Over 160 RNA modifications have been identified, including N7-methylguanine (m7G), N6-methyladenosine (m6A), and 5-methylcytosine (m5C). These modifications play key roles in regulating the fate of ...RNA. In eukaryotes, m6A is the most abundant mRNA modification, accounting for over 80% of all RNA methylation modifications. Highly dynamic m6A modification may exert important effects on organismal reproduction and development. Significant advances in understanding the mechanism of m6A modification have been made using immunoprecipitation, chemical labeling, and site-directed mutagenesis, combined with next-generation sequencing. Single-molecule real-time and nanopore direct RNA sequencing (DRS) approaches provide additional ways to study RNA modifications at the cellular level. In this review, we explore the technical history of identifying m6A RNA modifications, emphasizing technological advances in detecting m6A modification. In particular, we discuss the challenge of generating accurate dynamic single-base resolution m6A maps and also strategies for improving detection specificity. Finally, we outline a roadmap for future research in this area, focusing on the application of RNA epigenetic modification, represented by m6A modification.
SUMMARY
Australian pine (Casuarina spp.) is extensively planted in tropical and subtropical regions for wood production, shelterbelts, environmental protection, and ecological restoration due to ...their superior biological characteristics, such as rapid growth, wind and salt tolerance, and nitrogen fixation. To analyze the genomic diversity of Casuarina, we sequenced the genomes and constructed de novo genome assemblies of the three most widely planted Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana. We generated chromosome‐scale genome sequences using both Pacific Biosciences (PacBio) Sequel sequencing and chromosome conformation capture technology (Hi‐C). The total genome sizes for C. equisetifolia, C. glauca, and C. cunninghamiana are 268 942 579 bp, 296 631 783 bp, and 293 483 606 bp, respectively, of which 25.91, 27.15, and 27.74% were annotated as repetitive sequences. We annotated 23 162, 24 673, and 24 674 protein‐coding genes in C. equisetifolia, C. glauca, and C. cunninghamiana, respectively. We then collected branchlets from male and female individuals for whole‐genome bisulfite sequencing (BS‐seq) to explore the epigenetic regulation of sex determination in these three species. Transcriptome sequencing (RNA‐seq) revealed differential expression of phytohormone‐related genes between male and female plants. In summary, we generated three chromosome‐level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.
Significance Statement
In this study we generated three chromosome‐level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.
This study examined the storage of smoked bacon. Gas chromatography-mass spectrometry (GC–MS) and single-molecule real-time DNA sequencing technology (SMRT) were used to quantitate volatile ...constituents and microbial communities. The results indicated that the quality of bacon and its volatile constituents at room temperature are higher than those stored at 4 °C (P < 0.05). The bacterial abundance of smoked bacon in the H group that were hung from a beam at room temperature was higher than those of the L group, which were packaged under vacuum conditions and stored at 4 °C. The fungal diversity was lower in the H group than in the L group. Staphylococcus equorum was the most prominent bacterial species in the two groups, while both the fungal species Candida zeylanoides and Debaryomyces prosopidis were in all groups of smoked bacon. Pearson correlation coefficients indicated that Staphylococcus equorum positively correlated with heptanal in both groups. Lactobacillus sakei was positively (P < 0.01) related to heptanal in refrigeration, and Debaryomyces prosopidis positively correlated with hexanal in both groups. This study should help to improve the quality of meat by mediating microbial communities in Sichuan smoked bacon.
•Volatile constituents of smoked bacon at 25 °C were significantly higher than those at 4 °C.•Analysis of microbial diversity in Sichuan bacon using single-molecule real-time DNA sequencing.•Storage at room temperature facilitated maintaining the diversity of the microbiota.•Pearson correlation coefficient analysis was performed on the microbiology and quality of bacon.•Microbial diversity and quality of the bacon were significantly affected by different storage methods.
The modular Snow Microwave Radiative Transfer (SMRT) model simulates microwave scattering behavior in snow via different selectable theories and snow microstructure representations, which is well ...suited to intercomparisons analyses. Here, five microstructure models were parameterized from X-ray tomography and thin-section images of snow samples and evaluated with SMRT. Three field experiments provided observations of scattering and absorption coefficients, brightness temperature, and/or backscatter with the increasing complexity of snowpack. These took place in Sodankylä, Finland, and Weissfluhjoch, Switzerland. Simulations of scattering and absorption coefficients agreed well with observations, with higher errors for snow with predominantly vertical structures. For simulation of brightness temperature, difficulty in retrieving stickiness with the Sticky Hard Sphere microstructure model resulted in relatively poor performance for two experiments, but good agreement for the third. Exponential microstructure gave generally good results, near to the best performing models for two field experiments. The Independent Sphere model gave intermediate results. New Teubner-Strey and Gaussian Random Field models demonstrated the advantages of SMRT over microwave models with restricted microstructural geometry. Relative model performance is assessed by the quality of the microstructure model fit to micro-computed tomography (CT) data and further improvements may be possible with different fitting techniques. Careful consideration of simulation stratigraphy is required in this new era of high-resolution microstructure measurement as layers thinner than the wavelength introduce artificial scattering boundaries not seen by the instrument.
The ability to rapidly change gene expression patterns is essential for differentiation, development, and functioning of the brain. Throughout development, or in response to environmental stimuli, ...gene expression patterns are tightly regulated by the dynamic interplay between transcription activators and repressors. Nuclear receptor corepressor 1 (NCoR1) and silencing mediator for retinoid or thyroid‐hormone receptors (SMRT) are the best characterized transcriptional co‐repressors from a molecular point of view. They mediate epigenetic silencing of gene expression in a wide range of developmental and homeostatic processes in many tissues, including the brain. For instance, NCoR1 and SMRT regulate neuronal stem cell proliferation and differentiation during brain development and they have been implicated in learning and memory. However, we still have a limited understanding of their regional and cell type‐specific expression in the brain. In this study, we used fluorescent immunohistochemistry to map their expression patterns throughout the adult mouse brain. Our findings reveal that NCoR1 and SMRT share an overall neuroanatomical distribution, and are detected in both excitatory and inhibitory neurons. However, we observed striking differences in their cell type‐specific expression in glial cells. Specifically, all oligodendrocytes express NCoR1, but only a subset express SMRT. In addition, NCoR1, but not SMRT, was detected in a subset of astrocytes and in the microglia. These novel observations are corroborated by single cell transcriptomics and emphasize how NCoR1 and SMRT may contribute to distinct biological functions, suggesting an exclusive role of NCoR1 in innate immune responses in the brain.
Schematic representation of NCoR1 and SMRT distribution in neuronal and non‐neuronal cell types. Five major types of brain cells (glutamatergic and GABAergic neurons, astrocytes, oligodendrocytes, and microglia) are depicted within a representative coronal slice of the mouse brain. Both NCor1 and SMRT broadly localize within the nuclei of most neuronal cells. However, a diverse pattern of expression is detected in non‐neuronal cells. While NCoR1 is expressed in a small fraction of astrocytes, SMRT is not expressed in GFAP+ astrocytes or in Iba1+ microglia.
Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way ...transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses.
Post-replicative DNA methylation is essential for diverse biological processes in both eukaryotes and prokaryotes. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, ...remains one of the most formidable threats worldwide. Although DNA methylation of M. tuberculosis has been documented, little information is available for clinical drug-resistant M. tuberculosis. Single-molecule real-time (SMRT) sequencing was used to profile the core methylome of three clinical isolates, namely multidrug-resistant (MDR), extensively drug-resistant (XDR) and extremely drug-resistant (XXDR) strains. 3812, 6808 and 6041 DNA methylated sites were identified in MDR-MTB, XDR-MTB and XXDR-MTB genome, respectively. There are two types of methylated motifs, namely N
6
-methyladenine (m6A) and N
4
-methylcytosine (m4C). A novel widespread 6 mA methylation motif 5′-CACGCAG-3′ was found in XDR-MTB and XXDR-MTB. The methylated genes are involved in multiple cellular processes, especially metabolic enzymes engaged in glucose metabolism, fatty acid and TCA cycle. Many methylated genes are involved in mycobacterial virulence, antibiotic resistance and tolerance. This provided a comprehensive list of methylated genes in drug-resistant clinical isolates and the basis for further functional elucidation.
The mite
is distributed worldwide and parasitism the ear canals of cats and dogs, causing otitis externa. Molecular biology of
is poorly understood, with only a few genes being deposited in public ...databases. In the present study, we aimed to perform transcriptome analysis of
using SMRT and Illumina sequencing of RNA from different development stages. SMRT-Seq of
demonstrated 5,431 final transcripts, including 406 long non-coding RNAs and 2,698 differentially expressed genes (DEGs), including 1,357 up-regulated genes and 1,341 down-regulated genes between adult mites and nymph/larva. A total of 397 putative allergen genes were detected, 231 of which were DEGs. Among them, 77 were homologous of known mite allergens. The expression level of allergen genes hints at the pathogenicity of mites in different life stages, and the protein interaction network analysis could identify possible key genes in the pathogenic mechanism. Intriguingly, Gene Ontology analysis showed that most of the (DEGs) were associated with the terms hydrolase activity and proteolysis. Kyoto Encyclopedia of genes and genomes (KEGG) analysis identified drug metabolism-cytochrome P450 signal pathway as one of the top pathways. SMRT-Seq of the full-length transcriptome of
was performed first, and a valuable resource was acquired through the combination analysis with the Illumina sequencing data. The results of our analyses provide new information for further research into
.
Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the methyl-CpG–binding protein 2 (MeCP2) gene. The majority of RTT missense mutations disrupt the interaction of the ...MeCP2 with DNA or the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid receptors (SMRT) corepressor complex. Here, we show that the “NCoR/SMRT interaction domain” (NID) of MeCP2 directly contacts transducin beta-like 1 (TBL1) and TBL1 related (TBLR1), two paralogs that are core components of NCoR/SMRT. We determine the cocrystal structure of the MeCP2 NID in complex with the WD40 domain of TBLR1 and confirm by in vitro and ex vivo assays that mutation of interacting residues of TBLR1 and TBL1 disrupts binding to MeCP2. Strikingly, the four MeCP2-NID residues mutated in RTT are those residues that make the most extensive contacts with TBLR1. Moreover, missense mutations in the gene for TBLR1 that are associated with intellectual disability also prevent MeCP2 binding. Our study therefore reveals the molecular basis of an interaction that is crucial for optimal brain function.
A circular consensus sequencing (CCS) strategy involving single molecule, real‐time (SMRT) DNA sequencing technology was applied to de novo assembly and single nucleotide polymorphism (SNP) detection ...of chloroplast genomes. Chloroplast DNA was purified from enriched chloroplasts of pooled individuals to construct a shotgun library for each species. The sequencing reactions were performed on a PacBio RS platform. CCS sub‐reads were generated from polymerase reads that passed the native dumbbell‐shaped DNA templates multiple times. The complete chloroplast genome sequence was generated by mapping all reads to the draft sequence constructed in a step‐by‐step manner. The full‐chain, PCR‐free approach eliminates the possible context‐specific biases in library construction and sequencing reaction. The chloroplast genome was easily and completely assembled using the data generated from one SMRT Cell without requiring a reference genome. Comparisons of the three assembled Fritillaria genomes to 34.1 kb of validation Sanger sequences revealed 100% concordance, and the detected intraspecies SNPs at a minimum variant frequency of 15% were all confirmed. This simple approach with potential for parallel sequencing yields high‐quality chloroplast genomes for sensitive SNP detection and comparative analyses. We recommend this approach for its powerful applicability for evolutionary genetics and genomics studies in plants based on the sequences of chloroplast genomes.
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