🧬 PubMed RNA Editing Daily Digest

Two ADAR1 isoforms, p150 and p110, are involved in adenosine-to-inosine RNA editing. ADAR1p150-mediated hyper-editing of endogenous dsRNAs prevents their activation of type I interferon signaling-mediated via Melanoma Differentiation-Associated Protein 5 (MDA5), which enables cancer resistance to immune checkpoint blockade. ADAR1p150 also inhibits Z-RNA-mediated activation of Z-DNA Binding Protein 1 (ZBP1) and induction of necroptosis. ADAR1p110 suppresses the formation of telomeric repeat R-loops, which would otherwise induce apoptosis in telomerase-reactivated cancer cells. Together, ADAR1 inhibitors could serve as novel cancer therapeutics. Here, we identified, ADAR1i-124, which inhibits the catalytic activities of both ADAR1p150 and ADAR1p110. ADAR1i-124 activated MDA5 and ZBP1 pathways and dose-dependently inhibited viability across different types of cancer cell lines. Some cancer cell lines, unresponsive to ADAR1i-124 alone, became responsive when co-treated with 5-Aza-CdR. The DNA methylase inhibitor reactivated endogenous retroviruses, leading to the formation of retrovirus dsRNAs and the emergence of a new ADAR1 dependency. Our study establishes the potential of ADAR1i-124 as a future cancer therapeutic.

RNA editing is a post-transcriptional modification in metazoans and plays a crucial role in environmental adaptation. To elucidate the species specificity of RNA editing in marine mollusks and advance the comprehension of their post-transcriptional modifications, we selected three Tegulidae gastropods, Rochia conus, R. maxima, and Tectus pyramis, from coral reefs in the South China Sea and performed transcriptome-wide RNA editing analysis. The results showed that adenosine-to-inosine (A-to-I) RNA editing was the most prevalent editing type among these three Tegulidae species. The number of A-to-I editing sites (per Gb) and the editing level of these sites varied significantly among species (p < 0.05). Notably, the A-to-I editing density (number of A-to-I edited sites per Gb) of R. conus clustered with that of R. maxima, consistent with their clustering in the adenosine deaminases acting on RNA (ADAR) phylogeny. Furthermore, Gene Ontology analysis revealed that the edited coding genes of R. conus, R. maxima, and T. pyramis were mainly enriched in "neuron projection membrane," "cellular response to toxic substance," and "threonine catabolic process," respectively. These results suggest that the RNA editing patterns of the family Tegulidae are species-specific and that A-to-I RNA editing density is correlated with the ADAR phylogenetic history.

Mutations in the human ADAR gene, encoding adenosine deaminase acting on RNA 1 (ADAR1), cause Aicardi-Goutières syndrome 6, which is a severe auto-inflammatory encephalopathy with aberrant interferon (IFN) induction. AdarΔ2-13 null mutant mouse embryos lacking ADAR1 protein die with high levels of IFN-stimulated gene (ISG) transcripts. In Adar Mavs double mutants also lacking the Mitochondrial antiviral signalling (MAVS) adaptor, the aberrant IFN induction is prevented. Live pups are born and survive for 2 weeks, allowing ADAR1 function to be investigated. We have shown that early death of Adar Mavs mutants is rescued by the deletion of the Eif2ak2 gene encoding the antiviral dsRNA sensor protein kinase R (PKR). Here, we focused on characterizing the brain defects in Adar Mavs mutants and their dependencies on PKR. Mouse brains were collected on postnatal Days 8 and 14, then analysed by mass spectrometry, immunohistochemistry and RT-qPCR. The proteomic analyses showed upregulation of ISG-encoded proteins in the Adar Mavs double mutant, and the morphological analyses confirmed aberrant microgliosis in the brains. Both are prevented in Adar Mavs Eif2ak2 triple mutants, indicating the key role of aberrant PKR activation; PKR expression is also increased by IFN signalling. Altered expression levels of transcripts encoding differentially expressed proteins and of ADAR-edited transcripts were confirmed by RT-qPCR. Analysis of the expression levels of transcripts in the brains of mutants expressing a catalytically inactive ADAR E861A protein revealed that the levels of some but not all altered transcripts are restored. A further group of proteins, downregulated in Adar Mavs double mutants, are not rescued by removal of PKR and might result from effects of loss of the widespread ADAR1 RNA editing known to occur in brain transcripts. This group includes several motor proteins, some of which have been reported to be encoded by ADAR-edited transcripts. In this study, we show that Adar Mavs double mutants exhibit an aberrant IFN response in the brain, probably attributable to reactive microglia and astrocytes. Microgliosis, which is rescued in the triple mutant, is mostly dependent on aberrant PKR activation and is partly dependent on RNA editing.

CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.

CRISPR-Cas9 is natively present in the adaptive immune systems of a multitude of bacteria and has been adapted as an effective genome engineering tool. The Cas9 effector enzyme, which is composed of a single Cas9 protein and a single-guide RNA (sgRNA), identifies and cleaves double-stranded DNA targets through a series of conformational changes that require DNA distortion and unwinding. While most studies of Cas9 specificity have focused on the DNA sequence, the role of intrinsic DNA physical properties ("DNA shape") in modulating Cas9 activity remains insufficiently defined. We previously showed that with a 16-nucleotide (-nt) truncated guide, the intrinsic DNA duplex dissociation energy at the PAM+(17-20) segment beyond the RNA-DNA hybrid tunes Cas9 cleavage rates of linear substrates. Here, we examined the impact of DNA supercoiling on Cas9 cleavage with the 16-nt truncated guide. Enzyme kinetic analysis revealed that PAM+(17-20) DNA sequences beyond the RNA/DNA hybrid preserve their effects on Cas9 cleavage in the supercoiled state. Furthermore, combining a novel asymmetric hairpin construct with a parallel-sequential kinetics model, rates for first-step nicking and second-step cleavage by Cas9 were obtained for both supercoiled and linear substrates. With both topologies, it was found that first-step nicking is clearly impacted by PAM+(17-20) DNA sequences, and the effects can be correlated with DNA unwinding, which dictates R-loop dynamics. This work expands our understanding of DNA target recognition by Cas9, and the methods developed, in particular those for analyzing the progression of Cas9-induced nicks, will aid in further in-depth mechanistic investigation.