ABSTRACT Ribozyme-based permuted intron-exon (PIE) systems offer a protein-independent route to circRNA production, but existing platforms require elevated temperatures that promote RNA degradation. Here we report the first application of the Candida albicans mitochondrial large subunit ( C.a .mtLSU) group I intron as a PIE platform for circRNA synthesis, which we term P Can PIE (Pyle lab Candida PIE). We evaluated three peripheral stems, P5, P6b, and P8, as permutation sites and demonstrated that all three support circularization under near-physiological conditions (25°C, 6 mM MgCl 2 ), without the 55°C heating step required by existing PIE systems. Kinetic analysis revealed that permutation site does not affect the observed splicing rate constant but does influence P Can PIE folding and therefore influences circularization efficiency. The P6b permutation yielded the highest circularization efficiency, with 95 % of the precursor splicing to produce circRNA. Optimization of spacer sequences flanking the circRNA payload eliminated interference from structured native exon sequences and enabled efficient circularization of RNAs up to 1,657 nt, including structured, repetitive, and naturally occurring sequences. Together, these results establish P Can PIE as a versatile and near-physiologically active addition to the group I intron PIE toolkit.

Pathogenic fungi represent an expanding threat to public health, and yet resistance to current antifungals is surging due to the longstanding use of drugs with limited target diversity. T2307 is a promising bis-arylamidine (BARA) antifungal with novel target specificity, broad-spectrum activity, and low toxicity, but the clinical development of which was discontinued after Phase I trials. To further advance the therapeutic development of this promising class of compounds, we designed and synthesized BARA analogues and systematically evaluated their antifungal activity against clinically important and drug-resistant strains of two very different classes of fungi: Candida and Aspergillus . Many analogues showed potency comparable to or exceeding that of T2307 , and somewhat divergent structure–activity relationships (SARs) observed between Candida and Aspergillus provide useful insights into the molecular mechanisms for efficacy of the drugs . For example, compounds Z3, Z15, and Z17 were the most active against Candida spp. , whereas Z4 – Z6 and Z14 showed the best activity against Aspergillus strains. Spermine/spermidine competition experiments support that these compounds share a carrier-mediated uptake pathway with T2307 . The leading anti-yeast and anti-mold analogues exhibited a favorable safety profile and ADME properties and robust pharmacokinetic behavior, supporting the potential of BARAs as drug candidates suitable for further development of selective, broad-spectrum antifungal agents.

The RNA genome of West Nile Virus (WNV) folds into an elaborate series of RNA structural elements that are crucial for viral function. Among these elements, four pseudoknots (PKs) at the viral 3'-terminus, designated as SLII, SLIV, DBI, and DBII, are among the most crucial players in the overall flaviviral lifecycle. While many studies have focused on exploring the behavior of individual PKs, we investigated the collective role of all four PKs in viral growth and small flaviviral RNA (sfRNA) formation. Through mutational analyses and infectious models, we establish that the four PKs are interdependent and work synergistically to aid in the folding and compaction of the WNV 3'-terminal region. A striking hierarchy is observed in PK contributions to global folding and sfRNA formation, whereby SLIV plays the largest role, followed by DBI, DBII, and SLII. We also discover highly conserved RNA tertiary motifs within the PK assembly that are shared across flaviviruses, suggesting a new type of druggable target that may be of value in the search for pan-flaviviral therapeutics.

Long noncoding RNAs (lncRNAs) constitute most of the human transcriptome and perform essential roles in chromatin organization and transcriptional regulation. Because lncRNA genes are not constrained by protein-coding ability, they tend to exhibit more rapid evolutionary divergence. Their poor nucleotide sequence conservation among mammals often led to the assumption that lncRNAs lack conserved structures. However, emerging evidence indicates that many noncoding RNAs adopt secondary and tertiary folds critical for protein recruitment, chromatin binding, and regulation of gene expression. Nevertheless, there are few experimental secondary structures for lncRNAs, hindering mechanistic insight into lncRNA structure-function relationships. Even without available structural data, covariation, in which two nucleotides co-evolve, can provide evidence for conserved structures. This requires sequence alignments with sufficient divergence to detect covariation but enough similarity to maintain alignment quality. Here we report the development of a novel computational pipeline to mine 190 unannotated primate genomes to generate high-quality multiple sequence alignments of noncoding RNAs. This pipeline performs sequence searching, locus extraction, cross-species alignment, and downstream analyses, including assessment of covariation and primary sequence conservation. Ultimately, we demonstrate that because many noncoding elements, such as lncRNAs evolve at a more rapid rate than protein-coding genes, phylogenetic analyses constrained within a narrower evolutionary span can be used to identify conservation of primary sequence and secondary structure. By focusing our alignments on the primate lineage, our method overcomes the limitations of broad phylogenetic analyses, enabling high-resolution detection of subtle conservation patterns and conserved secondary structural motifs of long noncoding RNAs.

The innate immune receptor MDA5 serves as a first line of defense against RNA viruses by recognizing viral double-stranded RNA (dsRNA) and initiating antiviral signaling. MDA5 is upregulated by another RLR family member known as LGP2, which has been proposed to enhance MDA5 activity by recruiting MDA5 to dsRNA. However, it has remained unclear whether LGP2 functions solely as a transient recruitment factor or actively participates in signaling-competent MDA5 filaments. Here, we demonstrate that LGP2 does more than recruiting MDA5 to dsRNA, and that LGP2 becomes an integral component of MDA5 filaments. Mechanistically, LGP2 stabilizes MDA5 filaments and reduces the number of MDA5 molecules required to nucleate stable signaling-competent filaments. This interplay between the two RLR proteins is mediated by specific dsRNA-dependent interactions between the C-terminal tail of LGP2 and MDA5, resulting in a network of contacts that is essential for LGP2-mediated enhancement of MDA5 signaling. Together, these findings provide new mechanistic insight into how LGP2 regulates the assembly and persistence of signaling-competent MDA5 filaments, establishing filament architecture and stability as key determinants of MDA5 signaling. Collectively, this work provides a mechanistic framework for understanding LGP2-mediated regulation of MDA5 signaling and advances our knowledge of the molecular mechanisms governing antiviral immunity.

Long noncoding RNAs (lncRNAs) regulate diverse cellular processes and are frequently implicated in disease, but their functional mechanisms often remain elusive. One such lncRNA, HOTAIR (Hox transcript antisense intergenic RNA), is a ~2.1 kb mammalian transcript whose overexpression promotes invasion and metastasis in breast cancer. However, the mechanisms by which HOTAIR influences gene regulation in cancer are poorly understood. To approach this problem through a structural lens, we determined the full-length in cellulo secondary structure of HOTAIR using chemical probing in a metastatic breast cancer cell line. The resulting structure shows that HOTAIR adopts a multidomain architecture and has local structural features unique to the cellular context. Comparison between in vitro and in cellulo chemical probing identifies regions of differential accessibility that may indicate context-dependent molecular interactions or folding. Conservation analyses further reveal that HOTAIR is conserved across primates with evidence of structural covariation in specific domains. Together, these results provide a roadmap for future mechanistic studies of structure-function relationships in HOTAIR and its contribution to gene regulation in cancer.

In most cells, Malat1 long noncoding RNA localizes to the nucleus where it affects splicing and chromatin function. In neurons Malat1 is exported to the cytoplasm where it is translated to generate the M1 micropeptide. Here we characterize an internal ribosome entry site (IRES) required for Malat1 translation. Although preceded by a long Malat1 5' RNA segment this element induces translation at the M1 AUG. In vivo chemical probing and structural modeling identified a 135 nt RNA secondary structure consisting of three stem loops that is sufficient for IRES activity. Using this minimal element for affinity purification from cell extracts, the IRES RNA selectively binds ribosomal subunits and translation factors. Depletion of the binding proteins Rack1 and hnRNP A2/B1 inhibits downstream IRES-dependent translation without affecting translation of an upstream ORF. Our study identifies an unexpected functional unit hidden within a widely studied long noncoding RNA.

Viral genomes encode regulatory RNA structures that orchestrate key steps of viral replication and gene expression. Although these structures are increasingly recognized as critical regulators of viral function, their systematic characterization in an infection context and roles in regulating viral fitness and immune recognition in vivo remain limited. Here, we systematically map and functionally interrogate structured RNA elements across the murine norovirus genome using orthogonal in-cell chemical probing, revealing conserved motifs that regulate viral function. Targeted disruption of specific structural elements reduces viral replication in cell culture, modulates translation in cis, and decreases viral RNA levels in animal infection models. These findings enabled the rational design of a genetically stable, attenuated virus that elicits protective immunity and limits viral replication upon secondary challenge. Together, this work uncovers essential roles for RNA structure in norovirus biology and establishes a generalizable framework for RNA structure-guided design of antiviral vaccines and therapeutics.

Long noncoding RNA (lncRNA) Pnky is a trans--acting regulator of neural stem cell (NSC) differentiation, but the molecular mechanisms by which Pnky regulates neurogenesis are unknown. A fundamental step toward mechanistic understanding is to determine whether lncRNA structure underlies biological function. Using chemical probing and high-throughput analysis, we determined the secondary structure of Pnky folded in vitro and in cellulo. In vitro-transcribed Pnky RNA adopts a compact, highly structured conformation with evidence of tertiary interactions. In cellulo, Pnky secondary structure is similar to the in vitro conformation. We used locked nucleic acid (LNA) oligonucleotides to interrogate the entire Pnky transcript for function in NSCs and identified regions that when targeted increase neurogenesis-phenocopying Pnky knockdown-without decreasing transcript abundance. Our findings implicate specific structured regions of Pnky in the regulation of neurogenesis and illustrate how structural maps combined with phenotypic data can advance our understanding of lncRNA function.

Glioblastoma (GBM), the most frequent and aggressive primary brain tumor, remains refractory to all current therapies including surgical resection, chemotherapy, radiotherapy and immunotherapy. Immunosuppressive mechanisms in the GBM tumor microenvironment contribute to the lack of anti-tumor adaptive immunity. We found that a subset of tumor associated macrophages (TAMs) can be repolarized into an anti-tumor phenotype via agonist stimulation of the retinoic acid-inducible gene I (RIGI), a cytosolic double-stranded RNA pattern recognition receptor (PRR). In silico analysis of adult GBM datasets available in the public domain revealed that RIGI expression by a subset of activated TAMs positively correlated with patient survival. Studies in syngeneic mouse models of GBM showed that intratumoral delivery of stem-loop RNA 14 (SLR14), a RIG-I agonist, improved the efficacy of chemotherapy, radiotherapy and immunotherapy treatments, beyond the effects of other nuclei acid sensor agonists. We found that RIGI + macrophages are the main drivers of SLR14 effect, combining activation of TAMs and priming of functional cytotoxic CD8+ T lymphocytes and NK cells. The anti-GBM effect of SLR14 is opening a significant new avenue for adult GBM treatment.

Circular RNAs (circRNAs) are covalently closed RNA molecules known for their increased stability compared to linear RNAs. Synthetic circRNAs are being developed as RNA therapeutics, while natural circRNAs are being investigated for their biological roles in eukaryotes and their potential as disease biomarkers. Consequently, the accurate detection and validation of circRNAs is crucial for advancements in both fundamental RNA research and biotechnological applications. Common methods for circRNA validation involve RT-PCR using divergent primers, followed by sequencing across the circRNA junction. However, most described methods are high-throughput approaches that require time-consuming RNA processing steps, and they are unable to detect highly structured circRNAs. Additionally, methods for low-throughput sequencing of small circRNAs (<150 nt) require cloning prior to sequencing. A simplified protocol for the validation of circRNA sequences irrespective of structure, sequence complexity, and length has not yet been described. In this method, we describe an improved RT-PCR protocol for circRNA detection by using UltraMarathonRT® (uMRT), a highly processive reverse transcriptase. Unlike other reverse transcriptases, uMRT can reverse-transcribe large, structured circRNAs of varying sizes, at ambient temperatures, enabling sequencing of the resulting concatemeric amplicons generated by RT-PCR and other methods. Using this method, we sequenced circRNAs containing highly structured internal ribosome entry sites commonly utilized in synthetic circRNAs, natural circRNAs containing repetitive elements, and small circRNAs, all without the need for cloning. With this new platform, we offer a protocol for the precise detection of nearly any circRNA species. Key features • This protocol shortens current methods for circRNA detection and sequencing by sequencing RT-PCR products directly, without the need for cloning or processing the PCR product. • This protocol describes a simple and cost-effective RT-PCR method for single circRNAs. • The highly processive UltraMarathonRT (uMRT) functions at ambient conditions, reducing circRNA degradation. • This protocol enables sequencing of circRNAs that are below 150 nt as well as larger circRNAs. • This protocol enables the detection of structured and very large circRNAs.

Despite the promise of vastly expanding the druggable genome, rational design of RNA-targeting ligands remains challenging as it requires the rapid identification of hits and visualization of the resulting cocomplexes for guiding optimization. Here, we leveraged high-throughput screening, medicinal chemistry, and structural biology to identify a de novo splicing inhibitor against a large and highly folded fungal group I intron. High-resolution cryoEM structures of the intron in different liganded states not only reveal molecular interactions that rationalize experimental structure-activity relationship but also shed light on a unique strategy whereby RNA-associated metal ions and RNA conformation exhibit exceptional plasticity in response to small-molecule binding. This study reveals general principles that govern RNA-ligand recognition, the interplay between chemical bonding specificity, and dynamic responses within an RNA target.

Targeting RNA with small molecules has emerged as a promising approach in drug development, offering the potential to expand the druggable genome and enable pharmacological targeting of non-coding genes or difficult-to-target gene products. However, the identification of functionally active RNA binders faces low hit rates in routine chemical space exploration and lacks robust high-throughput screening assays. The visualization of atomic details of RNA-small molecule interactions also poses a challenge due to the dynamic nature of RNA molecules. To address these challenges, we targeted a structured RNA, the self-splicing intron from C. albicans, and established a molecular beacon-based platform for high-throughput intron splicing inhibitor discovery. Through a medicinal chemistry campaign and cryo-EM visualization, we identified a potent splicing inhibitor and provided the first high-resolution de novo ligand-bound RNA cryo-EM structure, shedding light on molecular interactions and dynamical strategies governing RNA-ligand recognition in drug development.

LncRNA Pnky is a trans-acting regulator of neural stem cell (NSC) differentiation, but the molecular mechanisms by which Pnky regulates neurogenesis is unknown. A fundamental step towards mechanistic understanding is to determine whether a lncRNA has folded structure that underlies biological function. Using chemical probing and high-throughput analysis, we determined the secondary structure of Pnky folded in vitro and in cellulo. Pnky adopts a compact structure in vitro with distinct modules and evidence of tertiary interactions. In cellulo, Pnky structure is remarkably similar to the in vitro conformation. We used locked nucleic acid oligonucleotides to interrogate the entire Pnky transcript for function in NSCs and identified regions that when targeted increased neurogenesis - phenocopying Pnky knockdown - without decreasing transcript abundance. Our findings provide a structural basis for the role of Pnky in neurogenesis and, more broadly, illustrate how structural maps combined with phenotypic data can advance fundamental understanding of lncRNA mechanism.

We report the discovery of small molecules that target the RNA tertiary structure of self-splicing group II introns and display potent antifungal activity against yeasts, including the major public health threat Candida parapsilosis. High-throughput screening efforts against a yeast group II intron resulted in an inhibitor class which was then synthetically optimized for enhanced inhibitory activity and antifungal efficacy. The most highly refined compounds in this series display strong, gene-specific antifungal activity against C. parapsilosis. This work demonstrates the utility of combining advanced RNA screening methodologies with medicinal chemistry pipelines to identify high-affinity ligands targeting RNA tertiary structures with important roles in human health and disease.

Recent studies have uncovered a number of functional RNA structures in RNA viruses, yet their regulatory roles remain poorly understood. Here, using an unbiased proteomic approach alongside targeted biochemical assays, we investigate a previously uncharacterized functional pseudoknot (pk1) within the hepatitis C virus coding region and show that it stably interacts with host ribosomes, inhibiting translation and potentially acting as a regulator between viral translation and RNA genome replication. Comparative structural analysis identifies pk1-like elements in related RNA viruses, suggesting a conserved regulatory mechanism. This study expands the known functions of pseudoknots in viral coding regions beyond frameshifting, highlights the critical role of RNA structure-mediated regulation within viral open reading frames, and provides insight into the design of viral therapeutics and vaccines.

The ability to map messenger RNA (mRNA) molecules from individual cells using next-generation sequencing technologies, known as single-cell RNA-seq (scRNA-seq), is transforming biology by redefining cellular identities with unmatched detail. However, all current protocols depend on copying RNA into complementary DNA with a single reverse transcriptase (RT) derived from murine leukemia virus, which is an RT enzyme known for low processivity and limited ability to unfold complex RNA structures. Here, for the first time, we introduce a group II intron reverse transcriptase, UltraMarathonRT (uMRT), to perform scRNA-seq. We demonstrate that this enzyme reveals an unexpected transcriptomics landscape by capturing additional genes and other genomic features that conventional RTs miss. We also combined uMRT with metabolic RNA labeling, nucleoside conversion and scRNA-seq to explore genome-wide transcriptome dynamics at the single-cell level. Overall, we establish uMRT as a transformative biotechnological tool for single-cell transcriptomics.

Systemic fungal infections are a growing public health threat, and yet viable antifungal drug targets are limited as fungi share a similar proteome with humans. However, features of RNA metabolism and the noncoding transcriptomes in fungi are distinctive. For example, fungi harbor highly structured RNA elements that humans lack, such as self-splicing introns within key housekeeping genes in the mitochondria. However, the location and function of these mitochondrial riboregulatory elements has largely eluded characterization. Here we used an RNA-structure-based bioinformatics pipeline to identify the group I introns interrupting key mitochondrial genes in medically relevant fungi, revealing their fixation within a handful of genetic hotspots and their ubiquitous presence across divergent phylogenies of fungi, including all highest priority pathogens such as Candida albicans, Candida auris, Aspergillus fumigatus and Cryptococcus neoformans. We then biochemically characterized two representative introns from C. albicans and C. auris, demonstrating their exceptionally efficient splicing catalysis relative to previously-characterized group I introns. Indeed, the C. albicans mitochondrial intron displays extremely rapid catalytic turnover, even at ambient temperatures and physiological magnesium ion concentrations. Our results unmask a significant new set of players in the RNA metabolism of pathogenic fungi, suggesting a promising new type of antifungal drug target.

Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.