Rui Zhang

Aging impairs intestinal stem cell (ISC) function, disrupting epithelial homeostasis and regenerative repair. Loss of ISC quiescence promotes intestinal dysfunction and contributes to organismal aging. Here, we report an epitranscriptomic mechanism through which a decrease in adenosine-to-inosine (A-to-I) RNA editing by the adenosine deaminase ADAR in ISCs during aging disrupts a conserved signaling axis that maintains ISC quiescence. In Drosophila melanogaster , ISC-specific loss of dADAR triggered hyperproliferation and tissue aging, whereas dADAR overexpression reduced age-related gut dysfunction and extended life span. Intestinal injury induced a decrease in dADAR, which was necessary for tissue regeneration, and in the zinc finger protein dZn72D, which stabilized dADAR to support intestinal homeostasis during aging. dADAR edited transcripts encoding the RNA binding protein Pumilio (dPUM) to generate an isoform that inhibited translation of two components of the mitogen-activated protein kinase (MAPK), dEGFR and dERK, thereby maintaining ISC quiescence. The abundance of ADAR2 and of the dZn72D ortholog ZFR decreased in human colonic crypts with aging and in mouse colon after injury, suggesting that attenuation of ADAR activity may play a conserved role in ISC aging and in facilitating colonic regeneration. Our work identifies a conserved mechanism of epitranscriptomic regulation that safeguards tissues and the decay of ADAR-mediated RNA editing as a form of age-related epigenetic information loss that disrupts ISC homeostasis.

The ability to selectively edit specific RNA modifications is needed to understand their roles in cellular function and disease. However, existing approaches, particularly catalytically inactive Cas (dCas)-based systems, have limited applicability owing to their reliance on eraser proteins, and their performance can vary depending on the modification type and target context. Here we introduce the RNA Modification-Blocking (RModBlock) strategy, which uses chemically modified antisense oligonucleotides (ASOs) with locked nucleic acid to precisely inhibit modifications at targeted sites. Most RNA modification writers, including those for m⁵C and pseudouridine, require specific structural contexts. Using representative modifications with and without eraser proteins, m⁵C and pseudouridine, respectively, we demonstrated that RModBlock ASOs blocked the formation of their structural context, inhibiting modified bases by up to 97% in human cells. RModBlock ASOs also inhibited m⁶A, despite its writer protein not requiring a strict structural context, suggesting broad applicability. Moreover, this strategy achieves comparable or superior performance when dCas13-eraser-based systems are applicable. Finally, by inhibiting cancer-relevant modifications and through in vivo delivery to the mouse liver, we highlight its therapeutic potential. This showcases the RModBlock strategy as a precise, efficient and versatile approach for manipulating RNA modifications, with broad applicability in basic and translational research.

Metastasis accounts for the vast majority of tumor-related mortality. Certain populations of tumor cells exhibit organotropism by preferentially colonizing specific distant organs. The organ specificity of metastatic cells is determined by unique interactions between tumor cells and the microenvironment in target organs. Tumor extracellular vesicles (EVs), particularly exosomes, delivering tumor cell components including nucleic acid complexes, proteins, and lipids, play a crucial role in mediating intercellular communication between tumor cells and their microenvironment. ADAR-mediated microRNA (miRNA) editing has emerged as a crucial mechanism influencing miRNA stability, processing, and target specificity. Although EVs are increasingly recognized as important vehicles of intercellular signaling and promising biomarkers for cancer, the landscape of miRNA editing within EVs remains largely unexplored. Here, we present EVmiRED (Extracellular Vesicle miRNA Editing Database), a resource that integrates miRNA expression and editing profiles from tumor-derived EVs. The current release includes data from 683 samples across 12 tumor types and cell lines. EVmiRED provides detailed information on miRNA abundance, editing frequency, and the predicted functional impact of specific editing events. EVmiRED enables users to query individual miRNAs, visualize expression and editing patterns, and access raw datasets for customized analyses. Together, EVmiRED offers a valuable platform to advance our understanding of RNA editing-mediated regulation in intercellular communication, tumor progression, and cancer immunology.

Recent advancements in molecular engineering have established RNA-based technologies as powerful tools for both fundamental research and translational applications. Among the various RNA-based technologies developed, RNA base editing has recently emerged as a groundbreaking advancement. It primarily involves the conversion of adenosine (A) to inosine (I) and cytidine (C) to uridine (U), which are mediated by ADAR and APOBEC enzymes, respectively. RNA base editing has been applied in both biological research and therapeutic contexts. It enables site-directed editing within target transcripts, offering reversible, dose-dependent effects, in contrast to the permanent or heritable changes associated with DNA base editing. Additionally, RNA editing-based profiling of RNA-binding protein (RBP) binding sites facilitates transcriptome-wide mapping of RBP-RNA interactions in specific tissues and at the single-cell level. Furthermore, RNA editing-based sensors have been utilized to express effector proteins in response to specific RNA species. As RNA base editing technologies continue to evolve, we anticipate that they will significantly drive advancements in RNA therapeutics, synthetic biology, and biological research.

Adenosine deaminase acting on RNA (ADAR)-mediated RNA base editing offers a safer alternative to genome editing for specific clinical applications because of nonpermanent editing of targets. Current guide RNA (gRNA) designs feature a fully complementary specificity domain with an A–C mismatch at the targeted adenosine. However, perfectly matched dsRNA is not the most effective ADAR substrate. Here we introduce MIRROR (mimicking inverted repeats to recruit ADARs using engineered oligoribonucleotides), an approach that implements structural motifs derived from highly edited inverted Alu repeats in human tissues to enable rational gRNA design for ADAR recruitment. We demonstrated that MIRROR is applicable to both short chemically synthesized gRNAs with modifications and long biologically generated gRNAs and surpasses current state-of-the-art approaches in both gRNA forms. It enhances editing efficiency by up to 5.7-fold in multiple human cell types and primary hepatocytes from an alpha-1 antitrypsin deficiency mouse model. Our findings improve programmable RNA editing in vitro and in vivo by rational design through the screening of highly edited natural substrate mimics.

Advances in sequencing technologies have empowered epitranscriptomic profiling at the single-base resolution. Putative RNA modification sites identified from a single high-throughput experiment may contain one type of modification deposited by different writers or different types of modifications, along with false positive results because of the challenge of distinguishing signals from noise. However, current tools are insufficient for subtyping, visualization, and denoising these signals. Here, we present iMVP, which is an interactive framework for epitranscriptomic analysis with a nonlinear dimension reduction technique and density-based partition. As exemplified by the analysis of mRNA m⁵C and ModTect variant data, we show that iMVP allows the identification of previously unknown RNA modification motifs and writers and the discovery of false positives that are undetectable by traditional methods. Using putative m⁶A/m⁶Am sites called from 8 profiling approaches, we illustrate that iMVP enables comprehensive comparison of different approaches and advances our understanding of the difference and pattern of true positives and artifacts in these methods. Finally, we demonstrate the ability of iMVP to analyze an extremely large human A-to-I editing dataset that was previously unmanageable. Our work provides a general framework for the visualization and interpretation of epitranscriptomic data.

The significance of A-to-I RNA editing in neurological development is widely recognized, however, its influence on retina development remains to be thoroughly understood. In this study, we aimed to characterize the temporal landscape of A-to-I editing in mouse retina development using total RNA-seq and Ribosome profiling, with a specific emphasis on its effect on gene translation. Our findings revealed that the editing underwent plastic changes and distinct editing contents facilitated stage-specific functions. Further analysis showed a dynamic interplay between A-to-I editing and alternative splicing, with A-to-I editing having a significant impact on splicing efficiency and increasing the quantity of splicing events. A-to-I editing held the potential of enhancing the translatome’s diversity, but this came at the expense of reduced translational efficiency. When coupled with splicing, it could produce a coordinated regulatory effect on translatome dynamics. Overall, our study presents a temporally resolved atlas of A-to-I editing, connecting its dynamic changes with the regulatory impact on splicing and translation.

Red blood cells (RBCs) of the Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) in routine testing. RhD-positive (D+) RBC transfusion for Asian-type DEL patients has been proposed but has not been generally adopted due to a lack of direct evidence regarding its safety and underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in Asian-type DEL patients; none of the recipients (0/42; 95% confidence interval, 0%-8.40%) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4,045 serologic D- pregnant women throughout China; alloanti-D was found only in true D- individuals (2.63%, 79/3,009), but not in those with Asian-type DEL (0/1,032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs following transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in Asian-type DEL patients, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as NCT03727230.