RNA Editing Research Articles

Oncometabolite D-2HG drives tumor metastasis and protumoral macrophage polarization by targeting FTO/m6A/ANGPTL4/integrin axis in triple-negative breast cancer

BackgroundD-2-hydroxyglutarate (D-2HG), an oncometabolite derived from the tricarboxylic acid cycle. Previous studies have reported the diverse effects of D-2HG in pathophysiological processes, yet its role in breast cancer remains largely unexplored.MethodsWe applied an advanced biosensor approach to detect the D-2HG levels in breast cancer samples. We then investigated the biological functions of D-2HG through multiple in vitro and in vivo assays. A joint MeRIP-seq and RNA-seq strategy was used to identify the target genes regulated by D-2HG-mediated N6-methyladenosine (m6A) modification. RNA pull-down assays were further applied to identify the reader that could specifically recognize the m6A modification on angiopoietin like 4 (ANGPTL4) mRNA and RNA immunoprecipitation was used to confirm the findings.ResultsWe found that D-2HG accumulated in triple-negative breast cancer (TNBC), exerting oncogenic effects both in vitro and in vivo by promoting TNBC cell growth and metastasis. Mechanistically, D-2HG enhanced global m6A RNA modifications in TNBC cells, notably upregulating m6A modification on ANGPTL4 mRNA, which was mediated by the inhibition of Fat-mass and obesity-associated protein (FTO), resulting in increased recognition of m6A-modified ANGPTL4 by YTH N6-methyladenosine RNA binding protein F1 (YTHDF1), thereby promoting the enhanced translation of ANGPTL4. As a secretory protein, ANGPTL4 subsequently activated the integrin-mediated JAK2/STAT3 signaling cascade in TNBC cells through autocrine signaling. Notably, the knockdown of ANGPTL4 or treatment with GLPG1087 (an integrin antagonist) significantly reduced D-2HG-induced proliferation and metastasis in TNBC cells. Additionally, ANGPTL4 was found to promote macrophage M2 polarization within the tumor microenvironment via paracrine signaling, further driving TNBC progression. The association of ANGPTL4 with poor prognosis in TNBC patients underscores its clinical relevance.ConclusionsOur study unveils a previously unrecognized role for D-2HG-mediated RNA modification in TNBC progression and targeting the D-2HG/FTO/m6A/ANGPTL4/integrin axis can serve as a promising therapeutic target for TNBC patients.

The role of FTO in m6A RNA methylation and immune regulation in Staphylococcus aureus infection-related osteomyelitis

BackgroundRegulators of n6-methyladenosine (m6A) RNA modification play important roles in many diseases; however, their involvement in Staphylococcus aureus (S. aureus)-related osteomyelitis remains inadequately explored. Therefore, this study aims to investigate the role of m6A in S. aureus infection-related osteomyelitis and elucidate its underlying mechanisms.MethodsWe downloaded the S. aureus infection-related osteomyelitis infection dataset GSE30119 from the Gene Expression Omnibus database. Initially, we constructed a diagnostic model based on m6A genes and predicted the hub node miRNAs and transcription factors by constructing a protein–protein interaction network. Subsequently, a prognostic model was built using LASSO regression, the receiver operating characteristic curve of the model was plotted, and the predictive performance of the diagnostic model was validated. Further, unsupervised clustering analysis, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were employed to assess immune cell infiltration. Additionally, we validated the expression of fat mass and obesity-associated protein (FTO) in S. aureus-infected Raw264.7 macrophages using qPCR and western blotting. Moreover, we conducted si-FTO experiments on mouse Raw264.7 macrophages to investigate the anti-inflammatory regulatory role of si-FTO during S. aureus infection.ResultsWe identified 19 co-expressed genes closely related to FTO were identified, along with 206 related transcription factor regulatory genes and 589 miRNAs. Enrichment analyses suggested that these genes were involved in pathways related to the proliferation and oxidation of various immune cells, cellular senescence, and various tumors and immune cells, as well as cell cycle-related functions. GSEA revealed that PD-1, TH1, TH2, CTLA4, and other pathways were significantly enriched in patients with high FTO expression. GSVA indicated that the differentially enriched pathways were related to included amino acid metabolism, immunity, and infection. Correlation analysis of immune infiltration revealed that monocytes, M2 macrophages, resting mast cells, and neutrophils were present in normal and diseased samples. Differences in expression were observed between the groups. The western blotting and qPCR analyses confirmed that the protein expression of FTO was reduced in macrophages after infection with S. aureus, consistent with the observed changes in mRNA expression. Furthermore, we validated that FTO may influence the regulation of inflammation through the FoxO1/NF-kB pathway.ConclusionThe m6A RNA methylation regulator FTO may serve as a potential diagnostic marker and therapeutic target, involved in the pathogenesis of S. aureus infection-related osteomyelitis. This finding provides new insights into the relationship between FTO-mediated m6A RNA methylation and osteomyelitis.

Dynamic, Single‐cell Monitoring of RNA Modifications Response to Viral Infection Using a Genetically Encoded Live‐cell RNA Methylation Sensor

AbstractRNA modifications, such as N6‐methylation of adenosine (m6A), serve as key regulators of cellular behaviors, and are highly dynamic; however, tools for dynamic monitoring of RNA modifications in live cells are lacking. Here, we develop a genetically encoded live‐cell RNA methylation sensor that can dynamically monitor RNA m6A level at single‐cell resolution. The sensor senses RNA m6A in cells via affinity‐induced cytoplasmic retention using a nuclear location sequence‐fused m6A reader. It allows for simultaneously measure RNA m6A dynamics and viral invasion at single‐cell level. Based on the single‐cell analytical tool, we found that SARS‐CoV‐2 infection enhances host‐cell RNA m6A level, and high‐level RNA m6A modification in host cells, in turn, facilitates viral infection. Particularly, Omicron, a variant of SARS‐CoV‐2, that features as high infection capacity, however, exhibits a reduced facilitation of m6A modification in host cells. In addition, the sensor can estimate viral inhibition via measuring cellular m6A level, that was explored for screening potential antiviral drugs. The methylation sensor can serve for elucidating the interplay between pathogens and host‐cell epigenetics at single‐cell level.

Targeting RNA Modification and Mitochondrial Metabolism Crosstalk in Leukemic Stem Cells with CDK7 Inhibitor TGN-1062

Targeting RNA Modification and Mitochondrial Metabolism Crosstalk in Leukemic Stem Cells with CDK7 Inhibitor TGN-1062

WD repeat domain 4 in tumorigenesis: Molecular mechanisms, cancer-type specific roles, and therapeutic potential

WD repeat domain 4 (WDR4) is an essential member of the WD-repeat protein family, known for its regulatory roles in cellular processes critical to cancer development, including RNA modification, protein stability, cell cycle progression, and apoptosis. Studies have shown that WDR4 plays a pivotal role in tumorigenesis across various cancer types, with a particular focus on breast cancer in this study, where its overexpression is closely associated with aggressive tumor characteristics and poorer patient outcomes. As a scaffold protein, WDR4 is involved in N7-methylguanosine tRNA methylation and ubiquitin-mediated protein degradation, thereby regulating RNA stability, protein synthesis, and cell survival. This review provides a comprehensive analysis of WDR4’s molecular mechanisms, its oncogenic functions across different cancer types, and its interactions with other key factors in the tumor microenvironment, further exploring its potential role in tumor progression. As research on WDR4 progresses, we not only gain a deeper understanding of its complex role in tumor biology but also uncover new therapeutic avenues. In particular, the potential of WDR4 as a biomarker and therapeutic target is increasingly recognized. Despite the challenges faced in its clinical application, such as the difficulty in developing targeted therapies and managing side effects, the future prospects of WDR4 in cancer diagnosis and treatment remain promising, and it is expected to emerge as an effective therapeutic target in the near future.

YTHDF1 and YTHDC1 m6A reader proteins regulate HTLV-1 tax and hbz activity.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive neurodegenerative disease. Regulation of viral gene expression plays a key role in viral persistence and pathogenesis. However, the molecular mechanisms underlying this fine-tuned regulation remain poorly understood. Little is known regarding RNA chemical modifications of HTLV-1 RNA and how these affect viral biology and disease development. Post-transcriptional chemical modification of RNA is common in eukaryotes, with N6-methyladenosine (m6A) being the most prevalent. In this study, we investigated the role of m6A RNA modifications on HTLV-1 gene expression. Using MeRIP-Seq, we mapped the sites of m6A modification to the 3' end of the viral genome. We found HTLV-1 RNA, as well as viral oncogene transcripts tax and hbz, contained m6A modifications. m6A-depletion in HTLV-1-transformed cells decreased sense-derived viral genes (Tax, Gag, and Env) and increased antisense-derived Hbz expression. Tax and hbz transcripts were bound by reader proteins YTHDF1 and YTHDC1 in a panel of HTLV-1 T-cell lines. Using expression vectors and shRNA-mediated knockdown, we found that YTHDF1 had opposing effects on viral gene expression, decreasing sense-derived viral genes and increasing antisense-derived Hbz. Upon further study, the YTHDF1 effects on tax abundance were dependent on tax m6A deposition. The nuclear m6A reader protein YTHDC1 affected the abundance of both sense- and antisense-derived viral transcripts and specifically enhanced the nuclear export of tax transcript. Collectively, our results demonstrate global m6A levels and m6A reader proteins YTHDF1 and YTHDC1 regulate HTLV-1 gene expression.IMPORTANCEHuman T-cell leukemia virus type 1 (HTLV-1) persistence and pathogenesis are controlled through tight regulation of viral gene expression. The fate of RNA can be controlled by epigenetic modifications that impact gene expression without altering the DNA sequence. Our study details the impact of N6-methyladenosine (m6A) RNA chemical modifications on HTLV-1 gene expression. We found that reductions in global m6A levels affected viral gene expression, decreasing Tax and other sense-derived viral genes, whereas increasing the antisense-derived Hbz. Our results suggest the oncogenic viral transcripts, tax and hbz, are m6A-modified in cells. We found that these viral RNA modifications are interpreted by reader proteins YTHDF1 and YTHDC1, which dictate the fate of the viral RNA. Understanding HTLV-1 RNA chemical modifications offers potential insights into novel therapeutic strategies for HTLV-1-associated diseases.

Transcriptomic Regulation by Astrocytic m6A Methylation in the mPFC.

Astrocytes, the most prevalent type of glial cells, have been found to play a crucial part in numerous physiological functions. By offering metabolic and structural support, astrocytes are vital for the proper functioning of the brain and regulating information processing and synaptic transmission. Astrocytes located in the medial prefrontal cortex (mPFC) are highly responsive to environmental changes and have been associated with the development of brain disorders. One of the primary mechanisms through which the brain responds to environmental factors is epitranscriptome modification. M6-methyladenosine methylation is the most prevalent internal modification of eukaryotic messenger RNA (mRNA), and it significantly impacts transcript processing and protein synthesis. However, the effects of m6A on astrocyte transcription and function are still not well understood. Our research demonstrates that ALKBH5, an RNA demethylase of m6A found in astrocytes, affects gene expression in the mPFC. These findings suggest that further investigation into the potential role of astrocyte-mediated m6A methylation in the mPFC is warranted.

Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication.

Clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13), an RNA editing technology, has shown potential in combating RNA viruses by degrading viral RNA within mammalian cells. In this study, we demonstrate the effective inhibition of porcine epidemic diarrhoea virus (PEDV) replication and spread using CRISPR-Cas13. We analysed the sequence similarity of the pseudoknot region between PEDV and severe acute respiratory syndrome coronavirus 2, both belonging to the Coronaviridae family, as well as the similarity of the RNA-dependent RNA polymerase (RdRp) gene region among three different strains of the PED virus. Based on this analysis, we synthesized three CRISPR RNAs (crRNAs) targeting the pseudoknot region and the nonpseudoknot region, each for comparison. In cells treated with crRNA #3 targeting the pseudoknot region, RdRp gene expression decreased by 95%, membrane (M) gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid (N) and M protein expression levels decreased by 83 and 98%, respectively. The optimal concentration for high antiviral efficacy without cytotoxicity was determined. Treating cells with 1.5 µg of Cas13b mRNA and 0.5 µg of crRNA resulted in no cytotoxicity while achieving over 95% inhibition of PEDV replication. The Cas13b mRNA therapeutics approach was validated as significantly more effective through a comparative study with merafloxacin, a drug targeting the pseudoknot region of the viral genome. Our results indicate that the pseudoknot region plays a crucial role in the degradation of the PEDV genome through the CRISPR-Cas13 system. Therefore, targeting Cas13b to the pseudoknot offers a promising new approach for treating coronavirus infections.

Writers, readers, and erasers of N6-Methyladenosine (m6A) methylomes in oilseed rape: identification, molecular evolution, and expression profiling

Backgroundm6A RNA modifications are the most prevalent internal modifications in eukaryotic mRNAs and are crucial for plant growth and development, as well as for responses to biotic or abiotic stresses. The modification is catalyzed by writers, removed by erasers, and decoded by various m6A-binding proteins, which are readers. Brassica napus is a major oilseed crop. The dynamic regulation of m6A modifications by writers, erasers, and readers offers potential targets for improving the quality of this crop.ResultsIn this study, we identified 92 m6A-regulatory genes in B. napus, including 13 writers, 29 erasers, and 50 readers. A phylogenetic analysis revealed that they could be further divided into four, three, and two clades, respectively. The distribution of protein motifs and gene structures among members of the same clade exhibited notable similarity. During the course of evolution, whole genome duplication (WGD) and segmental duplication were the primary drivers of the expansion of m6A-related gene families. The genes were subjected to rigorous purification selection. Additionally, several sites under positive selection were identified in the proteins. RNA-seq and quantitative real-time PCR (qRT-PCR) expression analyses revealed that the identified Bnam6As exhibit tissue-specific expression patterns, as well as their expression patterns in response to various abiotic and biotic stresses. The 2000 bp sequence upstream of Bnam6As contained a number of cis-acting elements that regulate plant growth and environmental response. Furthermore, the protein interaction network revealed their interactions with a number of proteins of significant functional importance.ConclusionThe identification of m6A modifiers in oilseed rape and their molecular evolution and expression profiling have revealed potential functions and molecular mechanisms of m6A, thus establishing a foundation for further functional validation and molecular breeding.

Clinical Impact of Cellular Senescence and RNA Dysregulation in HCC Is Associated With MASLD and HCV-SVR.

The incidence of hepatocellular carcinoma (HCC) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatitis C virus sustained virologic response (HCV-SVR) are increasing. However, the mechanisms driving HCC development in these patients remain unclear. This study aimed to evaluate the role of cellular senescence and RNA editing in HCC by examining cyclin-dependent kinase inhibitor 2A (p16) and adenosine deaminase acting on RNA (ADAR1) expression. HCC specimens from patients with MASLD or HCV-SVR were analyzed by immunohistochemistry to assess p16 and ADAR1 expression. Statistical analyses were conducted using the Chi-squared test, Fisher's exact test, and Mann-Whitney U-test. Survival analyses were performed using the Kaplan-Meier method, the log-rank test, and Cox regression analysis. Among 122 patients, 59 (48.4%) had MASLD and 63 (51.6%) had HCV-SVR. p16 expression was observed in 69 cases (57.0%) in the noncancerous areas and 53 cases (44.5%) in the cancerous areas. ADAR1 expression was positive in 28 cases (23.5%) in the cancerous areas and significantly associated with p16 expression in the cancerous areas (p=0.039). Patients with p16 expression in the noncancerous areas were older (p=0.045) and had elevated serum ALT levels (p=0.024). p16 expression in the cancerous areas were correlated with a shorter recurrence-free survival (HR=1.65, 95%CI=1.00-2.73, p=0.046). Cellular senescence and RNA editing may play a key role in MASLD- and HCV-SVR-related HCC. p16 expression in the cancerous areas may serve as a prognostic biomarker for surgical outcomes.

ERNIE-ac4C: A novel deep learning model for effectively predicting N4-acetylcytidine sites.

RNA modifications are known to play a critical role in gene regulation and cellular processes. Specifically, N4-acetylcytidine (ac4C) modification has emerged as a significant marker involved in mRNA translation efficiency, stability, and various diseases. Accurate identification of ac4C modification sites is essential for unraveling its functional implications. However, currently available experimental methods suffer from drawbacks such as lengthy detection times, complexity, and high costs, resulting in low efficiency and accuracy in prediction. Although several bioinformatics methods have been proposed and have advanced the prediction of ac4C modification sites, there is still ample room for improvement. In this research, we propose a novel deep learning model, ERNIE-ac4C, which combines the ERNIE-RNA language model and a two-dimensional Convolutional Neural Network (CNN). ERNIE-ac4C utilizes the fusion of sequence features and attention map features to predict ac4C modification sites. ERNIE-ac4C surpasses other state-of-the-art deep learning methods, demonstrating superior accuracy and effectiveness. The availability of the code on GitHub (https://github.com/lrlbcxdd/ERNIEac4C.git) and our openness to feedback from the research community contribute to the model's accessibility and its potential for further advancements. Our study provides valuable insights into ac4C research and enhances our understanding of the functional consequences of RNA modifications.

NAT10 drives endometriosis progression through acetylation and stabilization of TGFB1 mRNA.

Endometriosis, a gynecological disorder marked by pelvic pain and infertility, has its pathogenesis and pathophysiology significantly influenced by epigenetics, as these factors have been well characterized. However, the role of RNA-mediated epigenetic regulation in endometriosis remains to be elucidated. In our study, we found that N4-acetylcytidine (ac4C) RNA modification and N-acetyltransferase 10 (NAT10) were significantly upregulated in endometrial lesions compared to eutopic endometrium. Knockdown of NAT10 suppressed endometrial epithelial cell proliferation, epithelial-to-mesenchymal transition (EMT), and cell cycle processes in vitro. RNA-seq and acRIP-seq analyses revealed that the knockdown of NAT10 impaired cell proliferation and the TGF-beta signaling pathway. We further identified that ac4C RNA modification enhanced TGFB1 mRNA stability and expression levels, and inhibition of NAT10 activity by Remodelin effectively suppressed the growth of ectopic lesions in an endometriosis mouse model. Collectively, our findings reveal that increased NAT10-mediated ac4C modification enhances TGFB1 mRNA stability, thereby promoting the development of endometriosis. This discovery lays the molecular foundation for future therapeutic approaches targeting endometriosis.

Role of the m6A demethylase ALKBH5 in gastrointestinal tract cancer (Review).

N6‑methyladenosine (m6A) is one of the most universal, abundant and conserved types of internal post‑transcriptional modifications in eukaryotic RNA, and is involved in nuclear RNA export, RNA splicing, mRNA stability, gene expression, microRNA biogenesis and long non‑coding RNA metabolism. AlkB homologue 5 (ALKBH5) acts as a m6A demethylase to regulate a wide variety of biological processes closely associated with tumour progression, tumour metastasis, tumour immunity and tumour drug resistance. ALKBH5 serves a crucial role in human digestive system tumours, mainly through post‑transcriptional regulation of m6A modification. The present review discusses progress in the study of the m6A demethylase ALKBH5 in gastrointestinal tract cancer, summarizes the potential molecular mechanisms of ALKBH5 dysregulation in gastrointestinal tract cancer, and discusses the significance of ALKBH5‑targeted therapy, which may provide novel ideas for future clinical prognosis prediction, biomarker identification and precise treatment.

Exploration of the mechanism of 5-Methylcytosine promoting the progression of hepatocellular carcinoma.

5-Methylcytosine (m5C) is a ubiquitous RNA modification that is closely related to various cellular functions. However, no studies have comprehensively demonstrated the role of m5C in hepatocellular carcinoma (HCC) progression. In this study, six pairs of HCC and adjacent tissue samples were subjected to methylated RNA immunoprecipitation sequencing to identify precise m5C loci. Non-negative matrix factorization (NMF) was used to identify HCC subtypes in TCGA-LIHC cohort. Immune, metabolic, and tumor-related pathways in HCC subtypes with differences in methylation status were analyzed and a prognostic model based on m5C-related genes was constructed. Finally, using RIP and molecular interaction analysis, we demonstrated that YBX1 binds to TPM3 in an m5C dependent manner and regulates HCC progression. Widespread m5C sites were identified and found to be differentially distributed in HCC compared with adjacent tissues. Metabolic processes were inhibited in hypermethylated HCC, whereas immune checkpoint and multiple classical tumor pathways were significantly upregulated. More importantly, we have identified an m5C dependent regulatory axis. The m5C reader YBX1 binds to TPM3 in an M5C dependent manner and promotes the progression of hepatocellular carcinoma. These results provide new evidence for further understanding the comprehensive role of m5C in HCC and the regulatory mechanism of m5C.

Novel insights into the unique characterization of N6-methyladenosine RNA modification and regulating cold tolerance in winter Brassica rapa

Novel insights into the unique characterization of N6-methyladenosine RNA modification and regulating cold tolerance in winter Brassica rapa

CRISPR editing of candidate host factors that impact influenza A virus infection.

Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined three host factors for their contributions to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated the impact on IAV and other RNA viruses. We confirmed that knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. Although ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insights into how host factors can be further explored to understand the dynamics of pro- and anti-viral factors.IMPORTANCEInfluenza A virus (IAV) remains a global threat due to its ability to cause pandemics, making the identification of host factors essential for developing new antiviral strategies. In this study, we utilized CRISPR-based techniques to investigate host factors that impact IAV infectivity. Knockout of CMAS, a key enzyme in sialic acid biosynthesis, significantly reduced IAV binding and infection by disrupting sialic acid production on the cell surface. Overexpression of B4GALNT2 had similar effects, conferring resistance to IAV infection through diminished cell-surface binding. Overexpression of ADAR1, known for its role in RNA editing and immune regulation, impacted IAV replication minimally but enhanced coxsackie B virus replication. Such findings reveal the diverse roles of host factors in viral infection, offering insights for targeted therapeutic development against IAV and other pathogens.

Inhibition of FTO expression by thiodiphenol impairs Leydig cell maturation in pubertal male rats.

This study investigated the multifaceted effects of 4,4'-thiodiphenol (TDP) on male reproductive health, focusing on its toxicity, hormonal modulation, and impact on steroidogenesis. Male rats were exposed to TDP at varying doses (0, 1, 10, and 100 mg/kg) from day 35-56 postpartum. Overt toxicity was evident as TDP significantly reduced inhibited spermatogenesis at 10 and 100 mg/kg. TDP exhibited anti-androgenic properties by inhibiting testosterone biosynthesis at 10 and 100 mg/kg, correlating with increased LH levels at 10 mg/kg and altered T/LH ratios at ≥ 1 mg/kg. Immunohistochemical analysis revealed a dose-dependent increase in Leydig cell (LC) numbers, particularly at 100 mg/kg, attributed to enhanced LC proliferation as evidenced by elevated PCNA labeling. RNA-seq and qPCR analyses demonstrated the impact of TDP on LC gene expression, notably downregulating steroidogenesis-related genes such as Lhcgr, Star, Cyp11a1, and Hsd3b1, and inducing oxidative stress pathway genes. Protein expression studies confirmed reduced levels of key steroidogenic proteins and increased oxidative stress, evidenced by elevated malondialdehyde levels and reduced SOD1 expression. In vitro studies further elucidated the inhibition of TDP on steroidogenesis and induction of reactive oxygen species (ROS), independent of cytotoxicity. Additionally, the modulation of m6A RNA methylation was explored, showing an increase in m6A levels and a reduction in FTO and ALKBH5 expression and overexpression of Fto reversed TDP-mediated suppression of testosterone and steroidogenic and antioxidative genes, suggesting a novel regulatory mechanism involving RNA modification. Collectively, these findings underscore the potential of TDP as an endocrine disruptor with significant implications for male reproductive health.

Rapid and accurate demultiplexing of direct RNA nanopore sequencing datasets with SeqTagger.

Nanopore direct RNA sequencing (DRS) enables direct measurement of RNA molecules, including their native RNA modifications, without prior conversion to cDNA. However, commercial methods for molecular barcoding of multiple DRS samples are lacking, and community-driven efforts, such as DeePlexiCon, are not compatible with newer RNA chemistry flowcells and the latest-generation GPU cards. To overcome these limitations, we introduce SeqTagger, a rapid and robust method that can demultiplex direct RNA sequencing datasets with 99% precision and 95% recall. We demonstrate the applicability of SeqTagger in both RNA002/R9.4 and RNA004/RNA chemistries and show its robust performance both for long and short RNA libraries, including custom libraries that do not contain standard poly(A) tails, such as Nano-tRNAseq libraries. Finally, we demonstrate that increasing the multiplexing up to 96 barcodes yields highly accurate demultiplexing models. SeqTagger can be executed in a standalone manner or through the MasterOfPores NextFlow workflow. The availability of an efficient and simple multiplexing strategy improves the cost-effectiveness of this technology and facilitates the analysis of low-input biological samples.

Advances in RNA editing in hematopoiesis and associated malignancies.

Adenosine-to-inosine (A-to-I) RNA editing is a prevalent RNA modification essential for cell survival. The process is catalyzed by the Adenosine Deaminase Acting on RNA (ADAR) enzyme family that converts adenosines in double-stranded RNAs (dsRNAs) into inosines, which are read as guanosines during translation. Deep sequencing has helped to reveal that A-to-I editing occurs across various types of RNAs to affect their functions. RNA editing detection is now so sophisticated that we can achieve a high level of accuracy and sensitivity to identify low abundance edited events. Consequently, A-to-I editing has been implicated in various biological processes, including immune and stress responses, cancer progression and stem cell fate determination. In particular, a crucial role for this process has been recently reported in hematopoietic cell development and hematologic malignancy progression. Results from genetic mouse models have demonstrated the impact of ADARs' catalytic activity on hematopoietic cells, complemented by insights from human cell studies. Meanwhile clinical studies have implicated ADAR enzymes and RNA editing events in hematologic malignancies and highlighted their potential as prognostic indicators. In this review, we outline the regulatory mechanisms of RNA editing in both normal hematopoiesis and hematologic malignancies. We then speculate on how targeting ADAR expression and site-specific RNA substrates might serve as a therapeutic avenue for affected patients.

M6A -mediated lncRNA SCIRT stability promotes NSCLC progression through binding to SFPQ and activating the PI3K/Akt pathway

Non-small cell lung cancer (NSCLC) has emerged as one of the most prevalent malignancies worldwide. N6-methyladenosine (m6A) methylation, a pervasive epigenetic modification in long noncoding RNAs (lncRNAs), plays a crucial role in NSCLC progression. Here, we report that m6A modification and the expression of the lncRNA stem cell inhibitory RNA transcript (SCIRT) was significantly upregulated in NSCLC tissues and cells. Functional analysis revealed that SCIRT enhanced NSCLC cell proliferation, migration, invasion, and epithelial‒mesenchymal transition. The m6A modification of SCIRT can be installed by METTL3, which enhanced the stability of this lncRNA. Notably, SCIRT overexpression in response to DNA double-strand breaks (DSBs) sensitized cells to camptothecin (CPT) and impairs DNA homologous recombination repair. SCIRT directly interacted with SFPQ in vitro and was primarily localized in the nucleus. Furthermore, ectopic SCIRT expression upregulated SFPQ and activated the PI3K/Akt pathway following CPT treatment, suggesting an unexpected role of SCIRT in facilitating SFPQ-mediated DSB repair. In brief, our findings highlight the oncogenic role of SCIRT in NSCLC by binding SFPQ and activating PI3K/Akt signaling, presenting a promising therapeutic target for personalized NSCLC treatment.