, which underlie the selective binding of compound 11r to AChE and BChE. Moreover, docking and molecular dynamics simulations showed that compound 11r binds strongly to the L-S-shaped β-amyloid 1-42 (Aβ₄₂) fibril with a binding affinity of -11.3 kcal/mol, representing a structural barrier to Aβ₄₂ elongation. Additionally, compound 11r, selected as the representative scaffold, effectively disrupted Aβ aggregation, as demonstrated by in vitro studies, transmission electron microscopy (TEM), and cellular studies. It also displayed favorable drug-like properties, including predicted blood-brain barrier (BBB) permeability and an acceptable safety profile at active doses. The calcein-AM-assay also showed that this compound is unlikely to be actively effluxed from the brain. These findings underscore the therapeutic potential of chromeno[3,4-b]xanthone as multifunctional agents for AD, broadening the chemical space of small-molecule exploration.

Protein Phosphatase 1 (PP1) is a major serine/threonine phosphatase in eukaryotes, participating in several cellular processes and metabolic pathways. Due to their low substrate specificity, PP1’s catalytic subunits do not exist as free entities but instead bind to Regulatory Interactors of Protein Phosphatase One (RIPPO), which regulate PP1’s substrate specificity and subcellular localization. Most RIPPOs bind to PP1 through combinations of short linear motifs (4–12 residues), forming highly specific PP1 holoenzymes. These PP1-binding motifs may, hence, represent attractive targets for the development of specific drugs that interfere with a subset of PP1 holoenzymes. Several viruses exploit the host cell protein (de)phosphorylation machinery to ensure efficient virus particle formation and propagation. While the role of many host cell kinases in viral life cycles has been extensively studied, the targeting of phosphatases by viral proteins has been studied in less detail. Here, we compile and review what is known concerning the role of PP1 in the context of viral infections and discuss how it may constitute a putative host-based target for the development of novel antiviral strategies.

Influenza A virus (IAV) is one of the main causes of annual respiratory epidemics in humans. IAV employs multiple strategies to evade host immunity and hijack cellular mechanisms to support proper virion formation and propagation. Some of these strategies encompass the manipulation of pathways involved in protein homeostasis, leading to changes in the host proteome and protein distribution within the cell. In this study, we performed a detailed analysis of the interplay between IAV and the host cells’ proteostasis mechanisms throughout the entire infectious cycle. We reveal that IAV infection induces the activation of the inositol requiring enzyme 1 (IRE1) branch of the unfolded protein response (UPR), at an infection stage that coincides with high rates of viral protein translation. This activation is particularly important for infection, as attenuation of virus production was observed upon IRE1 inhibition. Concomitantly to UPR activation, we observed the accumulation of virus-induced insoluble protein aggregates, which contain both viral and host proteins and are associated with a dysregulation of the host cell RNA metabolism. We demonstrate that this accumulation is important for IAV propagation, as its prevention using a quinoline-steroid hybrid compound significantly reduces the number of produced infectious virus particles. Our data suggests that the formation of these insoluble protein aggregates favors the final steps of the infection cycle, more specifically the virion assembly. Our findings reveal additional mechanisms by which IAV disrupts the host cell proteostasis to favor infection and uncover new cellular targets that can be explored for the development of host-directed antiviral strategies.

Emerging evidence highlights the multifaceted roles of the RNA epitranscriptome during viral infections. By modulating the modification landscape of viral and host RNAs, viruses enhance their propagation and elude host surveillance mechanisms. Here, we discuss how specific RNA modifications, in either host or viral RNA molecules, impact the virus-life cycle and host antiviral responses, highlighting the potential of targeting the RNA epitranscriptome for novel antiviral therapies.