Internal m^(6)A and m^(7)G RNA modifications in hematopoietic system and acute myeloid leukemia

Epitranscriptomics focuses on the RNA-modification-mediated post-transcriptional regulation of gene expression.The past decade has witnessed tremendous progress in our understanding of the landscapes and biological functions of RNA modifications,as prompted by the emergence of potent analytical approaches.The hematopoietic system provides a lifelong supply of blood cells,and gene expression is tightly controlled during the differentiation of hematopoietic stem cells(HSCs).The dysregulation of gene expression during hematopoiesis may lead to severe disorders,including acute myeloid leukemia(AML).Emerging evidence supports the involvement of the mRNA modification system in normal hematopoiesis and AML pathogenesis,which has led to the development of small-molecule inhibitors that target N6-methyladenosine(m^(6)A)modification machinery as treatments.Here,we summarize the latest findings and our most up-to-date information on the roles of m^(6)A and N7-methylguanine in both physiological and pathological conditions in the hematopoietic system.Furthermore,we will discuss the therapeutic potential and limitations of cancer treatments targeting m^(6)A.

Adolescent alcohol exposure changes RNA modifications in adult brain by mass spectrometry-based comprehensive profiling analysis

Alcohol consumption is one of the leading causes of death worldwide.Adolescence is a critical period of structural and functional maturation of the brain.Adolescent alcohol use can alter epigenetic modifications.However,little is known on the long-term effects of alcohol consumption during adolescence on RNA epigenetic modifications in brain.Herein,we systematically explored the long-term effects of alcohol exposure during adolescence on small RNA modifications in adult rat brain tissues by comprehensive liquid chromatography-electrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS)analysis.We totally detected 26 modifications in small RNA of brain tissues.Notably,we observed most of these modifications were decreased in brain tissues.These results suggest that alcohol exposure during adolescence may impose a long-lasting impact on RNA modifications in brain tissues.This is the first report that alcohol use during adolescence can alter RNA modifications in adult brain.Collectively,this study suggests a long-term adverse effects of alcohol consumption on brain from RNA epigenetics angle by comprehensive mass spectrometry analysis.

Chronic sleep deprivation induces alterations in DNA and RNA modifications by liquid chromatography-mass spectrometry analysis

Sleep deprivation(SD)is a widespread issue that disrupts the lives of millions of people.These effects ini-tiate as changes within neurons,specifically at the DNA and RNA level,leading to disruptions in neuronal plasticity and the dysregulation of various cognitive functions,such as learning and memory.Nucleic acid epigenetic modifications that could regulate gene expression have been reported to play crucial roles in this process.However,there is a lack of comprehensive research on the correlation of SD with nucleic acid epigenetic modifications.In the current study,we aimed to systematically investigate the landscape of modifications in DNA as well as in small RNA molecules across multiple tissues,including the heart,liver,kidney,lung,hippocampus,and spleen,in response to chronic sleep deprivation(CSD).Using liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis,we characterized the dynamic changes in DNA and RNA modification profiles in different tissues of mice under CSD stress.Specifically,we ob-served a significant decrease in the level of 5-methylcytosine(5mC)and a significant increase in the level of 5-hydroxymethylcytosine(5hmC)in the kidney in CSD group.Regarding RNA modifications,we observed an overall increased trend for most of these significantly changed modifications across six tis-sues in CSD group.Our study sheds light on the significance of DNA and RNA modifications as crucial epigenetic markers in the context of CSD-induced stress.

RNA modifications in the progression of liver diseases:from fatty liver to cancer

Non-alcoholic fatty liver disease(NAFLD)has emerged as a prominent global health concern associated with high risk of metabolic syndrome,and has impacted a substantial segment of the population.The disease spectrum ranges from simple fatty liver to non-alcoholic steatohepatitis(NASH),which can progress to cirrhosis and hepatocellular carcinoma(HCC)and is increasingly becoming a prevalent indication for liver transplantation.The existing therapeutic options for NAFLD,NASH,and HCC are limited,underscoring the urgent need for innovative treatment strategies.Insights into gene expression,particularly RNA modifications such as N6methyladenosine(m^(6)A),hold promising avenues for interventions.These modifications play integral roles in RNA metabolism and cellular functions,encompassing the entire NAFLD-NASH-HCC progression.This review will encompass recent insights on diverse RNA modifications,including m^(6)A,pseudouridine(Ψ),N^(1)-methyladenosine(m^(1)A),and 5-methylcytidine(m^(5)C)across various RNA species.It will uncover their significance in crucial aspects such as steatosis,inflammation,fibrosis,and tumorigenesis.Furthermore,prospective research directions and therapeutic implications will be explored,advancing our comprehensive understanding of the intricate interconnected nature of these pathological conditions.

Regulations of m^(6)A and other RNA modifications and their roles in cancer

RNA modification is an essential component of the epitranscriptome,regulating RNA metabolism and cellular functions.Several types of RNA modifications have been identified to date;they include N^(6)-methyladenosine(m^(6)A),N^(1)-methyladenosine(m1A),5-methylcytosine(m5C),N^(7)-methylguanosine(m^(7)G),N^(6),2′-O-dimethyladenosine(m^(6)Am),N4-acetylcytidine(ac^(4)C),etc.RNA modifications,mediated by regulators including writers,erasers,and readers,are associated with carcinogenesis,tumor microenvironment,metabolic reprogramming,immunosuppression,immunotherapy,chemotherapy,etc.A novel perspective indicates that regulatory subunits and post-translational modifications(PTMs)are involved in the regulation of writer,eraser,and reader functions in mediating RNA modifications,tumorigenesis,and anticancer therapy.In this review,we summarize the advances made in the knowledge of different RNA modifications(especially m^(6)A)and focus on RNA modification regulators with functions modulated by a series of factors in cancer,including regulatory subunits(proteins,noncoding RNA or peptides encoded by long noncoding RNA)and PTMs(acetylation,SUMOylation,lactylation,phosphorylation,etc.).We also delineate the relationship between RNA modification regulator functions and carcinogenesis or cancer progression.Additionally,inhibitors that target RNA modification regulators for anticancer therapy and their synergistic effect combined with immunotherapy or chemotherapy are discussed.

Dysregulation of RNA modification systems in clinical populations with neurocognitive disorders

The study of modified RNA known as epitranscriptomics has become increasingly relevant in our understanding of disease-modifying mechanisms.Methylation of N6 adenosine(m^(6)A)and C5 cytosine(m^(5)C)bases occur on mRNAs,tRNA,mt-tRNA,and rRNA species as well as non-coding RNAs.With emerging knowledge of RNA binding proteins that act as writer,reader,and eraser effector proteins,comes a new understanding of physiological processes controlled by these systems.Such processes when spatiotemporally disrupted within cellular nanodomains in highly specialized tissues such as the brain,give rise to different forms of disease.In this review,we discuss accumulating evidence that changes in the m^(6)A and m^(5)C methylation systems contribute to neurocognitive disorders.Early studies first identified mutations within FMR1 to cause intellectual disability Fragile X syndromes several years before FMR1 was identified as an m^(6)A RNA reader protein.Subsequently,familial mutations within the m^(6)A writer gene METTL5,m^(5)C writer genes NSUN2,NSUN3,NSUN5,and NSUN6,as well as THOC2 and THOC6 that form a protein complex with the m^(5)C reader protein ALYREF,were recognized to cause intellectual development disorders.Similarly,differences in expression of the m^(5)C writer and reader effector proteins,NSUN6,NSUN7,and ALYREF in brain tissue are indicated in individuals with Alzheimer's disease,individuals with a high neuropathological load or have suffered traumatic brain injury.Likewise,an abundance of m^(6)A reader and anti-reader proteins are reported to change across brain regions in Lewy bodies diseases,Alzheimer's disease,and individuals with high cognitive reserve.m^(6)A-modified RNAs are also reported significantly more abundant in dementia with Lewy bodies brain tissue but significantly reduced in Parkinson's disease tissue,whilst modified RNAs are misplaced within diseased cells,particularly where synapses are located.In parahippocampal brain tissue,m^(6)A modification is enriched in transcripts associated with psychiatric disorders including conditions with clear cognitive deficits.These findings indicate a diverse set of molecular mechanisms are influenced by RNA methylation systems that can cause neuronal and synaptic dysfunction underlying neurocognitive disorders.Targeting these RNA modification systems brings new prospects for neural regenerative therapies.

Multilevel regulation of N^(6)-methyladenosine RNA modifications: Implications in tumorigenesis and therapeutic opportunities

N^(6)-methyladenosine(m^(6)A)RNA modification is widely perceived as the most abundant and common modification in transcripts.This modification is dynamically regulated by specific m^(6)A“writers”,“erasers”and“readers”and is reportedly involved in the occurrence and development of many diseases.Since m^(6)A RNA modification was discovered in the 1970s,with the progress of relevant research technologies,an increasing number of functions of m^(6)A have been reported,and a preliminary understanding of m^(6)A has been obtained.In this review,we summarize the mechanisms through which m^(6)A RNA modification is regulated from the perspectives of expression,posttranslational modification and protein interaction.In addition,we also summarize how external and internal environmental factors affect m^(6)A RNA modification and its functions in tumors.The mechanisms through which m^(6)A methylases,m^(6)A demethylases and m^(6)A-binding proteins are regulated are complicated and have not been fully elucidated.Therefore,we hope to promote further research in this field by summarizing these mechanisms and look forward to the future application of m^(6)A in tumors.

Sequencing methods and functional decoding of mRNA modifications

More than 160 types of post-transcriptional RNA modifications have been reported;there is substantial variation in modification type,abundance,site,and function across species,tissues,and RNA type.The recent development of high-throughput detection technology has enabled identification of diverse dynamic and reversible RNA modifications,including N6,2′-O-dimethyladenosine(m6Am),N1-methyladenosine(m1A),5-methylcytosine(m5C),N6-methyladenosine(m6A),pseudouridine(Ψ),and inosine(I).In this review,we focus on eukaryotic mRNA modifications.We summarize their biogenesis,regulatory mechanisms,and biological functions,as well as highthroughput methods for detection of mRNA modifications.We also discuss challenges that must be addressed in mRNA modification research.

Characterization of N6-methyladenosine long non-coding RNAs in sporadic congenital cataract and age-related cataract

AIM:To characterize the N6-methyladenosine(m6A)modification patterns in long non-coding RNAs(lncRNAs)in sporadic congenital cataract(CC)and age-related cataract(ARC).METHODS:Anterior capsule of the lens were collected from patients with CC and ARC.Methylated RNA immunoprecipitation with next-generation sequencing and RNA sequencing were performed to identify m6A-tagged lncRNAs and lncRNAs expression.Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and Gene Ontology annotation were used to predict potential functions of the m6A-lncRNAs.RESULTS:Large amount of m6A peaks within lncRNA were identified for both CC and ARC,while the level was much higher in ARC(49870 peaks)than that in CC(18688 peaks),yet those difference between ARC in younger age group(ARC-1)and ARC in elder age group(ARC-2)was quite slight.A total of 1305 hypermethylated and 1178 hypomethylated lncRNAs,as well as 182 differential expressed lncRNAs were exhibited in ARC compared with CC.On the other hand,5893 hypermethylated and 5213 hypomethylated lncRNAs,as well as 155 significantly altered lncRNA were identified in ARC-2 compared with ARC-1.Altered lncRNAs in ARC were mainly associated with the organization and biogenesis of intracellular organelles,as well as nucleotide excision repair.CONCLUSION:Our results for the first time present an overview of the m6A methylomes of lncRNA in CC and ARC,providing a solid basis and uncovering a new insight to reveal the potential pathogenic mechanism of CC and ARC.

Epitranscriptomics:Toward A Better Understanding of RNA Modifications

Ever since the first RNA nucleoside modification was charac- terized in 1957 [1], over 100 distinct chemical modifications have been identified in RNA to date [2]. Most of these modi- fications were characterized in non-coding RNAs （ncRNAs）, including tRNA, rRNA, and small nuclear RNA （snRNA） [3]. Studies in the past few decades have located various mod- ifications in these ncRNAs and revealed their functional roles [3]. For instance, NLmethyladenosine （mlA）, which is typically found at position 58 in the tRNA T-loop of eukaryotes, func- tions to stabilize tRNA tertiary structure [4] and affect transla- tion by regulating the associations between tRNA and polysome [5]. Pseudouridine （tp） in snRNA can fine-tune branch site interactions and affect mRNA splicing [6].

Special Issue on"RNA Modifications and Epitranscriptomics"

We are very pleased to announce a special issue,to be published in the Spring of 2022,on"RNA Modifications and Epitranscriptomics"in the journal Genomics,Proteomics&Bioinformatics(GPB).More than 100 distinct chemical modifications to RNA have been characterized so far.They are prevalent in non-coding RNA,including rRNA,tRNA,and small nuclear RNA(snRNA).

The occurrence order and cross-talk of different tRNA modifications

Chemical modifications expand the composition of RNA molecules from four standard nucleosides to over 160 modified nucleosides,which greatly increase the complexity and utility of RNAs.Transfer RNAs(tRNAs)are the most heavily modified cellular RNA molecules and contain the largest variety of modifications.Modification of tRNAs is pivotal for protein synthesis and also precisely regulates the noncanonical functions of tRNAs.Defects in tRNA modifications lead to numerous human diseases.Up to now,more than 100 types of modifications have been found in tRNAs.Intriguingly,some modifications occur widely on all tRNAs,while others only occur on a subgroup of tRNAs or even only a specific tRNA.The modification frequency of each tRNA is approximately 7% to 25%,with 5-20 modification sites present on each tRNA.The occurrence and modulation of tRNA modifications are specifically noticeable as plenty of interplays among different sites and modifications have been discovered.In particular,tRNA modifications are responsive to environmental changes,indicating their dynamic and highly organized nature.In this review,we summarized the known occurrence order,cross-talk,and cooperativity of tRNA modifications.

Adolescent alcohol exposure alters DNA and RNA modifications in peripheral blood by liquid chromatography-tandem mass spectrometry analysis

Alcohol consumption is a critical risk factor contributing to a verity of human diseases. The incidence of alcohol use disorder increases across adolescence in recent years. Accumulating line of evidence suggests that alcohol-induced changes of DNA cytosine methylation(5-methyl-2-deoxycytidine, 5 m C) in genomes play an important role in the development of diseases. However, systemic investigation of the effects of adolescent alcohol exposure on DNA and RNA modifications is still lacked. Especially, there hasn’t been any report to study the effects of alcohol exposure on RNA modifications. Similar to DNA modifications,RNA modifications recently have been identified to function as new regulators in modulating numbers of biological processes. In the current study, we systematically investigated the effects of alcohol exposure on both DNA and RNA modifications in peripheral blood of adolescent rats by liquid chromatographyelectrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS) analysis. The developed LC-ESI-MS/MS method enabled the sensitive and accurate determination of 2 DNA modifications and 12 RNA modifications. As for the alcohol exposure experiments, the adolescent rats were intraperitoneally injected with ethanol with an interval of one day for a total 14 days. The quantification results by LC-ESI-MS/MS analysis showed that adolescent alcohol exposure could alter both DNA and RNA modifications in peripheral blood. Specifically, we observed an overall decreased trend of RNA modifications. The discovery of the significant alteration of the levels of DNA and RNA modifications under alcohol exposure indicates that alcohol consumption may increase the risk of the incidence and development of diseases through dysregulating DNA and RNA modifications.

Comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by liquid chromatography-tandem mass spectrometry

RNA molecules contain diverse modifications that display important functions in a variety of physiological and pathological processes.So far over 150 chemical modifications have been characterized to be present in various RNA species,such as in messenger RNA(mRNA),ribosomal RNA(rRNA),and transfer RNA(tRNA).Previous studies revealed that certain RNA modifications were correlated to specific human diseases,indicating RNA modifications could serve as the potential indicator of human diseases.However,systemic investigation of the alteration of RNA modifications in different RNA species of carcinoma tissues are still lacked.Herein,we carried out the comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by liquid chromatography-tandem mass spectrometry(LC-ESIMS/MS)analysis.The developed method allowed us to simultaneously detect 48 different types of RNA modifications.Using this method,we detected 10,15,14,and 25 modifications in m RNA,18 S r RNA,28 S rRNA and small RNA(<200 nt),respectively.Compared to the normal tissues,we revealed a total of 14 RNA modification exhibited significant increase and 2 RNA modifications showed significant decrease in thyroid carcinoma tissues.Our study provided the first comprehensive profile as well as the alteration of modifications in different RNA species in thyroid carcinoma and matched tumor-adjacent normal tissues.The altered pattern RNA modifications may serve as the indicator of thyroid carcinoma.Moreover,this study may promote the in-depth understanding of the regulatory roles of RNA modifications in thyroid carcinoma.

The landscape of epigenetic regulation and therapeutic application of N^(6)-methyladenosine modifications in non-coding RNAs

RNA N^(6)-methyladenosine(m^(6)A)methylation is the most abundant and conserved RNA modification in eukaryotes.It participates in the regulation of RNA metabolism and various pathophysiological processes.Non-coding RNAs(ncRNAs)are defined as small or long transcripts which do not encode proteins and display numerous biological regulatory functions.Similar to mRNAs,m^(6)A deposition is observed in ncRNAs.Studying RNA m^(6)A modifications on ncRNAs is of great importance specifically to deepen our understanding of their biological roles and clinical implications.In this review,we summarized the recent research findings regarding the mutual regulation between RNA m^(6)A modification and ncRNAs(with a specific focus on microRNAs,long non-coding RNAs,and circular RNAs)and their functions.We also discussed the challenges of m^(6)A-containing ncRNAs and RNA m^(6)A as therapeutic targets in human diseases and their future perspective in translational roles.

RNA modification by M6A methylation in cardiovascular diseases: Current trends and future directions

N6-methyladenosine(M6A)is the most common modification in eukaryotic RNAs for the regulation of RNA transcription,processing,splicing,degradation,and translation.RNA modification by M6A is dynamically reversible,involving methylated transferase,demethylase,and methylated reading protein.M6A-mediated gene regulation involves cell differentiation,metastasis,apoptosis,and proliferation.Dysregulation of M6A can lead to various diseases.Cardiovascular disease(CVD)seriously endangers human health and brings great social burden.Seeking effective prevention and treatment strategies for CVD is a challenge to both fundamentalists and clinicians.Substantial evidence has suggested the key role of M6A modification in the development of CVDs.This review summarizes the mechanism of M6A RNA modification and the latest research progress in respect with its role in CVDs,including atherosclerosis,coronary artery disease,myocardial infarction and cardiac remodeling,myocardial ischemia-reperfusion injury,heart failure,hypertension,and aortic aneurysm,and the potential applications of the findings to CVDs,thereby providing new ideas and approaches for the diagnosis and therapy of CVDs.

A Bioorthogonal Post-modification of N^(6)-Isopentenyl Adenosine i^(6) A RNA in Live Cells

Selective methods for modulating RNA epigenetic modifications within living cells and organisms represent attractive techniques for investigating biological functions and medicinal application.In contrast to enzymatic methods,abiotic chemical modulation offers access to diverse new-to-natural functionalities.We herein report a visible light-assisted bioorthogonal reaction involving flavin mononucleotide,sodium azide,and blue light irradiation.In concert with previous chemical approaches mimicking the demodification pathway,our system functions as a powerful and selective post-modification enzyme that targets N^(6)-prenyl adenosine i^(6) A in RNA and enables the efficient construction of an artificial N^(6)-cyanomethyl adenosine(cnm^(6) A).Notably,most endogenous biomacromolecular functions,including other common RNA epigenetics,remained unaffected.Consequently,we have successfully modified i^(6) A in tRNA to cnm^(6) A in human cells and establish this system as a bona fide bioorthogonal reaction with potential applications in chemical biology and in-cell modulation.

Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs(rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation.In ribosome biogenesis,rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes.However,rRNA modifications in plants have yet to be fully defined.Herein,we proposed a method to purify rRNAs by a successive isolation with different strategies,including poly A-based m RNA depletion and agarose gel electrophoresis-based purification,with which highly pure rRNAs could be obtained.In addition,we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA.LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA,respectively.Notably,among these identified modifications,2 kinds of modifications of N^(2),N^(2)-dimethylguanosine(m^(2,2)G)and N^(6),N^(6)-dimethyladenosine(m^(6,6)A) in 18S rRNA,and 4 kinds of modifications of m^(2,2)G,m^(6,6)A,N7-methylguanosine(m^(7)G) and 3-methyluridin(m^(3)U) in 25S rRNA,were first reported to be present in plants.Moreover,exposure of Arabidopsis thaliana to cadmium(Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants,indicating that rRNA modifications play important roles in response to environmental stress.The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.

Complicated target recognition by archaeal box C/D guide RNAs

Box C/D RNAs guide the site-specific formation of 2′-O-methylated nucleotides(Nm)of RNAs in eukaryotes and archaea.Although C/D RNAs have been profiled in several archaea,their targets have not been experimentally determined.Here,we mapped Nm in r RNAs,t RNAs,and abundant small RNAs(s RNAs)and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus.The targets of C/D RNAs were assigned by analysis of base-pairing interactions,in vitro modification assays,and gene deletion experiments,revealing a complicated landscape of C/D RNA-target interactions.C/D RNAs widely use dual antisense elements to target adjacent sites in r RNAs,enhancing modification at weakly bound sites.Two consecutive sites can be guided with the same antisense element upstream of box D or D′,a phenomenon known as doublespecificity that is exclusive to internal box D′in eukaryotic C/D RNAs.Several C/D RNAs guide modification at a single non-canonical site.This study reveals the global landscape of RNA-guided 2′-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA.

The RNA m^(6)A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression

Emergency granulopoiesis and neutrophil mobilization that can be triggered by granulocyte colony-stimulating factor(G-CSF)through its receptor G-CSFR are essential for antibacterial innate defense.However,the epigenetic modifiers crucial for intrinsically regulating G-CSFR expression and the antibacterial response of neutrophils remain largely unclear.N6-methyladenosine(m^(6)A)RNA modification and the related demethylase alkB homolog 5(ALKBH5)are key epigenetic regulators of immunity and inflammation,but their roles in neutrophil production and mobilization are still unknown.We used cecal ligation and puncture(CLP)-induced polymicrobial sepsis to model systemic bacterial infection,and we report that ALKBH5 is required for emergency granulopoiesis and neutrophil mobilization.ALKBH5 depletion significantly impaired the production of immature neutrophils in the bone marrow of septic mice.In addition,Alkbh5-deficient septic mice exhibited higher retention of mature neutrophils in the bone marrow and defective neutrophil release into the circulation,which led to fewer neutrophils at the infection site than in their wild-type littermates.During bacterial infection,ALKBH5 imprinted production-and mobilization-promoting transcriptome signatures in both mouse and human neutrophils.Mechanistically,ALKBH5 erased m^(6)A methylation on the CSF3R mRNA to increase the mRNA stability and protein expression of G-CSFR,consequently upregulating cell surface G-CSFR expression and downstream STAT3 signaling in neutrophils.The RIP-qPCR results confirmed the direct binding of ALKBH5 to the CSF3R mRNA,and the binding strength declined upon bacterial infection,accounting for the decrease in G-CSFR expression on bacteria-infected neutrophils.Considering these results collectively,we define a new role of ALKBH5 in intrinsically driving neutrophil production and mobilization through m^(6)A demethylation-dependent posttranscriptional regulation,indicating that m^(6)A RNA modification in neutrophils is a potential target for treating bacterial infections and neutropenia.