The LRPPRC/SLIRP complex is a key post-transcriptional regulator of mitochondrial gene expression, stabilizing mitochondrial mRNAs and promoting their polyadenylation and translation. Mutations in LRPPRC cause mitochondrial disorders, including Leigh syndrome French-Canadian type (LSFC), primarily affecting oxidative phosphorylation. Here, we examined the RNA-binding properties of wild-type LRPPRC and three pathogenic variants (A354V, K909del, and R1276_K1300del) using electrophoretic mobility shift assays, acoustic force spectroscopy, and AlphaFold 3 modeling. All three mutations reduced intrinsic RNA binding, with R1276_K1300del showing no detectable interaction in the absence of SLIRP. Remarkably, SLIRP restored RNA binding of this mutant to near wild-type levels, likely through conformational stabilization, as supported by single-molecule and structural analyses. These findings highlight SLIRP's critical role in modulating LRPPRC function and suggest that enhancing SLIRP activity represents a potential therapeutic strategy for LRPPRC-related mitochondrial disorders.
Gapmer-type antisense oligonucleotides (Gapmers) are promising therapeutic agents. However, their clinical potential is frequently limited by off-target toxicities. To address this issue, Gapmers have been optimized by modifying the ribose moiety or internucleotide linkage, but toxicity has not always been reduced. The toxicity is due to the unintended interactions between the ribose-type modified nucleic acids with phosphorothioate backbones and the endogenous proteins. We therefore hypothesized that the use of acyclic nucleic acids, which possess an entirely distinct structure to ribose, would solve the aforementioned issue. In this study, we demonstrate that the incorporation of an acyclic analog, serinol nucleic acid (SNA) or L-threoninol nucleic acid (L-aTNA), provides an alternative approach. Notably, substitution with SNA or L-aTNA effectively mitigated toxicity, even when conventional 2'-O-methyl modification was unsuccessful. This approach reduced cytotoxicity across multiple Gapmer sequences and designs in a position-dependent manner. Mechanistically, our investigation into these acyclic nucleic acids revealed that reduced toxicity was associated with suppression of P54nrb protein mislocalization. Furthermore, representative SNA- or L-aTNA-modified Gapmers exhibited markedly reduced hepatotoxicity in vivo. Collectively, these findings suggest that acyclic nucleic acids have potential as a useful chemical strategy for the development of safer Gapmer therapeutics.
Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen with a remarkable ability to survive within its human host by balancing replication and persistence throughout the course of infection. The success of Mtb depends on its ability to adapt to diverse and hostile environments imposed by the host. This adaptation is primarily mediated through transcriptional regulation of gene expression. While Mtb encodes several transcription factors essential for growth, it remains unclear which transcription factors directly regulate the bacterial cell cycle. In this work, we characterized two essential transcription factors, WhiA and WhiB2, and defined their role in regulating Mtb replication. Using CRISPR interference, we demonstrated that whiA and whiB2 are essential for bacterial cell division. Results from functional genomic studies revealed that WhiA and WhiB2 regulate key genes involved in DNA replication and repair, ribosome function, cell wall synthesis, and septation. Subsequent in vitro studies demonstrated that WhiA and WhiB2 are dually required for activation of selected target genes, suggesting that they function cooperatively as regulators of the Mtb cell cycle.
Human endogenous retroviruses (HERVs), remnants of ancient viral infections, are increasingly recognized as potential regulatory elements influencing gene expression and immune responses. However, their role in infectious disease severity remains poorly understood. Here, we investigate the transcriptional impact of HERVs in dengue disease progression by leveraging RNA sequencing data from a large cohort of hospitalized patients across different severity subgroups. We identify differentially expressed HERVs and their co-expressed genes, revealing a severity-dependent transcriptional signature. Notably, genes co-expressed with HERVs in severe dengue were enriched in pathways related to immune modulation, phagocytosis, infectious disease response, and calcium signaling. We further demonstrate that HERVs may regulate genes through co-localization and chromatin interactions, with several HERV-gene pairs residing within same topologically associated domains (TADs). Using promoter interaction analysis, we identify potential regulatory mechanisms, exemplified by the interaction between HERV elements and MAGI3, an immune-modulatory gene. Validation in an independent cohort of 78 patients confirmed reproducibility of HERV expression patterns. Interestingly, genes encoding HERV envelope proteins, known for their immunomodulatory and antiviral properties, were consistently downregulated in severe dengue across both cohorts. These findings suggest a role for HERVs in shaping host transcriptional responses to dengue, with potential implications for disease progression and severity.
Three-prime Repair EXonuclease 1 (TREX1) is a 3 '-to-5 ' exonuclease that degrades cytoplasmic DNA to prevent aberrant innate immune and inflammatory responses. Loss of TREX1 function results in the accumulation of cytosolic DNA and autoimmune disorders in humans, whereas in cancer cells, TREX1 inhibition can promote antitumor immunity triggered by tumor-derived cytoplasmic DNA. Here, we show selective inhibition of human TREX1 (hTREX1) by small molecule inhibitors and reveal their mechanism of target engagement by X-ray crystallography. The structures show that these inhibitors competitively block the binding of the 3 '-terminal nucleotide of DNA substrates and make a hydrogen bond with a conserved Glu residue in the active site. Furthermore, binding of the inhibitors stabilizes Tyr129 of hTREX1 in an alternative conformation incompatible with DNA binding, generating a deep hydrophobic pocket that accommodates a key aromatic moiety of the inhibitors. The structural observations provide the basis for high selectivity of these inhibitors for hTREX1 over the closely related hTREX2 or other viral DEDDh-family nucleases. Our studies demonstrate that the conformational flexibility of the hTREX1 active site plays a crucial role in its selective inhibition, informing future design of hTREX1-selective ligands through this structural framework.
Arginine Vasopressin (AVP) modulates social and sexual behaviors in mammals through its receptor V1a. Differences in receptor expression and the resulting behavioral changes have been linked to population-level variation in the 5' flanking region of the AVPR1A gene, specifically, length polymorphism of the composite microsatellite RS3 ((CT)4TT(CT)n(GT)m). However, the source of this length variation is unknown. Here, using a genetically tractable system in Saccharomyces cerevisiae, we recapitulated the full spectrum of the RS3 length polymorphism observed in humans resulting from genetic recombination between two RS3 sequences. Our candidate gene analysis shows that transcription elevates the rate of these recombination events and implicates double-strand break repair via single-strand annealing.
Nonsense mutations introduce premature termination codons (PTCs) that trigger mRNA degradation and underlie genetic diseases, including Alport syndrome (AS). Suppressor tRNAs (sup-tRNAs) are potential therapies for such disorders, as they can precisely readthrough PTCs and restore full-length protein synthesis. For AS caused by COL4A5 nonsense-mediated deficiency, we developed a TDN-sup-tRNA delivery strategy using tetrahedral DNA nanostructures (TDNs) characterized by excellent biocompatibility and renal targeting. In a Col4a5-R471X mouse model, TDN-sup-tRNA achieved high readthrough efficiency, restoring COL4A5 protein expression and reconstituting glomerular basement membrane integrity. Notably, proteinuria was reduced to 35% of baseline within 4 weeks. Compared with AAV2/9-mediated delivery, this system exhibited enhanced renal targeting and superior therapeutic efficacy over a one-month treatment period, without detectable toxicity. Collectively, this strategy represents a safe and effective non-viral RNA therapeutic approach for precise correction of nonsense mutation-associated renal diseases.
Targets of RNA-binding proteins (RBPs) are often investigated by implementing variants of cross-linking and immunoprecipitation methodology, which can yield several disadvantages in target detection. The RBP and N6-methyladenosine (m6A) reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2/IMP2) exerts an essential pathophysiological role as a metabolic regulator and tumor promoter, impacting the stability, localization, and translation of its targets. Here, we employed HyperTRIBE as a method to identify RBP targets in native cells in vivo and identified targets of IMP2 in murine hepatocytes. IMP2-associated adenosine-to-inosine editing sites were identified by hydrodynamic transfection of mouse livers using an IMP2-ADAR (adenosine deaminase acting on RNA) construct. Functional enrichment and motif analysis results suggest IMP2-facilitated target stabilization and confirm presence of m6A-binding motifs. In addition, the overlap with data of a TRIBE experiment employing murine embryonic fibroblasts and with those of differential gene expression was investigated. Comparative transcriptomics between IMP2, wild-type, and control samples (mCherry-ADAR) revealed an enrichment of IMP2-bound mRNAs associated with autophagy, which could be validated by RNA immunoprecipitation in a human liver cancer cell line. A functional knockdown of IMP2 demonstrated an increased autophagic flux, providing further evidence for the involvement of IMP2 in autophagy.
Recent genetic studies have shown somatic expansion of the CAG repeat is the key process driving Huntington's disease (HD) pathogenesis. Recognition of insertion-deletion loops (IDLs), lesions prone to form within the CAG repeat, by Mutsβ (MSH3/MSH2) is thought to be the primary event in the expansion process. This starts a cascade that leads to error-prone repair and incorporation of additional CAG units into the repeat. In vitro data shows MSH3 binds IDLs through a DNA-binding pocket formed by MSH3 residues Y245/K246. In this study, we investigated the significance of this DNA-binding motif in CAG repeat expansion using cell lines harbouring long, unstable HTT CAG repeats. Genetic disruption of the MSH3 Y245/K246 motif significantly reduced DNA interaction, exhibited MMR deficiency in a frameshift mutator assay, and abrogated repeat expansion in a U2OS cell line expressing mutant HTT exon 1. Pharmacological blockade of this site using a small molecule targeting the DNA-binding pocket similarly reduced DNA binding and repeat expansion in a U2OS cell line. Crucially, this molecule also slowed CAG repeat expansion in medium spiny neurones derived from HD patient-iPSCs. Targeting of the MSH3 IDL binding pocket may represent a possible therapeutic strategy.
Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene, resulting in the loss of FMRP, an RNA-binding protein regulating translation of hundreds of mRNAs. The Fmr1 knock-out mouse models this deficiency and is used to study molecular perturbations in the FXS brain. Omics analysis shows that FMRP loss disrupts coordination between transcriptomic and translatomic layers. But limited sample availability and dataset heterogeneity hinder detection of subtle, coordinated multi-omic dysregulations. To address this, we trained a Multi-Channel Variational Autoencoder (MCVAE) on wild-type samples to learn a shared latent representation of transcriptomic and translatomic modalities via cross-modal reconstruction. Testing MCVAE on Fmr1-knock-out samples revealed deviations from wild-type as anomalies, uncovering known and novel perturbations. Compared to alternative methods, MCVAE shows stronger enrichment for FMRP mRNA targets and improved genotype discriminative power in post-hoc tests. Translatomic anomalies exhibited coordinated relationships with transcriptomic anomalies, as supported by publicly available databases exploration. Moreover, these anomalies mapped to validated FMRP regulators and neurodevelopmental pathways, establishing MCVAE as a framework to uncover coordinated molecular perturbations underlying the FXS pathophysiology and guide biomarker and therapeutic target identification.
Plasminogen activator inhibitor-1 (PAI-1) is the principal regulator of fibrinolysis, acting by inhibiting tissue-type and urokinase-type plasminogen activators (tPA/uPA). Dysregulated PAI-1 activity contributes to diverse pathologies, including cancer, cardiovascular disease, and fibrosis, underscoring its therapeutic potential. We previously identified R10-4, an RNA aptamer that attenuates PAI-1's antiproteolytic activity against tPA by destabilizing covalent complex formation. To elucidate its inhibitory mechanism, we combined plasminogen activation assays with in silico modeling to map residue-level interactions. Systematic truncation of R10-4 revealed two regions critical for inhibition: (i) the reactive center loop, encompassing the s5A strand, and (ii) the vitronectin-binding domain. Removal of 17 nucleotides from the 3' end disrupted aptamer engagement, significantly altering R10-4's inhibitory activity. These findings define the structural basis for R10-4 modulation of PAI-1 function, revealing a novel aptamer-mediated mechanism of inhibition. By targeting key functional domains, R10-4 emerges as a promising prototype therapeutic for correcting fibrinolytic dysregulation in thrombotic and fibrotic disease.
Zika virus (ZIKV) has evolved from a sporadically circulating pathogen into a global health threat associated with severe neurological complications. While research has been focused on amino acid substitutions, the impact of non-coding RNA evolution on ZIKV fitness remains largely unexplored. Here, we characterize the structural and functional evolution of stem-loop A (SLA), the essential 5' terminal promoter for the viral polymerase. We identified four distinct evolutionary variants, ancestral (Anc), intermediate (Int), contemporary (Con), and alternative (Alt) that exhibit a progressive shift in thermodynamic stability. Notably, SLACon-dominant in recent outbreaks-displays increased binding affinity for the NS5 polymerase, whereas the divergent SLAAlt variant markedly increases 5' exoribonuclease resistance at the cost of replicative efficiency. Although the SLAAlt variant proved lethal in a contemporary ZIKV backbone, 5' rapid amplification of cDNA ends (RACE) and phylogenetic analysis confirmed its viability in nature. These findings reveal a previously unrecognized plasticity in the ZIKV promoter and demonstrate that non-coding architecture undergoes adaptive refinement to balance genome stability with kinetic accessibility. Ultimately, this work illustrates how the structural evolution of RNA serves as a key determinant of viral fitness and the emergence of isolate-specific replication strategies.
Emerging and evolving viral diseases, such as SARS-CoV-2, continue to pose significant global health challenges, underscoring the urgent need for rapid and scalable antigen discovery pipelines. This work presents a computational pipeline that integrates diverse computational tools and machine learning models to accelerate the identification and optimization of antigen candidates. The pipeline employs efficient filtering and consensus-based strategies to highlight epitopes with high therapeutic potential. We demonstrate its utility by significantly narrowing the antigen search space for Rift Valley fever virus (RVFV) and Mayaro virus (MAYV), and by effectively identifying conserved neutralizing epitopes in SARS-CoV-2. Our proposed computational antigen pipeline offers a powerful framework for expediting the development of future vaccines and therapeutics in response to emerging pathogens.
Huntington's disease (HD) is the best-known example of a neurodegenerative disorder caused by the expansion of a glutamine-encoding CAG repeat in the causative gene. Growing evidence indicates that somatic CAG expansions play a key role in disease progression, providing a strong rationale for therapeutic strategies directly targeting the repeat tract. However, achieving sufficient efficacy while maintaining allele selectivity and minimizing off-target effects remains a major challenge. Here, we developed allele-selective, CAG-targeting artificial microRNA (amiRNA) molecules that exhibit significantly reduced off-target risk. This was achieved by introducing specific substitutions at selected positions within the guide strand. These molecules effectively downregulated polyglutamine (polyQ) proteins in cellular models of HD, spinocerebellar ataxias types 1 and 3, and dentatorubral pallidoluysian atrophy. The most promising candidate, amiR136-13A, reduced mutant huntingtin levels in different brain regions of the HD mouse model and did not induce toxicity up to 28 weeks following a single administration of an AAV5 vector. Transcriptomic profiling of human HD neural stem cells treated with amiR136-13A revealed minor changes in gene expression. Moreover, amiR136-13A reduced the level of HTT1a, a short pathogenic isoform of huntingtin. Collectively, these findings identify amiR136-13A as a potent, selective, and safe therapeutic candidate for HD and potentially other polyQ disorders.
Skeletal muscle aging is accompanied by deterioration in metabolic flexibility, neuromuscular connectivity, and structural integrity, all of which contribute to frailty and the loss of functional independence. Ames dwarf mice exhibit postnatal growth hormone deficiency and an exceptionally long lifespan, providing a unique model for revealing transcriptional programs that support healthy aging. Here, we present the first comprehensive transcriptomic and functional profile of hindlimb skeletal muscle in middle-aged and old-aged Ames dwarf mice. We show that Ames dwarf muscle maintains a transcriptional profile enriched for vascular remodeling, synaptic communication, extracellular matrix organization, and structural resilience while suppressing lipid metabolic pathways and age-associated transcriptional drift. Advanced age in Ames mice is marked by a substantial shift in transcription factors associated with downregulated genes and a temporally coordinated activation of senescence-associated and inflammatory-response signatures that appear to support, rather than impair, tissue maintenance. Functionally, Ames dwarf mice maintain neuromuscular coordination, grip strength, and endurance with age. Collectively, these findings indicate a distinct transcriptional drift in Ames dwarf skeletal muscle that integrates vascular, neuronal, and senescence-related signals to preserve structural and functional resilience. This work implicates molecular mediators, including Apelin, Klotho, and Notch1 that may underlie exceptional healthspan and modulate resistance to frailty.
Epstein-Barr virus (EBV) is a major risk factor for multiple sclerosis (MS), yet the contribution of specific viral variants remains unclear. Complete EBV genome analysis of wild-type variants has not been performed previously. In a pilot-study, targeted EBV sequencing of B cells did not yield adequate coverages due to excess of human DNA. Thus, we developed an ex vivo 6-day leukocyte culture method to enrich EBV DNA into supernatant by stimulation with tetradecanoyl phorbol acetate. Using this method in 20 MS patients and 20 controls, we obtained near-complete EBV genomes in 9 MS patients (average coverage 97%) and in 4 controls (average 85%). We identified 1088 single-nucleotide variants (33% missense variants) outside repetitive regions that met stringent quality criteria. Overall, 94% of the variants were present in three or more subjects, thus unlikely generated during the culture and several were exclusive to MS, worth of further investigation. In silico analysis indicated that variants within the EBNA1 cross-reactive region change human leukocyte antigen (HLA) peptide binding affinity, suggesting altered antigen presentation. These results illustrate a method for enriching EBV genomes and present the first wild-type sequences from MS patients. The observed diversity highlights potential for future studies in EBV-associated diseases and vaccine development.
Galectin-9 (Gal9) is known for its cytoplasmic roles in vesicular damage responses, yet its active functional roles in gene delivery remain unclear. Here, we investigated Gal9 dynamics in electrotransfection (ET), a non-viral gene delivery method widely used for plasmid DNA (pDNA) and mRNA transfection. Confocal imaging revealed unexpected nuclear accumulation of Gal9 after pDNA ET. Gal9 colocalized with SC35, a biomarker of nuclear speckles, and immunogold TEM confirmed its enrichment within those electron-dense, subnuclear domains following pDNA ET. Functionally, Gal9 knockdown markedly reduced ET efficiency and reporter gene expression, whereas overexpression enhanced both. The timing and the extent of Gal9-pDNA association varied with delivery method and cargo type, being prominent for DNA but minimal for mRNA. Across different cell types, endogenous Gal9 expression correlated with ET efficiency. These findings revealed previously unrecognized, active functional roles of Gal9 in organizing exogenous DNA within transcriptionally active regions of the nucleus and regulating transgene expression following their non-viral delivery.
The immunometabolite itaconate, generated by immune-responsive gene 1 (IRG1/ACOD1), and its derivative 4-octyl itaconate (4OI) have been found to modulate inflammation and progression of viral infections, but their effects on the significant respiratory pathogen human metapneumovirus (HMPV) is unknown. Here, we demonstrate that HMPV induces IRG1 expression via a TANK-binding kinase 1, NF-κB-, and interferon (IFN)-dependent manner in human primary macrophages. We further show that the addition of a cell-permeable derivative of itaconate, 4OI, but not itaconate itself or its natural isomer citraconate, reduces HMPV and IFN-β levels in human macrophages. 4OI additionally activated Nrf2, while Nrf2 depletion enhanced HMPV levels, suggesting that Nrf2 mediates the antiviral effect of 4OI on HMPV. Also, we found that 4OI reduced expression of ATP-dependent citrate lyase, a lipid metabolic enzyme that supports HMPV replication. Our study suggests 4OI as a potential compound for targeting HMPV-IFN-β-driven disease and highlights Nrf2-dependent lipid reprogramming as a potential modulator of 4OI antiviral effects.
Antisense oligonucleotides (ASOs) are promising therapeutics, but safety concerns such as liver toxicity and off-target (OffT) effects necessitate thorough evaluation during the compound selection process. This study leverages time course global proteomics and transcriptomics to assess ASO-induced changes in vitro, comparing liver toxic versus non-liver toxic ASOs. The research confirms that ASOs perturb different cellular pathways at both RNA and protein levels, effectively discriminating between liver toxic and non-liver toxic ASOs. Contrary to expectations, protein level reduction isn't delayed relative to ASO-induced RNA reduction, highlighting the importance of understanding RNA and protein level relationships in specific model systems. Furthermore, many OffT effects observed at the RNA level do not directly translate to corresponding protein level changes. These findings suggest that current RNA-focused OffT assessment strategies capture predicted OffTs but could benefit from protein level studies that could potentially de-risk oligonucleotide drug (OND) candidates with seemingly problematic OffT profiles at the RNA level. The study underscores the value of global proteomics as a complement to RNAseq in ASO drug development, refining safety assessment and improving candidate selection.
Hybridization, the merging of distinct genomes, is increasingly recognized as a major evolutionary force among eukaryotic pathogens, including facultatively sexual protist parasites like Leishmania and Trypanosoma. While it may contribute to pathogen virulence and drug resistance, hybridization remains poorly characterized, particularly how genetic distance between parental cells influences genomic compatibility and which compensatory mechanisms ensure hybrid viability. Here, we report the in vitro generation of an unusual sexual hybrid between Leishmania species infecting mammals (L. infantum) and reptiles (the Sauroleishmania L. tarentolae), and used this unique genetic model system to address these open questions. Our data provide evidence of genomic compatibility between even highly divergent Leishmania species, offering new insights into the evolutionary potential of Leishmania and related pathogens. We demonstrate that the genomic shock caused by the fusion of distinct genomes can be mitigated by two key mechanisms: (i) at the genomic level, chromosome loss allows the establishment of mosaic aneuploidy in the newly formed hybrid, and (ii) at the post-transcriptional level, preferential mono-parental allelic expression acts as a secondary compensatory mechanism. Our findings establish genome instability and post-transcriptional regulation as central processes in Leishmania hybridization, which may be of broad relevance to other biological systems undergoing genetic exchange.
