SnoRNP is essential for thermospermine-mediated development in Arabidopsis thaliana

Polyamines have been discovered for hundreds of years and once considered as a class of phytohormones.Polyamines play critical roles in a range of developmental processes.However,the molecular mechanisms of polyamine signaling pathways remain poorly understood.Here,we measured the contents of main types of polyamines,and found that endogenous level of thermospermine(T-Spm)in Arabidopsis thaliana is comparable to those of classic phytohormones and is significantly lower than those of putrescine(Put),spermidine(Spd),and spermine(Spm).We further found a nodule-like structure around the junction area connecting the shoot and root of the T-Spm biosynthetic mutant acl5 and obtained more than 50 suppressors of acl5 nodule structure(san)through suppressor screening.An in-depth study of two san suppressors revealed that NAP57 and NOP56,core components of box H/ACA and C/D snoRNPs,were essential for T-Spm-mediated nodule-like structure formation and plant height.Furthermore,analyses of rRNA modifications showed that the overall levels of pseudouridylation and 2′-O-methylation were compromised in san1 and san2 respectively.Taken together,these results establish a strong genetic relationship between rRNA modification and T-Spm-mediated growth and development,which was previously undiscovered in all organisms.

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.

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.

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.

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].

Single-base resolution analysis of DNA epigenome via high-throughput sequencing

Epigenetic changes caused by DNA methylation and histone modifications play important roles in the regulation of various cellular processes and development. Recent discoveries of 5-methylcytosine(5m C) oxidation derivatives including 5-hydroxymethylcytosine(5hm C), 5-formylcytsine(5f C) and 5-carboxycytosine(5ca C) in mammalian genome further expand our understanding of the epigenetic regulation. Analysis of DNA modification patterns relies increasingly on sequencing-based profiling methods. A number of different approaches have been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing(BS-seq), TAB-seq(TET-assisted bisulfite sequencing), ox BS-seq(oxidative bisulfite sequencing) and etc. These methods have been used to generate base-resolution maps of 5m C and its oxidation derivatives in genomic samples. The focus of this review will be to discuss the chemical methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.

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.

Genome-wide Mapping of Cellular Protein–RNA Interactions Enabled by Chemical Crosslinking

RNA–protein interactions influence many biological processes. Identifying the binding sites of RNA-binding proteins（RBPs） remains one of the most fundamental and important challenges to the studies of such interactions. Capturing RNA and RBPs via chemical crosslinking allows stringent purification procedures that significantly remove the non-specific RNA and protein interactions. Two major types of chemical crosslinking strategies have been developed to date, i.e., UV-enabled crosslinking and enzymatic mechanism-based covalent capture. In this review, we compare such strategies and their current applications, with an emphasis on the technologies themselves rather than the biology that has been revealed. We hope such methods could benefit broader audience and also urge for the development of new methods to study RNA RBP interactions.

Liquid biopsies:DNA methylation analyses in circulating cell-free DNA

Analysis of patient＇s materials like cells or nucleic acids obtained in a minimally invasive or noninvasive manner through the sampling of blood or other body fluids serves as liquid biopsies, which has huge potential for numerous diagnostic applications. Circulating cell-free DNA（cfDNA） is explored as a prognostic or predictive marker of liquid biopsies with the improvements in genomic and molecular methods. DNA methylation is an important epigenetic marker known to affect gene expression. cfDNA methylation detection is a very promising approach as abnormal distribution of DNA methylation is one of the hallmarks of many cancers and methylation changes occur early during carcinogenesis. This re？view summarizes the various investigational applications of cfDNA methylation and its oxidized de？rivatives as biomarkers for cancer diagnosis, prenatal diagnosis and organ transplantation monitoring.The review also provides a brief overview of the technologies for cfDNA methylation analysis based on next generation sequencing.

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.

Metatranscriptomic analysis of host response and vaginal microbiome of patients with severe COVID-19

Dear Editor,The pandemic of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)has become a global public health threat.Here we use a TRACE-seq-based metatranscriptomic analysis to compare host responses and vaginal microbiome of postmenopausal female patients with underlying severe COVID-19 disease with those of healthy females,thereby providing insights into the changes in the microenvironment of women's reproductive system.