Biology subjects

Levanon, E. Y.

Publications and source records attributed to Levanon, E. Y..

7 recordsLinked to original sources

bioRxiv2026

Transcript-Specific Site-Directed RNA Editing Reveals Principles for Splice-aware guide RNA Design

Site-directed RNA editing (SDRE) utilizing the adenosine deaminase acting on RNA (ADAR) enzymes is commonly facilitated by guide RNAs (gRNAs) optimized to enhance on-target editing and minimize bystander effects. However, the impact of gRNA binding and ADAR-mediated SDRE on canonical pre-mRNA splicing remains poorly understood. Here, we developed an in vitro transcript-specific editing strategy that enables selective targeting and direct comparison of SDRE in pre-mRNA and mature mRNA. Using splice-relevant variants associated with inherited retinal diseases, we investigated the effects of SDRE on exonic, near-canonical intronic, and deep intronic splice variants. We identified gRNA-induced splice perturbation at exonic and intronic targets and observed that higher editing levels could be associated with increased splice disruption. Conversely, SDRE of two exonic splice variants and a deep intronic variant resulted in increased production of correctly spliced transcripts, demonstrating the potential of SDRE for splice modulation. Finally, by dissecting the effects of ADAR expression and gRNA design on editing and splicing outcomes, we established a system for identifying design principles that reduce splice interference and enhance the generation of correctly spliced, edited transcripts. These findings highlight the importance of considering transcript context and splicing consequences in the development of SDRE-based therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/741163v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@55e0cforg.highwire.dtl.DTLVardef@1a5c29org.highwire.dtl.DTLVardef@98aec7org.highwire.dtl.DTLVardef@1fab73b_HPS_FORMAT_FIGEXP M_FIG Created in BioRender. Schneider, N. (2026) https://BioRender.com/59i0tiy C_FIG

molecular biology↗

bioRxiv2026

Structure-aware Graph Learning Predicts RNA Editability Across Tissues and Species

Programmable A-to-I RNA editing using endogenous ADAR enzymes is emerging as a therapeutic strategy, but editability remains difficult to predict because ADAR recognition depends on double-stranded RNA geometry and stability rather than sequence alone. We present AdarEdit, a structure-explicit graph-attention framework that represents each dsRNA substrate as a nucleotide graph with backbone and base-pair edges. The framework includes a baseline model and a bio-aware model, with the latter augmenting this representation with typed interactions and a motif-sensitive sequence branch. We trained and evaluated both models on high-confidence inverted Alu duplexes (n = 884) with secondary structures predicted by RNAfold and editing levels measured across 8,603 GTEx RNA-seq samples spanning 47 tissues. Across five tissue contexts, the baseline and bio-aware models achieved strong held-out performance (test F1 = 0.814-0.869, AUROC = 0.869-0.933) and outperformed a matched structure-string baseline on the Liver split. The same graph representation retained predictive ability in evolutionarily distant non-Alu species (sea urchin, acorn worm, and octopus), suggesting conserved principles of ADAR substrate recognition. Finally, attention profiles and in silico mutagenesis recapitulated known biochemical constraints, including suppression by an upstream guanosine, and revealed longer-range asymmetric structural influences on editing. Because Alu duplexes are edited predominantly by ADAR1, AdarEdit is geared primarily to the ADAR1 regime. ADAR1 is particularly relevant to therapeutic editing given its broad tissue expression. The sources of this work are available at our repository: https://github.com/Scientific-Computing-Lab/AdarEdit

bioinformatics↗

bioRxiv2026

Inositol hexakisphosphate Functions as a Cofactor and Modulator of ADAR1 Activity

Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn{superscript 2}-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.

molecular biology↗

bioRxiv2025

A Cytoplasmic Index for Quantifying Immune-Related A-to-I RNA Editing

Distinguishing self from non-self is a major challenge for the immune system. Endogenous cytoplasmic double-stranded RNA (dsRNA) can mimic viral RNA and activate immune sensors like MDA5. ADAR1-mediated A-to-I editing disrupts base-pairing to suppress immunogenicity of these endogenous structures. Global editing indices are widely used to probe this crucial ADAR1 function. However, they are dominated by nuclear pre-mRNA edits with limited immune relevance. Here we present the Cytoplasmic Editing Index (CEI) that quantifies editing specifically within dsRNA structures in mature cytoplasmic transcripts, which carry higher immunological risk. Analyzing over 25,000 RNA-seq samples, we demonstrate CEI captures ADARp150 activity and outperforms the global editing index in terms of sensitivity and signal-to-noise, enabling sharper tissue-specific profiling, enhanced detection power of infection-induced editing changes, and stronger association with cancer prognoses. An open-source, cloud-native pipeline delivers end-to-end, reproducible analysis at very low cost, supporting immediate, scalable adoption. Micro-abstractThe Cytoplasmic Editing Index (CEI) quantifies immune-relevant A-to-I RNA editing in inverted Alu clusters within 3'UTRs, capturing interferon-inducible ADAR1p150-dependent events. Analysis of >25,000 RNA-seq samples demonstrates CEI outperforms the global editing index in sensitivity and specificity, resolving tissue- and infection-linked editing patterns. An open-source, cloud-native pipeline enables scalable, low-cost deployment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/692070v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@159ae9org.highwire.dtl.DTLVardef@6d6119org.highwire.dtl.DTLVardef@101b4a1org.highwire.dtl.DTLVardef@f9d96b_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration

The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a critical yet controversial role in the PKR pathway. We demonstrate that PACT is a direct and specific suppressor of PKR against endogenous dsRNA ligands like inverted-repeat Alu RNAs, which robustly activate PKR in the absence of PACT. PACT-mediated inhibition does not involve competition for dsRNA binding. Instead, PACT impairs PKRs ability to scan along dsRNA--a process necessary for PKR molecules to encounter and autophoshorylate each other for activation. By scanning along dsRNA and directly interacting with PKR, PACT restricts PKRs movement on dsRNA, reducing the likelihood of PKR molecular collisions and subsequent autophosphorylation, effectively inhibiting PKR without sequestering dsRNA. Consequently, PKR inhibition is more robust with longer and less abundant dsRNA, and minimal with abundant or short dsRNA. Thus, PACT functions to adjust the PKR activation threshold for long endogenous dsRNA without altering its inherent activity, revealing new mechanisms for establishing self-tolerance.

biochemistry↗

bioRxiv2024

The prevalent NR2E3 c.932G>A mutation induces aberrant splicing that can be rescued using splice-shifting antisense oligonucleotides

Mutations in NR2E3 have been implicated in several progressive retinal disease phenotypes such as enhanced S-cone syndrome, Goldmann-Favre syndrome and retinitis pigmentosa. One of the most frequent mutations in NR2E3 is c.932G>A (p.R311Q), where pathogenicity is thought to stem from the resulting amino acid substitution. However, multiple studies that evaluated the effect of this substitution on the protein, did not elucidate the molecular basis underlying the pathogenicity. Primed by bioinformatic analyses, we hypothesized and experimentally validated that the NR2E3 c.932G>A mutation leads to aberrant splicing which results in a short, non-functional protein isoform. Using cell models expressing WT and mutant constructs of the full NR2E3 sequence (including exonic and intronic regions), we observed that the mutated transcript exhibits a high level (75%) of aberrant splicing through gain of a novel splice acceptor site within exon 6. This mis-splicing results in the in-frame loss of 186 base pairs that code for a portion of the protein ligand binding domain. We further designed and evaluated splice-shifting antisense oligonucleotides (ASOs), that circumvented the aberrant splicing. The best performing ASO successfully restored 70% of the total NR2E3 full-length isoform levels and demonstrated rescue of nuclear localization and rhodopsin transcriptional activation. This study demonstrates the importance of understanding splicing consequences of pathogenic mutations, allowing the design and development of ASO-based therapies. Our findings set the stage for the potential treatment of NR2E3-related retinal degeneration caused by the c.932G>A mutation using splice-shifting ASOs.

genomics↗

bioRxiv2022

Sex-specific regulation of metabolic health and vertebrate lifespan by AMP biosynthesis

Energy homeostasis is disrupted with age, which then fuels multiple age-related pathologies. The AMP-activated protein kinase (AMPK) is the primary sensor of cellular energy in eukaryotes. However, the genetic regulation of vertebrate aging by AMPK remains poorly understood. Here, we manipulate energy levels in the turquoise killifish by mutating APRT, a key enzyme in AMP biosynthesis. These manipulations produced a male-specific lifespan extension and restored metabolic plasticity. Exploring the observed sex differences using an integrated omics approach implicated the mitochondria as an important player. Mechanistically, APRT regulated mitochondrial functions and AMPK activity, mimicking energy starvation in heterozygous cells. A fasting-like state was also detected, particularly in heterozygous males, which leads to resistance to high-fat diet. Finally, life-long intermittent fasting eliminated the male-specific longevity benefits mediated by the APRT mutation. These observations identify the AMP/AMPK axis as a sex-specific regulator of vertebrate longevity and metabolic health.

genetics↗