Dysregulation of the RNA-editing enzyme ADAR1 is associated with human diseases, including cancer, but its RNA-editing-independent roles in alternative splicing remain largely unexplored. Comprehending these molecular mechanisms is paramount, as they may unveil novel therapeutic targets. This study elucidates how the ADAR1p110 isoform influences alternative splicing independently of its canonical editing activity. Employing RNA-sequencing, proteomic analysis of ADAR1p110 interactors, and functional assays with wild-type and mutant ADAR1 in diverse human cell lines (including cancer models), we investigated its impact on the splicing landscape. Our findings indicate that ADAR1p110 interacts with pivotal spliceosome components and auxiliary splicing regulators. Notably, ADAR1 extensively modulates alternative-splicing events, with most of these alterations occurring independently of its RNA-editing activity and often its RNA-binding capacity. Furthermore, ADAR1 alters the isoform expression of other splicing factors (e.g., ACIN1), suggesting an indirect regulatory mechanism. Importantly, this splicing reprogramming affects genes that govern therapeutic response, positioning the ADAR-splicing axis as a potential driver of drug resistance. By revealing this predominantly editing-independent mechanism, we expand the understanding of ADAR1’s non-canonical functions and identify a new avenue for therapeutic intervention in cancer.

Dysregulated A>I(G) RNA editing, which is mainly catalyzed by ADAR1 and is a type of post-transcriptional modification, has been linked to cancer. A low response to therapy in breast cancer (BC) is a significant contributor to mortality. However, it remains unclear if there is an association between A>I(G) RNA-edited sites and sensitivity to genotoxic drugs. To address this issue, we employed a stringent bioinformatics approach to identify differentially RNA-edited sites (DESs) associated with low or high sensitivity (FDR 0.1, log2 fold change 2.5) according to the IC50 of PARP inhibitors, anthracyclines, and alkylating agents using WGS/RNA-seq data in BC cell lines. We then validated these findings in patients with basal subtype BC. These DESs are mainly located in non-coding regions, but a lesser proportion in coding regions showed predicted deleterious consequences. Notably, some of these DESs are previously reported as oncogenic variants, and in genes related to DNA damage repair, drug metabolism, gene regulation, the cell cycle, and immune response. In patients with BC, we uncovered DESs predominantly in immune response genes, and a subset with a significant association (log-rank test p < 0.05) between RNA editing level in LSR, SMPDL3B, HTRA4, and LL22NC03-80A10.6 genes, and progression-free survival. Our findings provide a landscape of RNA-edited sites that may be involved in drug response mechanisms, highlighting the value of A>I(G) RNA editing in clinical outcomes for BC.

ADAR3 is a catalytically inactive member of the family of adenosine deaminases acting on RNA (ADARs). Here we have investigated its function in the context of the developing mouse brain. The expression of ADAR3 gradually increases throughout embryogenesis and drops after birth. Using primary cortical neurons, we show that ADAR3 is only expressed in a subpopulation of in vitro differentiated neurons, which suggests specific functions rather than being a general regulator of ADAR editing in the brain. The analysis of the ADAR3 interactome suggested a role in mRNA stability and translation, and we show that expression of ADAR3 in a neuronal cell line that is otherwise ADAR3-negative changes the expression and stability of a large number of mRNAs. Notably, we show that ADAR3 associates with polysomes and inhibits translation. We propose that ADAR3 binds to target mRNAs and stabilizes them in non-productive polysome complexes. Interestingly, the expression of ADAR3 downregulates genes related to neuronal differentiation and inhibits neurofilament outgrowth in vitro. In summary, we propose that ADAR3 negatively regulates neuronal differentiation, and that it does so by regulating mRNA stability and translation in an editing-independent manner.

Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients’ A>I(G) RNA-editing profiles.

MicroRNAs (miRNAs) exert their gene regulatory effects on numerous biological processes based on their selection of target transcripts. Current experimental methods available to identify miRNA targets are laborious and require millions of cells. Here we have overcome these limitations by fusing the miRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing the detection of miRNA targets transcriptome-wide in single cells. miRNAs guide the fusion protein to their natural target transcripts, causing them to undergo A>I editing, which can be detected by sensitive single-cell RNA sequencing. We show that agoTRIBE identifies functional miRNA targets, which are supported by evolutionary sequence conservation. In one application of the method we study microRNA interactions in single cells and identify substantial differential targeting across the cell cycle. AgoTRIBE also provides transcriptome-wide measurements of RNA abundance and allows the deconvolution of miRNA targeting in complex tissues at the single-cell level.

miRNAs are short noncoding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, containing several miRNA precursors. The largest mammalian miRNA cluster, the miR-379-410 cluster, is expressed primarily during embryonic development and in the adult brain; however, downstream regulation of this cluster is not well understood. Here, we investigated adenosine deamination to inosine (RNA editing) in the miR-379-410 cluster by adenosine deaminase acting on RNA (ADAR) enzymes as a possible mechanism modulating the expression and activity of these miRNAs in a brain-specific manner. We show that the levels of editing in the majority of mature miRNAs are lower than the editing levels of the corresponding site in primary miRNA precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. We found miR-376b-3p maturation is negatively regulated by ADAR2 in an editing activity-independent manner, whereas ADAR1-mediated and ADAR2-mediated editing were observed to be competitive. In addition, the edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase, encoding the enzyme responsible for the catabolism of the neurotransmitter gamma aminobutyric acid (GABA). Expression of edited miR-376b-3p led to increased intracellular GABA levels as well as increased cell surface presentation of GABA type A receptors. Our results indicate that both editing and editing-independent effects modulate the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain.

Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/β-catenin pathway and activity of nuclear β-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3β, while increasing active β-catenin. It also increased the activity of nuclear β-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the β-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, where several miRNA precursors are present in the same transcript. The largest mammalian miRNA cluster, the miR-379-410 cluster, is expressed primarily during embryonic development and in the adult brain. We investigated adenosine-to-inosine (A-to-I) RNA editing by the adenosine deaminase acting on RNA (ADAR) enzymes of the miR-379-410 cluster as a possible mechanism of modulating expression and activity of the miRNAs in a brain-specific manner. We show that editing levels of the majority of mature miRNAs are lower than the editing level of the corresponding site in primary miRNA (pri-miRNA) precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. miR-376b-3p maturation is negatively regulated by ADAR2 in an editing-independent manner, while ADAR1 and ADAR2 editing was observed to be competitive. The edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase (Abat), the enzyme responsible for the catabolism of the neurotransmitter GABA. Expression of edited miR-376b-3p led to increased intracellular GABA levels, as well as increased cell surface presentation of GABAA receptors, previously shown to be dependent on intracellular GABA levels. Our results indicate that editing and editing-independent effects modulates the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain.