Emerging Roles of RNA Methylation in Development.

More than 170 different types of chemical modifications have been identified on diverse types of RNA, collectively known as the epitranscriptome. Among them, N6-methyladenine (m6A), 5-methylcytosine (m5C), N1-methyladenine (m1A), and N7-methylguanosine (m7G) as the ubiquitous post-transcriptional modification are widely involved in regulating the metabolic processes such as RNA degradation, translation, stability, and export, mediating important physiological and pathological processes such as stress regulation, immune response, development, and tumorigenesis. Recently, the regulatory role of RNA modification during developmental processes is getting more attention. Therefore, the development of low-input even single-cell and high-resolution sequencing technologies is crucial for the exploration of the regulatory roles of RNA modifications in these important biological events of trace samples.This account focuses on the roles of RNA modifications in various developmental processes. We describe the distribution characteristics of various RNA modifications, catalytic enzymes, binding proteins, and the development of sequencing technologies. RNA modification is dynamically reversible, which can be catalyzed by methyltransferases and eliminated by demethylases. RNA m6A is the most abundant post-transcriptional modification on eukaryote mRNA, which is mainly concentrated near the stop codon, and involves in RNA metabolism regulation. RNA m5C, another most studied RNA modification, has been identified in a various of organisms and RNA species, mainly enriched in the regions downstream of translation initiation sites and broadly distributes across the whole coding sequence (CDS) in mammalian mRNAs. RNA m1A, with a lower abundance than m6A, is widely distributed in various RNA types, mainly locates in the 5' untranslated region (5'UTR) of mRNA and regulates translation. RNA m7G, one of the most common RNA modifications in eukaryotes, has been identified at cap regions and internal positions of RNAs and recently gained considerable attention.Thanks to the development of sequencing technology, m6A has been found to regulate the tumorigenic process, including tumor proliferation, invasion, and metastasis by modulating oncogenes and tumor suppressor genes, and affect oocyte maturation and embryonic development through regulating maternal and zygotic genes. m5C related proteins have been identified to participate in embryonic development, plant growth, and neural stem cell differentiation in a m5C dependent manner. m1A also has been revealed to be involved in these developmental processes. m7G dysregulation mainly involves in neurodevelopmental disorders and neurodegenerative diseases.Collectively, we summarized the gradually exhibited roles of RNA methylation during development, and discussed the possibility of RNA modifications as candidate biomarkers and potential therapeutic targets. The technological development is anticipated as the major driving force to expand our knowledge in this field.

Differential Transcriptomic Landscapes of SARS-CoV-2 Variants in Multiple Organs from Infected Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the persistent coronavirus disease 2019 (COVID-19) pandemic, which has resulted in millions of deaths worldwide and brought an enormous public health and global economic burden. The recurring global wave of infections has been exacerbated by growing variants of SARS-CoV-2. In this study, the virological characteristics of the original SARS-CoV-2 strain and its variants of concern (VOCs; including Alpha, Beta, and Delta) in vitro, as well as differential transcriptomic landscapes in multiple organs (lung, right ventricle, blood, cerebral cortex, and cerebellum) from the infected rhesus macaques, were elucidated. The original strain of SARS-CoV-2 caused a stronger innate immune response in host cells, and its VOCs markedly increased the levels of subgenomic RNAs, such as N, Orf9b, Orf6, and Orf7ab, which are known as the innate immune antagonists and the inhibitors of antiviral factors. Intriguingly, the original SARS-CoV-2 strain and Alpha variant induced larger alteration of RNA abundance in tissues of rhesus monkeys than Beta and Delta variants did. Moreover, a hyperinflammatory state and active immune response were shown in the right ventricles of rhesus monkeys by the up-regulation of inflammation- and immune-related RNAs. Furthermore, peripheral blood may mediate signaling transmission among tissues to coordinate the molecular changes in the infected individuals. Collectively, these data provide insights into the pathogenesis of COVID-19 at the early stage of infection by the original SARS-CoV-2 strain and its VOCs.

scm6A-seq reveals single-cell landscapes of the dynamic m6A during oocyte maturation and early embryonic development.

N6-methyladenosine (m6A) has been demonstrated to regulate RNA metabolism and various biological processes, including gametogenesis and embryogenesis. However, the landscape and function of m6A at single cell resolution have not been extensively studied in mammalian oocytes or during pre-implantation. In this study, we developed a single-cell m6A sequencing (scm6A-seq) method to simultaneously profile the m6A methylome and transcriptome in single oocytes/blastomeres of cleavage-stage embryos. We found that m6A deficiency leads to aberrant RNA clearance and consequent low quality of Mettl3Gdf9 conditional knockout (cKO) oocytes. We further revealed that m6A regulates the translation and stability of modified RNAs in metaphase II (MII) oocytes and during oocyte-to-embryo transition, respectively. Moreover, we observed m6A-dependent asymmetries in the epi-transcriptome between the blastomeres of two-cell embryo. scm6A-seq thus allows in-depth investigation into m6A characteristics and functions, and the findings provide invaluable single-cell resolution resources for delineating the underlying mechanism for gametogenesis and early embryonic development.

mRNA m5C inhibits adipogenesis and promotes myogenesis by respectively facilitating YBX2 and SMO mRNA export in ALYREF-m5C manner.

Although 5-methylcytosine (m5C) has been identified as a novel and abundant mRNA modification and associated with energy metabolism, its regulation function in adipose tissue and skeletal muscle is still limited. This study aimed at investigating the effect of mRNA m5C on adipogenesis and myogenesis using Jinhua pigs (J), Yorkshire pigs (Y) and their hybrids Yorkshire-Jinhua pigs (YJ). We found that Y grow faster than J and YJ, while fatness-related characteristics observed in Y were lower than those of J and YJ. Besides, total mRNA m5C levels and expression rates of NSUN2 were higher both in backfat layer (BL) and longissimus dorsi muscle (LDM) of Y compared to J and YJ, suggesting that higher mRNA m5C levels positively correlate with lower fat and higher muscle mass. RNA bisulfite sequencing profiling of m5C revealed tissue-specific and dynamic features in pigs. Functionally, hyper-methylated m5C-containing genes were enriched in pathways linked to impaired adipogenesis and enhanced myogenesis. In in vitro, m5C inhibited lipid accumulation and promoted myogenic differentiation. Furthermore, YBX2 and SMO were identified as m5C targets. Mechanistically, YBX2 and SMO mRNAs with m5C modification were recognized and exported into the cytoplasm from the nucleus by ALYREF, thus leading to increased YBX2 and SMO protein expression and thereby inhibiting adipogenesis and promoting myogenesis, respectively. Our work uncovered the critical role of mRNA m5C in regulating adipogenesis and myogenesis via ALYREF-m5C-YBX2 and ALYREF-m5C-SMO manners, providing a potential therapeutic target in the prevention and treatment of obesity, skeletal muscle dysfunction and metabolic disorder diseases.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

OsNSUN2-Mediated 5-Methylcytosine mRNA Modification Enhances Rice Adaptation to High Temperature.

Extreme weather events can cause heat stress that decreases crop production. Recent studies have demonstrated that protein degradation and rRNA homeostasis as well as transcription factors are involved in the thermoresponse in plants. However, how RNA modifications contribute to temperature stress response in plant remains largely unknown. Herein, we identified OsNSUN2 as an RNA 5-methylcytosine (m5C) methyltransferase in rice. osnsun2 mutant displayed severe temperature- and light-dependent lesion-mimic phenotypes and heat-stress hypersensitivity. Heat stress enhanced the OsNSUN2-dependent m5C modification of mRNAs involved in photosynthesis and detoxification systems, such as ß-OsLCY, OsHO2, OsPAL1, and OsGLYI4, which increased protein synthesis. Furthermore, the photosystem of osnsun2 mutant was vulnerable to high ambient temperature and failed to undergo repair under tolerable heat stress. Thus, OsNSUN2 mutation reduced photosynthesis efficiency and accumulated excessive reactive oxygen species upon heat treatment. Our findings demonstrate an important mechanism of mRNA m5C-dependent heat acclimation in rice.

5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.

Although 5-methylcytosine (m5C) is a widespread modification in RNAs, its regulation and biological role in pathological conditions (such as cancer) remain unknown. Here, we provide the single-nucleotide resolution landscape of messenger RNA m5C modifications in human urothelial carcinoma of the bladder (UCB). We identify numerous oncogene RNAs with hypermethylated m5C sites causally linked to their upregulation in UCBs and further demonstrate YBX1 as an m5C 'reader' recognizing m5C-modified mRNAs through the indole ring of W65 in its cold-shock domain. YBX1 maintains the stability of its target mRNA by recruiting ELAVL1. Moreover, NSUN2 and YBX1 are demonstrated to drive UCB pathogenesis by targeting the m5C methylation site in the HDGF 3' untranslated region. Clinically, a high coexpression of NUSN2, YBX1 and HDGF predicts the poorest survival. Our findings reveal an unprecedented mechanism of RNA m5C-regulated oncogene activation, providing a potential therapeutic strategy for UCB.

Identification of entacapone as a chemical inhibitor of FTO mediating metabolic regulation through FOXO1.

Recent studies have established the involvement of the fat mass and obesity-associated gene (FTO) in metabolic disorders such as obesity and diabetes. However, the precise molecular mechanism by which FTO regulates metabolism remains unknown. Here, we used a structure-based virtual screening of U.S. Food and Drug Administration-approved drugs to identify entacapone as a potential FTO inhibitor. Using structural and biochemical studies, we showed that entacapone directly bound to FTO and inhibited FTO activity in vitro. Furthermore, entacapone administration reduced body weight and lowered fasting blood glucose concentrations in diet-induced obese mice. We identified the transcription factor forkhead box protein O1 (FOXO1) mRNA as a direct substrate of FTO, and demonstrated that entacapone elicited its effects on gluconeogenesis in the liver and thermogenesis in adipose tissues in mice by acting on an FTO-FOXO1 regulatory axis.

5-Hydroxymethylome in Circulating Cell-free DNA as A Potential Biomarker for Non-small-cell Lung Cancer.

Non-small-cell lung cancer (NSCLC), the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminating tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here, we employed a sensitive and reliable method to map genome-wide 5hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identified six potential biomarkers from 66 patients and 67 healthy controls (mean decrease accuracy >3.2, P < 3.68E-19) using machine-learning-based tumor classifiers with high accuracy. Thus, the unique signature of 5hmC in tumor patient's cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.

Weight-reducing effect of Acer truncatum Bunge may be related to the inhibition of fatty acid synthase.

In order to study the anti-obesity ability and inhibition towards fatty acid synthase (FAS) of the extract, a 70% ethanol extract of Acer truncatum Bunge (AT) leaves was further extracted with ethyl acetate. FAS is a very significant lipogenic enzyme, participating in energy metabolism in vivo; it has also been observed that FAS inhibitors might be potent anti-obesity agents. Experimental results on animals showed that the extract significantly reduced food intake and adipose, and effectively controlled weight evolution. Lipogenesis inhibition might be regarded as one of the reasons for the weight control and adipose reduction by AT. The extract was further isolated using a series of column chromatography that yielded 10 known compounds. 1,2,3,4,6-Penta-O-galloyl-ß-D-glucose was found to be one of the major active constituents in the extract of AT.

Up-regulation of licochalcone A biosynthesis and secretion by Tween 80 in hairy root cultures of Glycyrrhiza uralensis Fisch.

We evaluated the effect of Tween 80 as elicitor on licochalcone A from hairy root cultures of Glycyrrhiza uralensis Fisch. After a 15-days treatment with 2% Tween 80, hairy roots still grew well and produced higher levels of licochalcone A and total flavonoids than the control (without treatment). Licochalcone A content and total flavonoid content were 3.103 and 127.095 mg per flask (9- and 11-fold higher), respectively, compared with controls. Secretion of licochalcone A and total flavonoids into the culture medium was remarkably high, up to 98 and 94% of the total production, respectively. The enhanced flavonoid production was associated with elevated mRNA levels and enzyme activities of phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), and cinnamate-4-hydroxylase (C4H). These results clearly demonstrated that Tween 80 treatment permeabilized the roots to enhance secretion, but also acted as an efficient elicitor of licochalcone A and total flavonoid production in hairy roots of G. uralensis Fisch.

Evaluation of the inhibitory activities of aceraceous plants on fatty acid synthase.

Fatty acid synthase (FAS) is a very significant lipogenic enzyme participating in energy metabolism in vivo and has been reported as a potential new therapeutic target for cancer treatment. The extracts from sixteen Aceraceae were prepared to assay their inhibitory activities against duck liver FAS and their correlated antitumor bioactivity. Their inhibition of FAS was composed of a reversible fast-binding inhibition, by which 0.41 microg/mL of the A. campestre extract inhibits 50% FAS activity, and an irreversible slow-binding inhibition with inactivation rate constants, k(obs), ranging between 1.5 x 10(-3) and 10.6 x 10(-3) min(-1). Three Aceraceae extracts were selected from their smaller IC50 values to study different type of inhibitions against the three substrates in the FAS overall reaction. As compared with other reported FAS inhibitors including EGCG with regard to inhibition constant and IC50 value, the extracts appeared to be more efficient inhibitors, and exhibited a considerable inhibition against the growth of five types of cancer cells (China patent application number 200610088901.6), which may be related to the inhibition of lipogenesis in these cells.

The isolation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from Acer truncatum Bunge by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from the ethyl acetate extract of the leaves of Acer truncatum Bunge using a two-phase system composed of n-hexane-ethyl acetate-methanol-water at a volume ratio of (0.25:5:1:5, v/v/v/v) for the first time. Each injection of 80 mg crude extract yielded 7.25 mg of pure 1,2,3,4,6-penta-O-galloyl-beta-D-glucose. High-performance liquid chromatography (HPLC) analyses of the CCC fraction revealed that the purity of 1,2,3,4,6-penta-O-galloyl-beta-D- glucose was over 95%.