RNA Editing Research Articles

Ultrasensitive Biosensors Detecting m6A in Blood: Achieving Early Screening and Typing of Tumors.

N6-methyladenosine (m6A) modification is one of the most widespread RNA modifications in eukaryotes and is involved in cancer development and progression by regulating oncogene expression. Herein, a reticulated rolling circle amplification (RCA) cascade reaction was used to construct a novel electrochemical biosensor for ultrasensitive detection of m6A, employing ferrocene-tyramine (Fc-Tyr) molecules as electroactive probes. In this strategy, the RCA cascade reaction not only amplifies specific circular DNA in the designed template to reduce the binding with similar nucleic acid sequences but also generates a long ssDNA through multiple repetitions to capture a large number of electrochemical signal probes and achieve the amplification of electrochemical biosensing signals. The developed biosensor demonstrated high selectivity and sensitivity toward m6A in the range of 0.5 pM-150 nM, with a detection limit of 14.07 fM. Meanwhile, total RNA extracted from cell samples was analyzed for m6A expression levels using the developed biosensor and a commercial colorimetric immunoassay, the biosensor and immunoassay showed consistent results. In addition, m6A levels in clinical serum samples were assessed using the developed electrochemical biosensor, which showed that m6A expression was much lower in healthy individuals than in cancer patients, therefore the biosensor is promising for cancer typing. This study provides a new method for rapid and convenient tumor marker detection in clinical practice, as well as a new idea for sensitive detection of other biomolecules.

Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications.

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA using the Oxford Nanopore technology promises the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N6-methyladenosine (m6A) modifications in native mRNA. We utilized human and mouse cells with known genetic variants to assign allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses revealed the importance of sequences adjacent to the DRACH-motif in determining m6A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discovered allele-specific m6A modification (ASM) events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long reads and surpassing the capabilities of antibody-based short-read approaches. This technological advancement promises to advance our understanding of the role of genetics in determining mRNA modifications.

Investigating N6-methyladenosin RNA modification as a mediator of microRNA mechanical regulation in the heart

Abstract Background The heart is exposed to mechanical forces and continuously adapts to its environment in a process known as cardiac remodeling. Excessive mechanical load is a primary driver of pathological cardiac remodeling, leading to heart failure (HF), a prominent cause of morbidity and mortality worldwide. A set of microRNAs (miRs), termed mechano-miRs, has been identified to respond to mechanical forces and contribute to heart pathophysiology and HF. N6-methyladenosine (m6A) RNA modification is emerging as a novel regulatory layer impacting gene expression and occurring both in coding and non-coding RNAs. In this context, the N6-adenosine-methyltransferase METTL-3 regulates the processing of miR primary transcripts (pri-miRs) to functional miRs, promoting angiogenesis and cardiac repair after myocardial infarction. On the other side, dysregulation of m6A due to a defect in FTO, a m6A demethylase, has been implicated in HF. However, the role of m6A RNA modifications in heart mechanical responses and mechano-miR regulation remains unexplored. Purpose This project aims to: 1) investigate the impact of mechanical overload on the expression of m6A regulatory machinery components; 2) unveil the role of m6A-mechano-miRs axis in mechanically induced responses driving pathological cardiac remodeling leading to heart failure. Methods and Results Using living myocardial slices (LMS) technology, we modulate mechanical load in a multicellular environment while maintaining physiological behavior. Human LMS, obtained from the left ventricle of human donor non-failing hearts (NHS Blood and Transplant INOAR program, IRAS project ID: 189069) and rat LMS are subjected to two degrees of mechanical preload: physiological sarcomere length (SL) (SL = 2.2 μm) and overload (SL = 2.4 μm). This enables us to mimic the effects of mechanical stress on the heart during HF (volume overload model). LMS are maintained for 48 hours under electrical stimulation in circulating oxygenated media at 37°C. Mechanically overloaded LMS exhibit decreased contractility, accompanied by an increase in global m6A levels compared to physiological controls. Consistent with this finding, the analysis of the expression of m6A machinery reveals a downregulation of m6A demethylases (FTO and ALKBH5) in overloaded LMS. We identified putative cardiac mechano-miRs by small-RNA sequencing in both human and rat LMS and next predicted potential m6A sites on their primary transcripts via SRAMP (sequence-based RNA adenosine methylation site predictor). Conclusions Mechanical overload affects global m6A levels and the expression of m6A demethylases in the heart. Ongoing analysis of the m6A profile on the identified mechano-miR primary transcripts and mRNA targets holds promise for unveiling novel mechanically regulated m6A events, providing insights for transformative therapeutic strategies.

Novel balloon catheter containing therapeutic mRNA with smart RNA switch alleviate restenosis after vascular injury

Abstract Background Cardiovascular diseases are the leading cause of death worldwide, and vascular intervention is an effective method for ischemic heart diseases. However, vascular restenosis significantly affects the long-term therapeutic outcomes, which mainly caused by excessive proliferation of vascular smooth muscle cells (VSMCs). While drug-eluting stents and drug-coated balloons have successfully decreased the incidence of restenosis by inhibiting the VSMCs proliferation, they concurrently suppress the repair of endothelial cells (ECs) and re-endothelialization, thereby increasing the risk of late thrombotic events and mortality. Consequently, it is urged to develop novel high-selectively therapeutic devices to overcome vascular restenosis. In current mRNA therapies of cardiovascular diseases, there are few effective systems for cell-specific expression. Therefore, we propose to utilize RNA editing (based on ADAR1 RNA editing) to investigate a smart RNA molecular switch capable of cell-specific recognition and selectively releasing payload in targeted cells or tissues for cell precision therapy. However, the application of gene editing in the cardiovascular system faces challenge of low delivery efficiency. We innovatively take the balloon as a specific and efficient tool to delivery therapeutic mRNA, which may be a potential therapy for vascular restenosis. Objective We investigated therapeutic mRNA containing a smart RNA switch to selectively suppress the VSMCs proliferation while promoting re-endothelialization and delivered by balloon as a novel therapy in vascular restenosis animal models. Results For RNA switch high-throughput screening, we utilized PiggyBac transposon system to establish monoclonal overexpressing cell lines and chose the cell strain with expression levels closely mimicking VSMCs and ECs. We found the most effective RNA switch from dozens of potential candidates which could specifically recognize VSMCs but not ECs. Therapeutic mRNA sequences containing the RNA switch efficiently initiated payload translation in VSMCs while maintaining payload silence in primary vascular endothelial cells, which effectively and selectively inhibited VSMCs excessive proliferation and promoted endothelial repair. Additionally, we developed an interventional balloon delivery system for mRNA drugs, achieving in situ delivery to cardiovascular lesions in vivo, demonstrating therapeutic effects consistent with cellular phenotypes in animal models. Conclusion We suggest that the smart RNA switch can serve as a novel, cell-specific therapeutic tool for restenosis and other cardiovascular diseases. Additionally, utilizing a balloon for mRNA delivery represents a new paradigm for mRNA therapy and gene editing in the cardiovascular field.

A robust deep learning approach for identification of RNA 5-methyluridine sites

RNA 5-methyluridine (m5U) sites play a significant role in understanding RNA modifications, which influence numerous biological processes such as gene expression and cellular functioning. Consequently, the identification of m5U sites can play a vital role in the integrity, structure, and function of RNA molecules. Therefore, this study introduces GRUpred-m5U, a novel deep learning-based framework based on a gated recurrent unit in mature RNA and full transcript RNA datasets. We used three descriptor groups: nucleic acid composition, pseudo nucleic acid composition, and physicochemical properties, which include five feature extraction methods ENAC, Kmer, DPCP, DPCP type 2, and PseDNC. Initially, we aggregated all the feature extraction methods and created a new merged set. Three hybrid models were developed employing deep-learning methods and evaluated through 10-fold cross-validation with seven evaluation metrics. After a comprehensive evaluation, the GRUpred-m5U model outperformed the other applied models, obtaining 98.41% and 96.70% accuracy on the two datasets, respectively. To our knowledge, the proposed model outperformed all the existing state-of-the-art technology. The proposed supervised machine learning model was evaluated using unsupervised machine learning techniques such as principal component analysis (PCA), and it was observed that the proposed method provided a valid performance for identifying m5U. Considering its multi-layered construction, the GRUpred-m5U model has tremendous potential for future applications in the biological industry. The model, which consisted of neurons processing complicated input, excelled at pattern recognition and produced reliable results. Despite its greater size, the model obtained accurate results, essential in detecting m5U.

ALKBH5 Protects Against Hepatic Ischemia-Reperfusion Injury by Regulating YTHDF1-Mediated YAP Expression.

Ischemia/reperfusion (I/R) injury with severe cell death is a major complication involved in liver transplantation and resection. The identification of key regulators improving hepatocyte activity may provide potential strategies to clinically resolve I/R-induced injury. N6-methyladenosine (m6A) RNA modification is essential for tissue homeostasis and pathogenesis. However, the potential involvement of m6A in the regulation of hepatocyte activity and liver injury has not been fully explored. In the present study, we found that hepatocyte AlkB homolog H5 (ALKBH5) levels were decreased both in vivo and in vitro I/R models. Hepatocyte-specific ALKBH5 overexpression effectively attenuated I/R-induced liver necrosis and improved cell proliferation in mice. Mechanistically, ALKBH5-mediated m6A demethylation improved the mRNA stability of YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1), thereby increasing its expression, which consequently promoted the translation of Yes-associated protein (YAP). In conclusion, ALKBH5 is a regulator of hepatic I/R injury that improves hepatocyte repair and proliferation by maintaining YTHDF1 stability and YAP content. The ALKBH5-m6A-YTHDF1-YAP axis represents promising therapeutic targets for hepatic I/R injury to improve the prognosis of liver surgery.

The Mysterious World of Non-Canonical Caps - What We Know and Why We Need New Sequencing Techniques.

It was long believed that viral and eukaryotic mRNA molecules are capped at their 5' end solely by the N7-methylguanosine cap, which regulates various aspects of the RNA life cycle, from its biogenesis to its decay. However, the recent discovery of a variety of non-canonical RNA caps derived from metabolites and cofactors - such as NAD, FAD, CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleoside polyphosphates - has expanded the known repertoire of RNA modifications. These non-canonical caps are found across all domains of life and can impact multiple aspects of RNA metabolism, including stability, translation initiation, and cellular stress responses. The study of these modifications has been facilitated by sophisticated methodologies such as liquid chromatography-mass spectrometry, which have unveiled their presence in both prokaryotic and eukaryotic organisms. The identification of these novel RNA caps highlights the need for advanced sequencing techniques to characterize the specific RNA types bearing these modifications and understand their roles in cellular processes. Unravelling the biological role of non-canonical RNA caps will provide insights into their contributions to gene expression, cellular adaptation, and evolutionary diversity. This review emphasizes the importance of these technological advancements in uncovering the complete spectrum of RNA modifications and their implications for living systems.

RPS 2.0: an updated database of RNAs involved in liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) is a crucial process for the formation of biomolecular condensates such as coacervate droplets, P-bodies and stress granules, which play critical roles in many physiological and pathological processes. Increasing studies have shown that not only proteins but also RNAs play a critical role in LLPS. To host LLPS-associated RNAs, we previously developed a database named 'RPS' in 2021. In this study, we present an updated version RPS 2.0 (https://rps.renlab.cn/) to incorporate the newly generated data and to host new LLPS-associated RNAs driven by post-transcriptional regulatory mechanisms. Currently, RPS 2.0 hosts 171 301 entries of LLPS-associated RNAs in 24 different biomolecular condensates with four evidence types, including 'Reviewed', 'High-throughput (LLPS enrichment)', 'High-throughput (LLPS perturbation)' and 'Predicted', and five event types, including 'Expression', 'APA', 'AS', 'A-to-I' and 'Modification'. Additionally, extensive annotations of LLPS-associated RNAs are provided in RPS 2.0, including RNA sequence and structure features, RNA-protein/RNA-RNA interactions, RNA modifications, as well as diseases related annotations. We expect that RPS 2.0 will further promote research of LLPS-associated RNAs and deepen our understanding of the biological functions and regulatory mechanisms of LLPS.

Differential redox sensitivity of tRNA dihydrouridylation.

Various transfer RNA (tRNA) modifications have recently been shown to regulate stress-dependent gene expression by modulating messenger RNA translation. Among these modifications, dihydrouridine stands out for its increase of tRNA structural flexibility. However, whether and how dihydrouridine synthesis reacts to environmental stimuli is largely unknown. In this study, we manipulated the intracellular redox state of Escherichia coli using paraquat, revealing differential sensitivities of the three tRNA-dihydrouridine synthases towards oxidative stress. Using liquid chromatography-mass spectrometryquantification of dihydrouridine in various knockout strains, we validated the use of a specific RNA sequencing method, namely AlkAnilineSeq, for the precise mapping of dihydrouridines throughout E. coli tRNAs. We found DusA showing high activity, followed by DusB and DusC, whose activity was decreased under paraquat treatment. The relative sensitivity is most plausibly explained by a paraquat-dependent drop of NADPH availability. These findings are substantiated by in vitro kinetics, revealing DusA as the most active enzyme, followed by DusB, while DusC showed little activity, likely related to the efficacy of the redox reaction of the flavin coenzyme with NADPH. Overall, our study underscores the intricate interplay between redox dynamics and tRNA modification processes, revealing a new facet of the regulatory mechanisms influencing cellular responses to oxidative stress.

Coxsackievirus B3-Induced m6A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

N6-methyladenosine (m6A) is the most prevalent internal RNA modification. Here, we demonstrate that coxsackievirus B3 (CVB3), a common causative agent of viral myocarditis, induces m6A modification primarily at the stop codon and 3′ untranslated regions of its genome. As a positive-sense single-stranded RNA virus, CVB3 replicates exclusively in the cytoplasm through a cap-independent translation initiation mechanism. Our study shows that CVB3 modulates the expression and nucleo-cytoplasmic transport of the m6A machinery components—METTL3, ALKBH5 and YTHDFs—resulting in increased m6A modifications that enhance viral replication. Mechanistically, this enhancement is mediated through YTHDF-driven stress granule (SG) formation. We observed that YTHDF proteins co-localize with human antigen R (HuR), a protein facilitating cap-independent translation, in SGs during early infection. Later in infection, YTHDFs are cleaved, suppressing SG formation. Notably, for the first time, we identified that during early infection CVB3’s RNA-dependent RNA polymerase (3D) and double-stranded RNA (dsRNA) are stored in SGs, co-localizing with HuR. This early-stage sequestration likely protects viral components for use in late-phase replication, when SGs are disrupted due to YTHDF cleavage. In summary, our findings reveal that CVB3-induced m6A modifications enhance viral replication by regulating YTHDF-mediated SG dynamics. This study provides a potential therapeutic strategy for CVB3-induced myocarditis.

A de novo Missense Mutation in PPP2R5D Alters Dopamine Pathways and Morphology of iPSC-derived Midbrain Neurons.

Induced pluripotent stem cell (iPSC) models of neurodevelopmental disorders (NDDs) have promoted an understanding of commonalities and differences within or across patient populations by revealing the underlying molecular and cellular mechanisms contributing to disease pathology. Here, we focus on developing a human model for PPP2R5D-related NDD, called Jordan syndrome, which has been linked to Early-Onset Parkinson's Disease (EOPD). Here we sought to understand the underlying molecular and cellular phenotypes across multiple cell states and neuronal subtypes in order to gain insight into Jordan syndrome pathology. Our work revealed that iPSC-derived midbrain neurons from Jordan syndrome patients display significant differences in dopamine-associated pathways and neuronal architecture. We then evaluated a CRISPR-based approach for editing heterozygous dominant G-to-A mutations at the transcript level in patient-derived neural stem cells. Our findings show site-directed RNA editing is influenced by sgRNA length and cell type. These studies support the potential for a CRISPR RNA editor system to selectively edit mutant transcripts harboring G-to-A mutations in neural stem cells while providing an alternative editing technology for those suffering from NDDs.

Selective RNA pseudouridinylation in situ by circular gRNAs in designer organelles

RNA modifications play a pivotal role in the regulation of RNA chemistry within cells. Several technologies have been developed with the goal of using RNA modifications to regulate cellular biochemistry selectively, but achieving selective and precise modifications remains a challenge. Here, we show that by using designer organelles, we can modify mRNA with pseudouridine in a highly selective and guide-RNA-dependent manner. We use designer organelles inspired by concepts of phase separation, a central tenet in developing artificial membraneless organelles in living mammalian cells. In addition, we use circular guide RNAs to markedly enhance the effectiveness of targeted pseudouridinylation. Our studies introduce spatial engineering through optimized RNA editing organelles (OREO) as a complementary tool for targeted RNA modification, providing new avenues to enhance RNA modification specificity.

Coronavirus S protein alters dsRNA accumulation and stress granule formation through regulation of ADAR1-p150 expression.

The precise role of the highly variable coronavirus S protein in modulating innate immune responses remains unclear. In this study, we demonstrated that the mutant strain of swine coronavirus porcine enteric diarrhea virus induced significantly lower levels of double-stranded RNA (dsRNA) accumulation, inhibited protein kinase R (PKR) activation and suppressed stress granule (SG) formation compared with the classical strain. The 29th amino acid at N-terminus of S was identified as the key functional site for regulation of SG formation, and found that mutant S inhibited PKR phosphorylation and SG formation by upregulating adenosine deaminase acting on RNA 1 (ADAR1)-p150. Notably, the Zα domain of ADAR1-p150 was essential for inhibiting SG formation. Upregulation of ADAR1-p150 also reduced accumulation of dsRNA depending on its RNA editing function. Virus rescue confirmed that the mutant carrying a substitution at amino acid 29 failed to induce ADAR1-p150, leading to dsRNA accumulation, PKR activation and SG formation. Interestingly, the latest severe acute respiratory syndrome coronavirus-2 strains exhibit a novel 25PPA27 deletion at N-terminus of S that was also shown to lead to altered ADAR1-p150 expression and SG inhibition. The transcription factor TCF7L2 was identified as a player in S-mediated transcriptional enhancement of ADAR1-p150. This study is the first to clarify the crucial role of N-terminus of S in immune regulation of coronaviruses.

LoDEI: a robust and sensitive tool to detect transcriptome-wide differential A-to-I editing in RNA-seq data

RNA editing is a highly conserved process. Adenosine deaminase acting on RNA (ADAR) mediated deamination of adenosine (A-to-I editing) is associated with human disease and immune checkpoint control. Functional implications of A-to-I editing are currently of broad interest to academic and industrial research as underscored by the fast-growing number of clinical studies applying base editors as therapeutic tools. Analyzing the dynamics of A-to-I editing, in a biological or therapeutic context, requires the sensitive detection of differential A-to-I editing, a currently unmet need. We introduce the local differential editing index (LoDEI) to detect differential A-to-I editing in RNA-seq datasets using a sliding-window approach coupled with an empirical q value calculation that detects more A-to-I editing sites at the same false-discovery rate compared to existing methods. LoDEI is validated on known and novel datasets revealing that the oncogene MYCN increases and that a specific small non-coding RNA reduces A-to-I editing.

M7G-modified mt-tRF3b-LeuTAA regulates mitophagy and metabolic reprogramming via SUMOylation of SIRT3 in chondrocytes

N7-methylguanosine (m7G) modification is one of the most prevalent RNA modifications, and methyltransferase-like protein-1 (METTL1) is a key component of the m7G methyltransferase complex. METTL1-catalyzed m7G as a new RNA modification pathway that regulates RNA structure, biogenesis, and cell migration. Increasing evidence indicates that m7G modification has been implicated in the pathophysiological process of osteoarthritis (OA). However, the underlying molecular mechanisms of m7G modification remains incompletely elucidated during the progression of OA. Here we found that METTL1 and m7G levels were markedly increased in OA chondrocytes. In addition, METTL1-mediated m7G modification upregulated mt-tRF3b-LeuTAA expression to exacerbate chondrocyte degeneration. Mechanistically, mt-tRF3b-LeuTAA decreased the SUMO-specific protease 1 (SENP1) protein expression and upregulated the level of sirtuin 3 (SIRT3) SUMOylation to inhibit PTEN induced kinase 1 (PINK1)/Parkin-mediated mitochondrial mitophagy. Intra-articular injection of PMC-tRF3b-LeuTAA inhibitor (Polyamidoamine-polyethylene glycol surface-modified with Minimal self-peptides and Chondrocyte-affinity peptides, PMC) attenuated destabilization of the medial meniscus (DMM) mouse cartilage degeneration in vivo. Our study demonstrates that METTL1/m7G/mt-tRF3b-LeuTAA axis accelerate cartilage degradation by inhibiting mitophagy and promoting mitochondrial dysfunction through SIRT3 SUMOylation, and suggest that targeting METTL1 and its downstream signaling axis could be a promising therapeutic target for OA treatment.

RNA modification in normal hematopoiesis and hematologic malignancies.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic cells. Previous studies have shown that m6A plays a critical role under both normal physiological and pathological conditions. Hematopoiesis and differentiation are highly regulated processes, and recent studies on m6A mRNA methylation have revealed how this modification controls cell fate in both normal and malignant hematopoietic states. However, despite these insights, a comprehensive understanding of its complex roles between normal hematopoietic development and malignant hematopoietic diseases remains elusive. This review first provides an overview of the components and biological functions of m6A modification regulators. Additionally, it highlights the origin, differentiation process, biological characteristics, and regulatory mechanisms of hematopoietic stem cells, as well as the features, immune properties, and self-renewal pathways of leukemia stem cells. Last, the article systematically reviews the latest research advancements on the roles and mechanisms of m6A regulatory factors in normal hematopoiesis and related malignant diseases. More importantly, this review explores how targeting m6A regulators and various signaling pathways could effectively intervene in the development of leukemia, providing new insights and potential therapeutic targets. Targeting m6A modification may hold promise for achieving more precise and effective leukemia treatments.

The YTHDF Proteins Shape the Brain Gene Signatures of Alzheimer's Disease.

The gene signatures of Alzheimer's Disease (AD) brains reflect an output of a complex interplay of genetic, epigenetic, epi-transcriptomic, and post-transcriptional regulations. To identify the most significant factor that shapes the AD brain signature, we developed a machine learning model (DEcode-tree) to integrate cellular and molecular factors explaining differential gene expression in AD. Our model indicates that YTHDF proteins, the canonical readers of N6-methyladenosine RNA modification (m6A), are the most influential predictors of the AD brain signature. We then show that protein modules containing YTHDFs are downregulated in human AD brains, and knocking out YTHDFs in iPSC-derived neural cells recapitulates the AD brain gene signature in vitro . Furthermore, eCLIP-seq analysis revealed that YTHDF proteins influence AD signatures through both m6A-dependent and independent pathways. These results indicate the central role of YTHDF proteins in shaping the gene signature of AD brains.

RMVar 2.0: an updated database of functional variants in RNA modifications.

Evaluating the impact of genetic variants on RNA modifications (RMs) is crucial for identifying disease-associated variants and understanding the pathogenic mechanisms underlying human diseases. Previously, we developed a database called RMVar to catalog variants linked to RNA modifications in humans and mice. Here, we present an updated version RMVar 2.0 (http://rmvar.renlab.cn). In this updated version, we applied an enhanced analytical pipeline to the latest RNA modification datasets and genetic variant information to identify RM-associated variants. A notable advancement in RMVar 2.0 is our incorporation of allele-specific RNA modification analysis to identify RM-associated variants, a novel approach not utilized in RMVar 1.0 or other comparable databases. Furthermore, the database offers comprehensive annotations for various molecular events, including RNA-binding protein (RBP) interactions, RNA-RNA interactions, splicing events, and circular RNAs (circRNAs), which facilitate investigations into how RM-associated variants influence post-transcriptional regulation. Additionally, we provide disease-related information sourced from ClinVar and GWAS to help researchers explore the connections between RNA modifications and various diseases. We believe that RMVar 2.0 will significantly enhance our understanding of the functional implications of genetic variants affecting RNA modifications within the context of human disease research.

All the sites we cannot see: Sources and mitigation of false negatives in RNA modification studies.

RNA modifications are essential for human health - too much or too little of them leads to serious illnesses ranging from neurodevelopmental disorders to cancer. Technical advances in RNA modification sequencing are beginning to uncover the RNA targets of diverse RNA-modifying enzymes that are dysregulated in disease. However, the emerging transcriptome-wide maps of modified nucleosides installed by these enzymes should be considered as first drafts. In particular, a range of technical artefacts lead to false negatives- modified sites that are overlooked owing to technique-dependent, and often sequence-context-specific, 'blind spots'. In this Review, we discuss potential sources of false negatives in sequencing-based RNA modification maps, propose mitigation strategies and suggest guidelines for transparent reporting of sensitivity to detect modified sites in profiling studies. Important considerations for recognition and avoidance of false negatives include assessment and reporting of position-specific sequencing depth, identification of protocol-dependent RNA capture biases and applying controls for false negatives as well as for false positives. Despite their limitations, emerging maps of RNA modifications reveal exciting and largely uncharted potential for post-transcriptional control of all aspects of RNA function.

N6-Methyladenosine RNA Modification Regulates the Differential Muscle Development in Large White and Ningxiang Pigs.

N6-methyladenosine (m6A) is the most common modification in eukaryotic RNAs. Growing research indicates that m6A methylation is crucial for a multitude of biological processes. However, research on the m6A modifications in the regulation of porcine muscle growth is lacking. In this study, we identified differentially expressed genes in the neonatal period of muscle development between Large White (LW) and NingXiang (NX) pigs and further reported m6A methylation patterns via MeRIP-seq. We found that m6A modification regulates muscle cell development, myofibrils, cell cycle, and phosphatase regulator activity during the neonatal phase of muscle development. Interestingly, differentially expressed genes in LW and NX pigs were mainly enriched in pathways involved in protein synthesis. Furthermore, we performed a conjoint analysis of MeRIP-seq and RNA-seq data and identified 27 differentially expressed and m6A-modified genes. Notably, a typical muscle-specific envelope transmembrane protein, WFS1, was differentially regulated by m6A modifications in LW and NX pigs. We further revealed that the m6A modification accelerated the degradation of WFS1 in a YTHDF2-dependent manner. Noteworthy, we identified a single nucleotide polymorphism (C21551T) within the last exon of WFS1 that resulted in variable m6A methylation, contributing to the differing WFS1 expression levels observed in LW and NX pigs. Our study conducted a comprehensive analysis of the m6A modification on NX and LW pigs during the neonatal period of muscle development, and elucidated the mechanism by which m6A regulates the differential expression of WFS1 in the two breeds.