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Angiosarcoma (AS) is a rare and aggressive tumor arising within the endothelium, characterized by a high metastatic rate and poor prognosis. Our prior work established that endothelial loss of Dicer1, a key enzyme in microRNA (miRNA) processing, drives AS formation in mice, indicating a tumor suppressive role for miRNAs in tumorigenesis. Here, we corroborated this hypothesis by generating a novel conditional knockout model targeting Dgcr8, a core component of the microprocessor complex required for pri-miRNA processing. Conditional deletion of Dgcr8 phenocopies Dicer1 loss, resulting in spontaneous AS formation and global loss of mature miRNAs. We further demonstrate that treatment with enoxacin (ENX), a repurposed antibiotic known to enhance miRNA processing, reduces viability, migration, and clonogenicity of AS cells. ENX increases the abundance of tumor-suppressive miRNAs and downregulates oncogenic pathways, including pathways related to cell cycle progression, angiogenesis, and cell migration. These results establish the essential role of miRNA biogenesis in suppressing AS and reveal a pharmacologically targetable vulnerability via ENX-mediated enhancement of miRNA expression in tumors.

Detection of foreign RNAs is a crucial activation step for innate immunity pathways in response to viral infections. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic RNA sensor that triggers type I and III interferon (IFN) expression and activates the antiviral response in response to RNA virus infection. The activating ligand for RIG-I has been shown to be 5'-triphosphated, blunt-ended, double-stranded (ds)RNA, but questions remain on the impact of other RNA motifs on RIG-I activation. Here we show that immune-activating copy-back viral genomes (cbVGs) contain RNA stem loops away from the 5' end of the RNA that enhance RIG-I signaling and IFN expression. Importantly, the sequence of the terminal loops of the activating motifs impacts the strength of IFN expression. Additionally, we show that synthetic versions of these cbVG-derived stem loops trigger innate immune responses in mice demonstrating their potential as immunostimulants in vivo.

SCNα genes encode components of voltage-gated sodium channels that are crucial for generating electrical signals. Humans have ten paralogous SCNα genes, some of which contain duplicated mutually exclusive exons 5a and 5b. In reconstructing their evolutionary history, we found multiple unannotated copies of exon 5 in distant species and showed that exon 5 duplication goes back to a common ancestor of the SCNα gene family. We characterized splicing patterns of exons 5a and 5b across tissues, tumors, and developmental stages, and demonstrated that the nonsense mediated decay (NMD) system is not the major factor contributing to their mutually exclusive choice. Comparison of SCN2A, SCN3A, SCN5A, and SCN9A intronic nucleotide sequences revealed multiple Rbfox2 binding sites and two highly conserved intronic splicing regulatory elements (ISRE) that are shared between paralogs. Minigene mutagenesis and blockage by antisense oligonucleotides showed that the formation of RNA structure between ISRE promotes exon 5b skipping in SCN9A. The inclusion of exon 5b is also suppressed in siRNA-mediated knockdown of Rbfox2, which makes the collective action of RNA structure and Rbfox2 compatible with the model of a structural RNA bridge. ISRE sequences are conserved from human to elephant shark and may represent an ancient, evolutionarily conserved regulatory mechanism. Our results demonstrate the power of comparative sequences analysis in application to paralogs for elucidating splicing regulatory programs.

RNA-binding proteins (RBPs) are essential modulators in the regulation of mRNA processing. The binding patterns, interactions, and functions of most RBPs are not well-characterized. Previous studies have shown that motif context is an important contributor to RBP binding specificity, but its precise role remains unclear. Despite recent computational advances to predict RBP binding, existing methods are challenging to interpret and largely lack a categorical focus on RBP motif contexts and RBP-RBP interactions. There remains a need for interpretable predictive models to disambiguate the contextual determinants of RBP binding specificity in vivo. Here, we present a novel and comprehensive pipeline to address these knowledge gaps. We devise a Natural Language Processing-based method to deconstruct sequences into entities comprising a target k-mer and its flanking regions, then use this representation to formulate RBP binding prediction as a weakly supervised Multiple Instance Learning problem. To interpret our predictions, we introduce a deterministic motif discovery algorithm to leverage our data structure, recapitulating the established motifs of numerous RBPs as validation. Importantly, we characterize the binding motifs and binding contexts for 71 RBPs in HepG2 and 74 RBPs in K562, with many of them being novel. Finally, through feature integration, transitive inference, and a new cross-prediction approach, we propose novel cooperative and competitive RBP-RBP interaction partners and hypothesize their potential regulatory functions. In summary, we present a complete framework for investigating the contextual determinants of specific RBP binding, and we demonstrate the significance of our findings in delineating RBP binding patterns, interactions, and functions.

N ⁶-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes, yet whether m⁶A sites are functionally important or represent neutral byproducts remains unclear. Previous evolutionary analyses failed to detect consistent conservation signatures at m⁶A sites, and report conflicting patterns of conservation across genic regions, such as the coding sequence (CDS) and untranslated regions (UTRs). To reconcile these inconsistencies and definitively determine whether m⁶A sites are under selection, we developed novel motif-level conservation metrics that incorporate knowledge of m⁶A biogenesis to distinguish m⁶A-specific selection from other confounding sources. We analyzed ∼500,000 candidate sites with quantitative, single-nucleotide resolution m⁶A measurements across a phylogeny spanning 447 mammalian species. After controlling for proximity to exon-junctions, we observed a clear, dose-dependent relationship between m⁶A stoichiometry and evolutionary conservation in both CDS and UTRs. Highly methylated sites (>60%) exhibited significantly increased conservation compared to lowly methylated sites-with an effect size approximately one-third of the typical CDS-UTR difference-providing definitive evidence of purifying selection and supporting a model where highly modified sites contribute functionally to gene regulation. We established a methodological framework for evolutionary analysis of RNA modifications, highlighting the necessity of quantitative measurements, comprehensive phylogenetic sampling, and careful consideration of modification biogenesis.

This Perspective discusses seven frontiers in RNA modification research. The examples cited highlight technological advances, regulatory principles both unique and broad-spanning, and questions about how biological information is post-transcriptionally encoded in chemical marks comprising just a few atoms.

Environmental stress activates transposons and is proposed to generate genetic diversity that facilitates adaptive evolution. piRNAs guide germline transposon silencing, but the impact of stress on the piRNA pathway is not well understood. In Drosophila, the Rhino-Deadlock-Cuff complex (RDC) drives transcription of clusters composed of nested transposon fragments, generating precursors that are processed into mature piRNAs in the cytoplasm. We show that acute heat shock triggers rapid, reversible loss of RDC localization and cluster transcript expression with coordinate changes in the cytoplasmic processing machinery. Maternal piRNAs bound to Piwi are proposed to guide Rhino localization to clusters during early embryogenesis. However, RDC relocalization after heat shock is accelerated in piwi mutants and delayed in thoc7 mutants, which disrupt piRNA precursor binding to THO complex, and we show that maternally deposited piRNAs are dispensable for RDC localization to the major 42AB cluster. Cluster specification is reconsidered in light of these findings.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape strategies include general inhibition of host gene expression referred to as host shutoff. Viral nonstructural protein 1 (Nsp1) is the main host shutoff factor that blocks protein translation and induces messenger RNA (mRNA) cleavage and degradation. Viral mRNAs are resistant to the translation shutoff and cleavage induced by Nsp1, and the 5' leader sequence present in all viral mRNAs has been shown to confer resistance. However, the exact molecular mechanism for escape from Nsp1 host shutoff has not been demonstrated. In our previous work, we analyzed the effects of Nsp1 on the expression and function of cellular proteins important for stress granule formation. We discovered that the host transcript for the TIA1 cytotoxic granule-associated RNA-binding protein-like 1 (TIAL1, commonly referred to as TIAR) is resistant to SARS-CoV-2 Nsp1 host shutoff. In this work, using reporter shutoff assays, we examined sequence and structural features of the TIAR 5' untranslated region (UTR) and discovered that the first 23 nt of the TIAR transcript are both necessary and sufficient to confer resistance to the Nsp1. Furthermore, our work revealed that the lack of guanosines within a window of 10-18 nt downstream from the 5' end is a defining feature of Nsp1-resistant transcripts shared between the SARS-CoV-2 leader sequence and the TIAR 5' UTR. Our findings are consistent with the model in which sequence features of 5' UTRs, rather than their secondary structure, confer resistance to Nsp1 host shutoff to both viral and cellular mRNAs.

Ribosome profiling (Ribo-seq) is a next-generation, high-resolution sequencing technique that captures ribosome-protected mRNA fragments to map ribosome positions across the transcriptome. This method serves as a powerful proxy for global translational activity by revealing where ribosomes engage with mRNAs. Recent advances have expanded the utility of Ribo-seq to resolve distinct ribosome populations, including initiating ribosomes, small subunits, collided ribosomes, mitochondrial ribosomes, and those associated with specific translation factors or localized to subcellular compartments. These methodological advances have significantly broadened the scope of Ribo-seq, enabling new insights into the molecular mechanisms that govern translation across diverse eukaryotic systems. In this mini-review, we highlight key innovations in Ribo-seq technology and discuss how they have deepened our understanding of the spatial, temporal, and regulatory dimensions of translational control.

Small noncoding RNAs are versatile regulators of gene expression, capable of guiding the silencing of complementary mRNAs through their association with Argonaute proteins. This RNA-guided silencing, known as RNA interference (RNAi), is a conserved mechanism that shapes diverse biological processes, from development to genome defense. Central to the effectiveness of RNAi is the precise loading of small RNAs into their appropriate Argonaute partners, a step that ensures both specificity and fidelity in target recognition. Although most organisms harbor multiple classes of small RNAs and a corresponding repertoire of Argonautes, the rules that dictate their selective pairing remain only partially understood. Caenorhabditis elegans, with its expanded array of small RNA classes and Argonaute proteins, provides a powerful system to probe these mechanisms. In this review, we synthesize current knowledge on small RNA loading specificity, integrating insights from C. elegans with findings from other organisms. We focus on the interplay between small RNA biogenesis, biochemical properties of small RNAs, structural features of Argonautes, post-translational modifications, and the spatiotemporal coexpression patterns that together orchestrate precise Argonaute loading.