Chemical manipulation of m1A mediates its detection in human tRNA.

N 1-methyl adenosine (m1A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m1A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red-m1A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-GTT and 5.8S rRNA.

Biomarkers for predicting response to aspirin therapy in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin desensitization followed by daily aspirin use is an effective treatment for aspirin-exacerbated respiratory disease (AERD). OBJECTIVE: To assess clinical features as well as genetic, immune, cytological and biochemical biomarkers that might predict a positive response to high-dose aspirin therapy in AERD. METHODS: We enrolled 34 AERD patients with severe asthma who underwent aspirin desensitization followed by 52-week aspirin treatment (650 mg/d). At baseline and at 52 weeks, clinical assessment was performed; phenotypes based on induced sputum cells were identified; eicosanoid, cytokine and chemokine levels in induced sputum supernatant were determined; and induced sputum expression of 94 genes was assessed. Responders to high-dose aspirin were defined as patients with improvement in 5-item Asthma Control Questionnaire score, 22-item Sino-Nasal Outcome Test (SNOT-22) score and forced expiratory volume in 1 second at 52 weeks. RESULTS: There were 28 responders (82%). Positive baseline predictors of response included female sex (p = .002), higher SNOT-22 score (p = .03), higher blood eosinophil count (p = .01), lower neutrophil percentage in induced sputum (p = .003), higher expression of the hydroxyprostaglandin dehydrogenase gene, HPGD (p = .004) and lower expression of the proteoglycan 2 gene, PRG2 (p = .01). The best prediction model included Asthma Control Test and SNOT-22 scores, blood eosinophils and total serum immunoglobulin E. Responders showed a marked decrease in sputum eosinophils but no changes in eicosanoid levels. CONCLUSIONS AND CLINICAL RELEVANCE: Female sex, high blood eosinophil count, low sputum neutrophil percentage, severe nasal symptoms, high HPGD expression and low PRG2 expression may predict a positive response to long-term high-dose aspirin therapy in patients with AERD.

Anterior gradient 2 promotes tumorigenesis through upregulation of CCAAT-enhancer binding protein beta and hypoxia-inducible factor-2α and subsequent secretion of interleukin-6, interleukin-8, and vascular endothelial growth factor in the Caki-1 clear cell renal cell carcinoma cell line.

It has been previously established that hypoxia leads to tumor development, treatment resistance, and a poor prognosis. Under oxygen deprivation, hypoxia-inducible factors (HIFs) are stimulated to activate the genes necessary for tumor development in a low-oxygen environment. These genes encode regulators of angiogenesis, epithelial-mesenchymal transition, and cellular metabolism. A disulfide isomerase, anterior gradient 2 (AGR2), has been shown to increase hypoxia-inducible factor 1, alpha subunit (HIF-1α) stability in breast cancer. Our goal was to determine if AGR2 affects the level of transcription factor hypoxia-inducible factor 2, alpha subunit (HIF-2α). As a model, we used the clear cell renal cell carcinoma (ccRCC) cell line Caki-1. The cells were transduced with lentiviral vector (Tet-On) encoding AGR2. After induction of AGR2 expression, cells were grown under either hypoxic (0.5% O2 ) or normoxic (21% O2 ) conditions. Our data showed that AGR2 upregulated both HIF-1α and HIF-2α expression in Caki-1 cells increasing the expression of HIF-activated genes (glucose transporter 1, phosphoglycerate kinase 1, vascular endothelial growth factor A, and transforming growth factor-alpha) under the hypoxic conditions. Under the normoxic conditions, AGR2 strongly activated CCAAT-enhancer binding protein beta (C/EBPß). Upregulation of C/EBPß correlated with increased expression and secretion of the interleukin-6 and interleukin-8, inducing angiogenesis and inflammation in Caki-1 cells. In summary, our studies revealed that AGR2 has essential functions in ccRCC progression through upregulation of C/EBPß and HIF-2α expressions, which affects cell signaling and metabolism.

Artificial neural network identifies nonsteroidal anti-inflammatory drugs exacerbated respiratory disease (N-ERD) cohort.

BACKGROUND: To date, there has been no reliable in vitro test to either diagnose or differentiate nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (N-ERD). The aim of the present study was to develop and validate an artificial neural network (ANN) for the prediction of N-ERD in patients with asthma. METHODS: This study used a prospective database of patients with N-ERD (n = 121) and aspirin-tolerant (n = 82) who underwent aspirin challenge from May 2014 to May 2018. Eighteen parameters, including clinical characteristics, inflammatory phenotypes based on sputum cells, as well as eicosanoid levels in induced sputum supernatant (ISS) and urine were extracted for the ANN. RESULTS: The validation sensitivity of ANN was 94.12% (80.32%-99.28%), specificity was 73.08% (52.21%-88.43%), and accuracy was 85.00% (77.43%-92.90%) for the prediction of N-ERD. The area under the receiver operating curve was 0.83 (0.71-0.90). CONCLUSIONS: The designed ANN model seems to have powerful prediction capabilities to provide diagnosis of N-ERD. Although it cannot replace the gold-standard aspirin challenge test, the implementation of the ANN might provide an added value for identification of patients with N-ERD. External validation in a large cohort is needed to confirm our results.

Pseudouridine Detection and Quantification Using Bisulfite Incorporation Hindered Ligation.

Pseudouridine (Ψ) is a widespread RNA modification found in various RNA species, including rRNA, tRNA, snRNA, mRNA, and long noncoding RNA (lncRNA). Understanding the function of Ψ in these RNA types requires a robust method for the detection and quantification of the Ψ level at single-nucleotide resolution. A previously used method utilizes Ψ labeling by N-cyclohexyl-N'-ß-(4-methylmorpholinium)ethylcarbodiimide (CMC). The quantification of Ψ is based on the stop ratio after reverse transcription. However, the use of CMC followed by strong alkaline treatment causes severe RNA degradation, often requiring a large amount of RNA. The removal of CMC and recovery of RNA by ethanol precipitation are also time-consuming. Here, we introduce a Bisulfite Incorporation Hindered ligation-based method (BIHIND), which can detect and quantify Ψ sites on rRNA, mRNA, and noncoding RNA. BIHIND can be coupled with quantitative PCR (BIHIND-qPCR) for quantitative detection of Ψ fraction at individual modification sites, as well as with next-generation sequencing (BIHIND-seq) for high-throughput sequencing of Ψ without requiring reverse transcription. We validated the robustness of BIHIND with the elucidation of Ψ dynamics following pseudouridine synthase depletion.

The Role of SOX2 and SOX9 in Radioresistance and Tumor Recurrence.

Head and neck squamous cell carcinoma (HNSCC) exhibits considerable variability in patient outcome. It has been reported that SOX2 plays a role in proliferation, tumor growth, drug resistance, and metastasis in a variety of cancer types. Additionally, SOX9 has been implicated in immune tolerance and treatment failures. SOX2 and SOX9 induce treatment failure by a molecular mechanism that has not yet been elucidated. This study explores the inverse association of SOX2/SOX9 and their distinct expression in tumors, influencing the tumor microenvironment and radiotherapy responses. Through public RNA sequencing data, human biopsy samples, and knockdown cellular models, we explored the effects of inverted SOX2 and SOX9 expression. We found that patients expressing SOX2LowSOX9High showed decreased survival compared to SOX2HighSOX9Low. A survival analysis of patients stratified by radiotherapy and human papillomavirus brings additional clinical relevance. We identified a gene set signature comprising newly discovered candidate genes resulting from inverted SOX2/SOX9 expression. Moreover, the TGF-ß pathway emerges as a significant predicted contributor to the overexpression of these candidate genes. In vitro findings reveal that silencing SOX2 enhances tumor radioresistance, while SOX9 silencing enhances radiosensitivity. These discoveries lay the groundwork for further studies on the therapeutic potential of transcription factors in optimizing HNSCC treatment.

Dysregulation of the Epitranscriptomic Mark m1A in Ischemic Stroke.

Methylation of adenosine at N1 position yields N1-methyladenosine (m1A), which is an epitranscriptomic modification that regulates mRNA metabolism. Recent studies showed that altered m1A methylation promotes acute and chronic neurological diseases. We currently evaluated the effect of focal ischemia on cerebral m1A methylome and its machinery. Adult male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and the peri-infarct cortex was analyzed at 12 h and 24 h of reperfusion. The bulk abundance of m1A was measured by mass spectrometry and dot blot, and transcriptome-wide m1A alterations were profiled using antibody-independent m1A-quant-seq. Expression of the m1A writers and erasers was estimated by real-time PCR. Ischemia significantly decreased m1A levels and concomitantly upregulated m1A demethylase alkB homolog 3 at 24 h of reperfusion compared to sham. Transcriptome-wide profiling showed differential m1A methylation at 14 sites (8 were hypo- and 6 were hypermethylated). Many of those are located in the 3'-UTRs of unannotated transcripts proximal to the genes involved in regulating protein complex assembly, circadian rhythms, chromatin remodeling, and chromosome organization. Using several different approaches, we show for the first time that m1A epitranscriptomic modification in RNA is highly sensitive to cerebral ischemia.

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid leukaemia differentiation.

N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. However, the mechanism by which MTC is recruited to distinct genomic loci remains elusive. Here we identify RBFOX2, a well-studied RNA-binding protein, as a chromatin factor that preferentially recognizes m6A on caRNAs. RBFOX2 can recruit RBM15, an MTC component, to facilitate methylation of promoter-associated RNAs. RBM15 also physically interacts with YTHDC1 and recruits polycomb repressive complex 2 (PRC2) to the RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this RBFOX2/m6A/RBM15/YTHDC1/PRC2 axis plays a critical role in myeloid leukaemia. Downregulation of RBFOX2 notably inhibits survival/proliferation of acute myeloid leukaemia cells and promotes their myeloid differentiation. RBFOX2 is also required for self-renewal of leukaemia stem/initiation cells and acute myeloid leukaemia maintenance. Our study presents a pathway of m6A MTC recruitment and m6A deposition on caRNAs, resulting in locus-selective chromatin regulation, which has potential therapeutic implications in leukaemia.

Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.

N6-methyladenosine dynamics in neurodevelopment and aging, and its potential role in Alzheimer's disease.

BACKGROUND: N6-methyladenosine (m6A) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain. RESULTS: We show that m6A modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, m6A shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more m6A sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more m6A sites compared to mice. Differential m6A sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These m6A sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased m6A methylation in a mouse model of Alzheimer's disease, which is correlated with reduced protein levels. CONCLUSIONS: Our results suggest that m6A exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, m6A controls protein levels of key genes involved in Alzheimer's disease-associated pathways, suggesting that m6A plays an important role in aging and neurodegenerative disease.

METTL3-dependent RNA m6A dysregulation contributes to neurodegeneration in Alzheimer's disease through aberrant cell cycle events.

BACKGROUND: N6-methyladenosine (m6A) modification of RNA influences fundamental aspects of RNA metabolism and m6A dysregulation is implicated in various human diseases. In this study, we explored the potential role of RNA m6A modification in the pathogenesis of Alzheimer disease (AD). METHODS: We investigated the m6A modification and the expression of m6A regulators in the brain tissues of AD patients and determined the impact and underlying mechanism of manipulated expression of m6A levels on AD-related deficits both in vitro and in vivo. RESULTS: We found decreased neuronal m6A levels along with significantly reduced expression of m6A methyltransferase like 3 (METTL3) in AD brains. Interestingly, reduced neuronal m6A modification in the hippocampus caused by METTL3 knockdown led to significant memory deficits, accompanied by extensive synaptic loss and neuronal death along with multiple AD-related cellular alterations including oxidative stress and aberrant cell cycle events in vivo. Inhibition of oxidative stress or cell cycle alleviated shMettl3-induced apoptotic activation and neuronal damage in primary neurons. Restored m6A modification by inhibiting its demethylation in vitro rescued abnormal cell cycle events, neuronal deficits and death induced by METTL3 knockdown. Soluble Aß oligomers caused reduced METTL3 expression and METTL3 knockdown exacerbated while METTL3 overexpression rescued Aß-induced synaptic PSD95 loss in vitro. Importantly, METTL3 overexpression rescued Aß-induced synaptic damage and cognitive impairment in vivo. CONCLUSIONS: Collectively, these data suggested that METTL3 reduction-mediated m6A dysregulation likely contributes to neurodegeneration in AD which may be a therapeutic target for AD.

Sputum biomarkers during aspirin desensitization in nonsteroidal anti-inflammatory drugs exacerbated respiratory disease.

BACKGROUND: Aspirin desensitization (AD) is an effective and safe therapeutic option for patients with nonsteroidal anti-inflammatory drugs (NSAIDs)-exacerbated respiratory disease (N-ERD). The mechanisms driving its beneficial effects remain poorly understood. OBJECTIVE: To investigate the effect of long-term AD on clinical, biochemical and radiological changes in N-ERD patients. METHODS: The study group consisted of twenty-three individuals with N-ERD who underwent AD, followed by ingestion of 325 mg aspirin twice daily. Twenty patients completed the 52 weeks of AD. The following evaluations were conducted at baseline and in the 52nd week of AD: (i) clinical: asthma exacerbations, Asthma Control Test (ACT), Visual Analogue Scale (VAS) for the assessment of nasal symptoms; (ii) blood and induced sputum supernatant (ISS) periostin, (iii) phenotypes based on induced sputum (IS) cells, (iiii) ISS and nasal lavage (NL) concentration of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), tetranor-PGD-M, tetranor-PGE-M, 8-iso-PGE2, leukotriene B4 (LTB4), LTC4, LTD4 and LTE4, and urine LTE4. RESULTS: A significant improvement was observed in ACT (P = 0.02) and VAS score (P = 0.008) in the 52nd week of AD. ISS periostin and IS eosinophil count decreased significantly in the 52nd week of AD (P = 0.04 and P = 0.01, respectively). ISS and NL eicosanoid concentrations did not change following long-term AD. CONCLUSION: and Clinical Relevance: AD is associated with a decrease in sputum periostin biosynthesis, which may prevent the recruitment of eosinophils into respiratory tissue and be one of explanation of the clinical benefits of AD. Long-term aspirin administration does not lead to an imbalance between pro- and anti-inflammatory ISS eicosanoids.