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.

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.

RNA Modifications and Epitranscriptomics

More than 170 distinct chemical modifications have been identified in non-coding and coding RNAs.Accumulating evidence suggests that RNA modifications play pivotal roles at both the molecular and physiological levels.Dysregulation of RNAmodifying enzymes has been linked to various human cancers and developmental diseases.The expanding understanding of RNA modifications in molecular and cellular functions further suggests promising prospects for therapeutic applications.Recently,the creation of effective mRNA vaccines against coronavirus disease 2019(COVID-19),based on RNA base modification,was honored with the Nobel Prize in Physiology or Medicine 2023(https://www.nobelprize.org/prizes/medicine/2023/press-release/).Aiming to provide a forum for emerging advances in detection and functional studies of epitranscriptomic modifications,we have organized a special issue"RNA Modifications and Epitranscriptomics"for the journal Genomics,Proteomics&Bioinformatics(GPB).This special issue encompasses a wide range of topics,including:(1)dynamic landscapes of RNA modifications in various organisms,including animals,plants,and viruses;(2)mechanistic regulation of m^(6)A and m5 C modifications in human diseases and plant responses to stresses;(3)an online platform for unveiling the context-specific m^(6)A methylation and m^(6)Aaffecting mutation;and(4)the regulatory role of non-coding RNAs(ncRNAs),including tRNAs and circular RNAs(circRNAs),in gene expression regulation.

Epitranscriptomic technologies and analyses

RNA can interact with RNA-binding proteins(RBPs),mRNA,or other non-coding RNAs(ncRNAs)to form complex regulatory networks.High-throughput CLIP-seq,degradome-seq,and RNA-RNA interactome sequencing methods represent powerful approaches to identify biologically relevant ncRNA-target and protein-ncRNA interactions.However,assigning ncRNAs to their regulatory target genes or interacting RNA-binding proteins(RBPs)remains technically challenging.Chemical modifications to mRNA also play important roles in regulating gene expression.Investigation of the functional roles of these modifications relies highly on the detection methods used.RNA structure is also critical at nearly every step of the RNA life cycle.In this review,we summarize recent advances and limitations in CLIP technologies and discuss the computational challenges of and bioinformatics tools used for decoding the functions and regulatory networks of ncRNAs.We also summarize methods used to detect RNA modifications and to probe RNA structure.

Transformation of 5-Carboxylcytosine to Cytosine Through C-C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Active demethylation of 5-methylcytosine(5mC)can be realized through ten-eleven translocation(TET)dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC),and 5-carboxylcytosine(5caC),followed by thymine DNA glycosylase(TDG)-initiated base excision repair(BER).The TDG-BER pathwaymay lead to the generation of DNA strand breaks,potentially compromising genome integrity.Alternatively,direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks.However,cleavage of the C–C bond in 5caC in human cells remains an open question.We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate.After incubation with whole-cell protein extract or transfection into human cells,we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography–tandem mass spectrometry(LCMS/MS)analyses at both the mononucleotide and oligodeoxynucleotide levels.Our results clearly showed the direct conversion of 5caC to cytosine in human cells,providing evidence to support a novel pathway for active DNA demethylation.