Deciphering the epitranscriptome： A green perspective

The advent of high-throughput sequencing technol- ogies coupled with new detection methods of RNA modifica- tions has enabled investigation of a new layer of gene regulation - the epitranscriptome. With over loo known RNA modifications, understanding the repertoire of RNA modifications is a huge undertaking. This review summarizes what is known about RNA modifications with an emphasis on discoveries in plants. RNA ribose modifications, base methyl- ations and pseudouridylation are required for normal develop- ment in Arabidopsis, as mutations in the enzymes modifying them have diverse effects on plant development and stress responses. These modifications can regulate RNA structure, turnover and translation. Transfer RNA and ribosomal RNA modifications have been mapped extensively and their functions investigated in many organisms, including plants. Recent work exploring the locations, functions and targeting of N6-methyladenosine （m^6A）, 5-methylcytosine （m^5C）, pseudour- idine （up）, and additional modifications in mRNAs and ncRNAs are highlighted, as well as those previously known on tRNAs and rRNAs. Many questions remain as to the exact mechanisms of targeting and functions of specific modified sites and whether these modifications have distinct functions in the different classes of RNAs.

Analysis of N6-methyladenosine-modified mRNAs in diabetic cataract

BACKGROUND Cataracts remain a prime reason for visual disturbance and blindness all over the world,despite the capacity for successful surgical replacement with artificial lenses.Diabetic cataract(DC),a metabolic complication,usually occurs at an earlier age and progresses faster than age-related cataracts.Evidence has linked N6-methyladenosine(m6A)to DC progression.However,there exists a lack of understanding regarding RNA m6A modifications and the role of m6A in DC pathogenesis.AIM To elucidate the role played by altered m6A and differentially expressed mRNAs(DEmRNAs)in DC.METHODS Anterior lens capsules were collected from the control subjects and patients with DC.M6A epitranscriptomic microarray was performed to investigate the altered m6A modifications and determine the DEmRNAs.Through Gene Ontology and pathway enrichment(Kyoto Encyclopedia of Genes and Genomes)analyses,the potential role played by dysregulated m6A modification was predicted.Real-time polymerase chain reaction was further carried out to identify the dysregulated expression of RNA methyltransferases,demethylases,and readers.RESULTS Increased m6A abundance levels were found in the total mRNA of DC samples.Bioinformatics analysis predicted that ferroptosis pathways could be associated with m6A-modified mRNAs.The levels of five methylation-related genes-RBM15,WTAP,ALKBH5,FTO,and YTHDF1-were upregulated in DC samples.Upregulation of RBM15 expression was verified in SRA01/04 cells with high-glucose medium and in samples from DC patients.CONCLUSION M6a mRNA modifications may be involved in DC progression via the ferroptosis pathway,rendering novel insights into therapeutic strategies for DC.

The m^(6)A reader YTHDC2 maintains visual function and retinal photoreceptor survival through modulating translation of PPEF2 and PDE6B

Inherited retinal dystrophies (IRDs) are major causes of visual impairment and irreversible blindness worldwide, while the precise molecular and genetic mechanisms are still elusive. N6-methyladenosine (m^(6)A) modification is the most prevalent internal modification in eukaryotic mRNA. YTH domain containing 2 (YTHDC2), an m^(6)A reader protein, has recently been identified as a key player in germline development and human cancer. However, its contribution to retinal function remains unknown. Here, we explore the role of YTHDC2 in the visual function of retinal rod photoreceptors by generating rod-specific Ythdc2 knockout mice. Results show that Ythdc2 deficiency in rods causes diminished scotopic ERG responses and progressive retinal degeneration. Multi-omics analysis further identifies Ppef2 and Pde6b as the potential targets of YTHDC2 in the retina. Specifically, via its YTH domain, YTHDC2 recognizes and binds m^(6)A-modified Ppef2 mRNA at the coding sequence and Pde6b mRNA at the 5′-UTR, resulting in enhanced translation efficiency without affecting mRNA levels. Compromised translation efficiency of Ppef2 and Pde6b after YTHDC2 depletion ultimately leads to decreased protein levels in the retina, impaired retinal function, and progressive rod death. Collectively, our finding highlights the importance of YTHDC2 in visual function and photoreceptor survival, which provides an unreported elucidation of IRD pathogenesis via epitranscriptomics.

RNA Methylome Reveals the m^(6)A-mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-withering

The epitranscriptomic mark N6-methyladenosine(m^(6)A),which is the predominant internal modification in RNA,is important for plant responses to diverse stresses.Multiple environmental stresses caused by the tea-withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor.However,the effects of the m^(6)A-mediated regulatory mechanism on flavor-related metabolic pathways in tea leaves remain relatively uncharacterized.We performed an integrated RNA methylome and transcriptome analysis to explore the m^(6)Amediated regulatory mechanism and its effects on flavonoid and terpenoid metabolism in tea(Camellia sinensis)leaves under solar-withering conditions.Dynamic changes in global m^(6)A level in tea leaves were mainly controlled by two m^(6)A erasers(CsALKBH4A and CsALKBH4B)during solar-withering treatments.Differentially methylated peak-associated genes following solarwithering treatments with different shading rates were assigned to terpenoid biosynthesis and spliceosome pathways.Further analyses indicated that CsALKBH4-driven RNA demethylation can directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related transcripts and also indirectly influence the flavonoid,catechin,and theaflavin contents by triggering alternative splicing-mediated regulation.Our findings revealed a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a link between the m^(6)A-mediated regulatory mechanism and the formation of tea flavor under solar-withering conditions.

Single-base resolution mapping of 2′-O-methylation sites by an exoribonuclease-enriched chemical method

2′-O-methylation(Nm)is one of the most abundant RNA epigenetic modifications and plays a vital role in the post-transcriptional regulation of gene expression.Current Nm mapping approaches are normally limited to highly abundant RNAs and have significant technical hurdles in m RNAs or relatively rare non-coding RNAs(nc RNAs).Here,we developed a new method for enriching Nm sites by using RNA exoribonuclease and periodate oxidation reactivity to eliminate 2′-hydroxylated(2′-OH)nucleosides,coupled with sequencing(Nm-REP-seq).We revealed several novel classes of Nm-containing nc RNAs as well as m RNAs in humans,mice,and drosophila.We found that some novel Nm sites are present at fixed positions in different t RNAs and are potential substrates of fibrillarin(FBL)methyltransferase mediated by sno RNAs.Importantly,we discovered,for the first time,that Nm located at the 3′-end of various types of nc RNAs and fragments derived from them.Our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.

Detection,regulation,and functions of RNA N^(6)-methyladenosine modification in plants

N6-Methyladenosine(m^(6)A)is the most abundant internal chemical modification in eukaryotic mRNA and plays important roles in gene expression regulation,including transcriptional and post-transcriptional regulation.m^(6)A is a reversible modification that is installed,removed,and recognized by methyltransferases(writers),demethylases(erasers),and m^(6)A-binding proteins(readers),respectively.Recently,the breadth of research on m^(6)A in plants has expanded,and the vital roles of m^(6)A in plant development,biotic and abiotic stress responses,and crop trait improvement have been investigated.In this review,we discuss recent developments in research on m^(6)A and highlight the detection methods,distribution,regulatory proteins,and molecular and biological functions of m^(6)A in plants.We also offer some perspectives on future investigations,providing direction for subsequent research on m^(6)A in plants.

Transcriptome-wide profiling of RNA N^(4)-cytidine acetylation in Arabidopsis thaliana and Oryza sativa

Acetylation of N^(4)-cytidine(ac^(4)C)has recently been discovered as a novel modification of mRNA.RNA ac^(4)C modification has been shown to be a key regulator of RNA stability,RNA translation,and the thermal stress response.However,its existence in eukaryotic mRNAs is still controversial.In plants,the existence,distribution pattern,and potential function of RNA ac^(4)C modification are largely unknown.Here we report the presence of ac^(4)C in the mRNAs of both Arabidopsis thaliana and rice(Oryza sativa).By comparing two ac^(4)C sequencing methods,we found that RNA immunoprecipitation and sequencing(acRIP-seq),but not ac^(4)C sequencing,was suitable for plant RNA ac^(4)C sequencing.We present transcriptome-wide atlases of RNA ac^(4)C modification in A.thaliana and rice mRNAs obtained by acRIP-seq.Analysis of the distribution of RNA ac^(4)C modifications showed that ac^(4)C is enriched near translation start sites in rice mRNAs and near translation start sites and translation end sites in Arabidopsis mRNAs.The RNA ac^(4)C modification level is positively correlated with RNA half-life and the number of splicing variants.Similar to that in mammals,the translation efficiency of ac^(4)C target genes is significantly higher than that of other genes.Our in vitro translation results confirmed that RNA ac^(4)C modification enhances translation efficiency.We also found that RNA ac^(4)C modification is negatively correlated with RNA structure.These results suggest that ac^(4)C is a conserved mRNA modification in plants that contributes to RNA stability,splicing,translation,and secondary structure formation.

Emerging Regulatory Mechanisms of N6-Methyladenosine Modification in Cancer Metastasis

Cancer metastasis is the major cause of cancer-related deaths and accounts for poor therapeutic outcomes.A metastatic cas-cade is a series of complicated biological processes.N6-methyladenosine(m^(6)A)is the most abundant and conserved epi-transcriptomic modification in eukaryotic cells,which has great impacts on RNA production and metabolism,including RNA splicing,processing,degradation and translation.Accumulating evidence demonstrates that m^(6)A plays a critical role in regulating cancer metastasis.However,there is a lack of studies that review the recent advances of m^(6)A in cancer metastasis.Here,we systematically retrieved the functions and mechanisms of how the m^(6)A axis regulates metastasis,and especially summarized the organ-specific liver,lung and brain metastasis mediated by m^(6)A in various cancers.Moreover,we discussed the potential application of m^(6)A modification in cancer diagnosis and therapy,as well as the present limitations and future perspectives of m^(6)A in cancer metastasis.This review provides a comprehensive knowledge on the m^(6)A-mediated regulation of gene expression,which is helpful to extensively understand the complexity of cancer metastasis from a new epitranscriptomic point of view and shed light on the developing novel strategies to anti-metastasis based on m^(6)A alteration.

YTH Domain： A Family of N^6-methyladenosine (m^6A) Readers

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N^6-methyladenosine （m^6A） is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs （lncRNAs）. In mammalian cells, m^6A can be incorporated by a methyltransferase complex and removed by demethy- lases, which ensures that the m^6A modification is reversible and dynamic. Moreover, m^6A is recognized by the YT521-B homology （YTH） domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the m^6A recognition by YTH domaincontaining proteins, which would shed new light on m^6A-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.

Reversible RNA Modification N^1-methyladenosine(m^1A) in mRNA and tRNA

More than 100 modifications have been found in RNA. Analogous to epigenetic DNA methylation, epitranscriptomic modifications can be written, read, and erased by a complex network of proteins. Apart from Na-methyladenosine （m6A）, N1-methyladenosine （mXA） has been found as a reversible modification in tRNA and mRNA. mlA occurs at positions 9, 14, and 58 of tRNA, with m1A58 being critical for tRNA stability. Other than the hundreds of m1A sites in mRNA and long non-coding RNA transcripts, transcriptome-wide mapping of m1A also identifies 〉 20 m1A sites in mitochondrial genes, m1A in the coding region of mitochondrial transcripts can inhibit the translation of the corresponding proteins. In this review, we summarize the current understanding of mlA in mRNA and tRNA, covering high-throughput sequencing methods developed for m1A methylome, m1A-related enzymes （writers and erasers）, as well as its functions in mRNA and tRNA.

Comparative Analysis of Human Genes Frequently and Occasionally Regulated by m^6A Modification

The m^6A modification has been implicated as an important epitranscriptomic marker, which plays extensive roles in the regulation of transcript stability, splicing, translation, and localization. Nevertheless, only some genes are repeatedly modified across various conditions and the principle of m^6A regulation remains elusive. In this study, we performed a systems-level analysis of human genes frequently regulated by m^6A modification （m^6Afreq genes） and those occasionally regulated by m^6A modification （m^6Aocca genes）. Compared to the m^6Aocca genes, the m^6Afreq genes exhibit gene importance-related features, such as lower dN/dS ratio, higher protein-protein interaction network degree, and reduced tissue expression specificity. Signaling network analysis indicates that the m^6Afreq genes are associated with downstream components of signaling cascades, high-linked signaling adaptors, and specific network motifs like incoherent feed forward loops. Moreover, functional enrichment analysis indicates significant overlaps between the m^6Afreq genes and genes involved in various layers of gene expression, such as being the microRNA targets and the regulators of RNA processing. Therefore, our findings suggest the potential interplay between m^6A epitranscriptomic regulation and other gene expression regulatory machineries.

The RNA Modification N^6-methyladenosine and Its Implications in Human Disease

Impaired gene regulation lies at the heart of many disorders, including developmental diseases and cancer. Furthermore, the molecular pathways that control gene expression are often the target of cellular parasites, such as viruses. Gene expression is controlled through multiple mechanisms that are coordinated to ensure the proper and timely expression of each gene. Many of these mechanisms target the life cycle of the RNA molecule, from transcription to translation. Recently, another layer of regulation at the RNA level involving RNA modifications has gained renewed interest of the scientific community. The discovery that N6-methyladenosine （m6A）, a mod- ification present in mRNAs and long noncoding RNAs, can be removed by the activity of RNA demethylases, launched the field of epitranscriptomics; the study of how RNA function is regulated through the addition or removal of post-transcriptional modifications, similar to strategies used to regulate gene expression at the DNA and protein level. The abundance of RNA post-transcriptional modifications is determined by the activity of writer complexes （methylase） and eraser （RNA demethylase） proteins. Subsequently, the effects of RNA modifications materialize as changes in RNA structure and/or modulation of interactions between the modified RNA and RNA binding proteins or regulatory RNAs. Disruption of these pathways impairs gene expression and cellular function. This review focuses on the links between the RNA modification m6A and its implications in human diseases.

Mapping the epigenetic modifications of DNA and RNA

Over 17 and 160 types of chemical modifications have been identified in DNA and RNA,respectively.The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics.Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study.Here,we review the recent technological advances in this rapidly evolving field.We focus on high-throughput detection methods and biological findings for these modifications,and discuss questions to be addressed as well.We also summarize third-generation sequencing methods,which enable long-read and single-molecule sequencing of DNA and RNA modification.

Structural Insights into N^6-methyladenosine (m^6A) Modification in the Transcriptome

More than 100 types of chemical modifications in RNA have been well documented. Recently, several modifications, such as N6-methyladenosine （m^6A）, have been detected in mRNA, opening the window into the realm of epitranscriptomies. The m^6A modification is the most abundant modification in mRNA and non-coding RNA （ncRNA）. At the molecular level, m^6A affects almost all aspects of mRNA metabolism, including splicing, translation, and stability, as well as microRNA （miRNA） maturation, playing essential roles in a range of cellular processes. The m^6A modification is regulated by three classes of proteins generally referred to as the ＂writer＂ （adenosine methyltransferase）, ＂eraser＂ （m^6A demethylating enzyme）, and ＂reader＂ （m^6A-binding protein）. The m^6A modification is reversibly installed and removed by writers and erasers, respectively. Readers, which are members of the YT521-B homology （YTH） family proteins, selectively bind to RNA and affect its fate in an m^6A-dependent manner. In this review, we summarize the structures of the functional proteins that modulate the m^6A modification, and provide our insights into the m^6A-mediated gene regulation.

Arab/c/opste N^(6)-methyladenosine reader CPSF30-L recognizes FUE signals to control polyadenylation site choice in liquid-like nuclear bodies

The biological functions of the epitranscriptomic modification N^(6)-methyladenosine(m^(6)A)in plants are not fully understood.CPSF30-L is a predominant isoform of the polyadenylation factor CPSF30 and consists of CPSF30-S and an m^(6)A-binding YTH domain.Little is known about the biological roles of CPSF30-L and the molecular mechanism underlying its m^(6)A-binding function in alternative polyadenylation.Here,we charac-terized CPSF30-L as an Arabidopsis m^(6)A reader whose m^(6)A-binding function is required for the floral tran-sition and abscisic acid(ABA)response.We found that the m^(6)A-binding activity of CPSF30-L enhances the formation of liquid-like nuclear bodies,where CPSF30-L mainly recognizes m*A-modified far-upstream elements to control polyadenylation site choice.Deficiency of CPSF30-L lengthens the 3'untranslated region of three phenotypes-related transcripts,thereby accelerating their mRNA degradation and leading to late flowering and ABA hypersensitivity.Collectively,this study uncovers a new molecular mechanism for m^(6)A-driven phase separation and polyadenylation in plants.

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).