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N6-methyladenosine (m6A) RNA methylation is one of the most abundant modifications on mRNAs and plays an important role in various biological processes. The formation of m6A is catalysed by a methyltransferase complex containing a key factor methyltransferase-like 3 (Mettl3). However, the functions of Mettl3 and m6A modification in liver lipid and glucose metabolism remain unclear. Here, we show that both Mettl3 expression and m6A level increased in the liver of mice with High Fat Diet (HFD)-induced metabolic disorders, and overexpression of Mettl3 aggravated HFD-induced liver metabolic disorders and insulin resistance. Hepatocyte-specific knockout of Mettl3 significantly alleviated HFD-induced metabolic disorders by slowing weight gain, reducing lipid accumulation and improving insulin sensitivity. Mechanistically, Mettl3 depletion-mediated m6A loss causes extended RNA half-lives of metabolism-related genes, consequently protects mice against HFD-induced metabolic syndrome. Our findings reveal a critical role of Mettl3-mediated m6A in HFD-induced metabolic disorders and hepatogenous diabetes.

Exposure of airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5) is epidemiologically associated with lung dysfunction and respiratory symptoms, including pulmonary fibrosis. However, whether epigenetic mechanisms are involved in PM2.5-induced pulmonary fibrosis is currently poorly understood. Herein, using a PM2.5-induced pulmonary fibrosis mouse model, we found that PM2.5 exposure leads to aberrant mRNA 5-methylcytosine (m5C) gain and loss in fibrotic lung tissues. Moreover, we showed the m5C-mediated regulatory map of gene functions in pulmonary fibrosis after PM2.5 exposure. Several genes act as m5C gain-upregulated factors, probably critical for the development of PM2.5-induced fibrosis in mouse lungs. These genes, including Lcn2, Mmp9, Chi3l1, Adipoq, Atp5j2, Atp5l, Atpif1, Ndufb6, Fgr, Slc11a1, and Tyrobp, are highly related to oxidative stress response, inflammatory responses, and immune system processes. Our study illustrates the first epitranscriptomic RNA m5C profile in PM2.5-induced pulmonary fibrosis and will be valuable in identifying biomarkers for PM2.5 exposure-related lung pathogenesis with translational potential.

BackgroundDynamic N6-methyladenosine (m6A) RNA modification generated and erased by N6-methyltransferases and demethylases regulates gene expression, alternative splicing and cell fate. Ocular melanoma, comprising uveal melanoma (UM) and conjunctival melanoma (CM), is the most common primary eye tumor in adults and the 2nd most common melanoma. However, the functional role of m6A modification in ocular melanoma remains unclear.Methodsm6A assays and survival analysis were used to explore decreased global m6A levels, indicating a late stage of ocular melanoma and a poor prognosis. Multiomic analysis of miCLIP-seq, RNA-seq and Label-free MS data revealed that m6A RNA modification posttranscriptionally promoted HINT2 expression. RNA immunoprecipitation (RIP)-qPCR and dual luciferase assays revealed thatHINT2mRNA specifically interacted with YTHDF1. Furthermore, polysome profiling analysis indicated a greater amount ofHINT2mRNA in the translation pool in ocular melanoma cells with higher m6A methylation.ResultsHere, we show that RNA methylation significantly inhibits the progression of UM and CM. Ocular melanoma samples showed decreased m6A levels, indicating a poor prognosis. Changes in global m6A modification were highly associated with tumor progression in vitro and in vivo. Mechanistically, YTHDF1 promoted the translation of methylatedHINT2mRNA, a tumor suppressor in ocular melanoma.ConclusionsOur work uncovers a critical function for m6A methylation in ocular melanoma and provides additional insight into the understanding of m6A modification.

Over 150 types of RNA modifications are identified in RNA molecules. Transcriptome profiling is one of the key steps in decoding the epitranscriptomic panorama of these chemical modifications and their potential functions. N(7)-methylguanosine (m(7)G) is one of the most abundant modifications present in tRNA, rRNA and mRNA 5′cap, and has critical roles in regulating RNA processing, metabolism and function. Besides its presence at the cap position in mRNAs, m(7)G is also identified in internal mRNA regions. However, its transcriptome-wide distribution and dynamic regulation within internal mRNA regions remain unknown. Here, we have established m(7)G individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (m(7)G miCLIP-seq) to specifically detect internal mRNA m(7)G modification. Using this approach, we revealed that m(7)G is enriched at the 5′UTR region and AG-rich contexts, a feature that is well-conserved across different human/mouse cell lines and mouse tissues. Strikingly, the internal m(7)G modification is dynamically regulated under both H(2)O(2) and heat shock treatments, with remarkable accumulations in the CDS and 3′UTR regions, and functions in promoting mRNA translation efficiency. Consistently, a PCNA 3′UTR minigene reporter harboring the native m(7)G modification site displays both enriched m(7)G modification and increased mRNA translation upon H(2)O(2) treatment compared to the m(7)G site-mutated minigene reporter (G to A). Taken together, our findings unravel the dynamic profiles of internal mRNA m(7)G methylome and highlight m(7)G as a novel epitranscriptomic marker with regulatory roles in translation.

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.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

N6-methyladenosine (m6A), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of m6A modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced m6A levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. m6A immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that m6A was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, m6A was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated m6A modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2. Keywords: N6-methyladenosine (m6A), Mettl3, Neurogenesis, Neuronal development, Ezh2

Double-stranded RNA (dsRNA) is a virus-encoded signature capable of triggering intracellular Rig-like receptors (RLR) to activate antiviral signaling, but whether intercellular dsRNA structural reshaping mediated by the N6-methyladenosine (m6A) modification modulates this process remains largely unknown. Here, we show that, in response to infection by the RNA virus Vesicular Stomatitis Virus (VSV), the m6A methyltransferase METTL3 translocates into the cytoplasm to increase m6A modification on virus-derived transcripts and decrease viral dsRNA formation, thereby reducing virus-sensing efficacy by RLRs such as RIG-I and MDA5 and dampening antiviral immune signaling. Meanwhile, the genetic ablation of METTL3 in monocyte or hepatocyte causes enhanced type I IFN expression and accelerates VSV clearance. Our findings thus implicate METTL3-mediated m6A RNA modification on viral RNAs as a negative regulator for innate sensing pathways of dsRNA, and also hint METTL3 as a potential therapeutic target for the modulation of anti-viral immunity., N6-methyladenosine (m6A) RNA modification regulates RNA metabolism, and has been implicated in immune regulation. Here, the authors show that the m6A methyltransferase, METTL3, translocates into the cytoplasm to increase viral RNA m6A modification, decreases viral ds RNA content, and thereby dampens the RIG/MDA5-induced anti-viral immunity.

Anterior vaginal prolapse (AVP) is the most common form of pelvic organ prolapse (POP) and has deleterious effects on women’s health. Despite recent advances in AVP diagnosis and treatment, a cell atlas of the vaginal wall in AVP has not been constructed. Here, we employ single-cell RNA-seq to construct a transcriptomic atlas of 81,026 individual cells in the vaginal wall from AVP and control samples and identify 11 cell types. We reveal aberrant gene expression in diverse cell types in AVP. Extracellular matrix (ECM) dysregulation and immune reactions involvement are identified in both non-immune and immune cell types. In addition, we find that several transcription factors associated with ECM and immune regulation are activated in AVP. Furthermore, we reveal dysregulated cell–cell communication patterns in AVP. Taken together, this work provides a valuable resource for deciphering the cellular heterogeneity and the molecular mechanisms underlying severe AVP., Anterior vaginal prolapse (AVP), the most common form of pelvic organ prolapse, has deleterious effects on women’s health. Here the authors employ single-cell RNA-seq to construct a transcriptomic atlas of vaginal wall cells from AVP patients, and find that extracellular matrix dysregulation and immune reaction are associated with AVP.

Background Vertebrate early embryogenesis is initially directed by a set of maternal RNAs and proteins, yet the mechanisms controlling this program remain largely unknown. Recent transcriptome-wide studies on RNA structure have revealed its pervasive and crucial roles in RNA processing and functions, but whether and how RNA structure regulates the fate of the maternal transcriptome have yet to be determined. Results Here we establish the global map of four nucleotide-based mRNA structures by icSHAPE during zebrafish early embryogenesis. Strikingly, we observe that RNA structurally variable regions are enriched in the 3′ UTR and contain cis-regulatory elements important for maternal-to-zygotic transition (MZT). We find that the RNA-binding protein Elavl1a stabilizes maternal mRNAs by binding to the cis-elements. Conversely, RNA structure formation suppresses Elavl1a’s binding leading to the decay of its maternal targets. Conclusions Our study finds that RNA structurally variable regions are enriched in mRNA 3′ UTRs and contain cis-regulatory elements during zebrafish early embryogenesis. We reveal that Elavl1a regulates maternal RNA stability in an RNA structure-dependent fashion. Overall, our findings reveal a broad and fundamental role of RNA structure-based regulation in vertebrate early embryogenesis.

Review history.

While N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is linked to cell differentiation and tissue development, the biological significance of m6A modification in mammalian glial development remains unknown. Here, we identify a novel m6A reader, Prrc2a (Proline rich coiled-coil 2 A), which controls oligodendrocyte specification and myelination. Nestin-Cre-mediated knockout of Prrc2a induces significant hypomyelination, decreased lifespan, as well as locomotive and cognitive defects in a mouse model. Further analyses reveal that Prrc2a is involved in oligodendrocyte progenitor cells (OPCs) proliferation and oligodendrocyte fate determination. Accordingly, oligodendroglial-lineage specific deletion of Prrc2a causes a similar phenotype of Nestin-Cre-mediated deletion. Combining transcriptome-wide RNA-seq, m6A-RIP-seq and Prrc2a RIP-seq analysis, we find that Olig2 is a critical downstream target gene of Prrc2a in oligodendrocyte development. Furthermore, Prrc2a stabilizes Olig2 mRNA through binding to a consensus GGACU motif in the Olig2 CDS (coding sequence) in an m6A-dependent manner. Interestingly, we also find that the m6A demethylase, Fto, erases the m6A modification of Olig2 mRNA and promotes its degradation. Together, our results indicate that Prrc2a plays an important role in oligodendrocyte specification through functioning as a novel m6A reader. These findings suggest a new avenue for the development of therapeutic strategies for hypomyelination-related neurological diseases.

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

METTL3 catalyzes the formation of N6-methyl-adenosine (m6A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m6A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m6A modification in spermatogenesis and reproduction in mammals.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

The syncytiotrophoblast (STB) plays a key role in maintaining the function of the placenta during human pregnancy. However, the molecular network that orchestrates STB development remains elusive. The aim of this study was to obtain broad and deep insight into human STB formation via transcriptomics. We adopted RNA sequencing (RNA-Seq) to investigate genes and isoforms involved in forskolin (FSK)-induced fusion of BeWo cells. BeWo cells were treated with 50 μM FSK or dimethyl sulfoxide (DMSO) as a vehicle control for 24 and 48 h, and the mRNAs at 0, 24 and 48 h were sequenced. We detected 28,633 expressed genes and identified 1902 differentially expressed genes (DEGs) after FSK treatment for 24 and 48 h. Among the 1902 DEGs, 461 were increased and 395 were decreased at 24 h, whereas 879 were upregulated and 763 were downregulated at 48 h. When the 856 DEGs identified at 24 h were traced individually at 48 h, they separated into 6 dynamic patterns via a K-means algorithm, and most were enriched in down–even and up–even patterns. Moreover, the gene ontology (GO) terms syncytium formation, cell junction assembly, cell fate commitment, calcium ion transport, regulation of epithelial cell differentiation and cell morphogenesis involved in differentiation were clustered, and the MAPK pathway was most significantly regulated. Analyses of alternative splicing isoforms detected 123,200 isoforms, of which 1376 were differentially expressed. The present deep analysis of the RNA-Seq data of BeWo cell fusion provides important clues for understanding the mechanisms underlying human STB formation.

N6-methyladenosine (m6A) is one of the most abundant modifications on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex (MTC) containing a key factor methyltransferase-like 3 (Mettl3). However, the functions of Mettl3 and m6A modification in hepatic lipid and glucose metabolism remain unclear. Here, we showed that both Mettl3 expression and m6A level increased in the livers of mice with high fat diet (HFD)-induced metabolic disorders. Overexpression of Mettl3 aggravated HFD-induced liver metabolic disorders and insulin resistance. In contrast, hepatocyte-specific knockout of Mettl3 significantly alleviated HFD-induced metabolic disorders by slowing weight gain, reducing lipid accumulation, and improving insulin sensitivity. Mechanistically, Mettl3 depletion-mediated m6A loss caused extended RNA half-lives of metabolism-related genes, which consequently protected mice against HFD-induced metabolic syndrome. Our findings reveal a critical role of Mettl3-mediated m6A in HFD-induced metabolic disorders and hepatogenous diabetes.

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 β-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.

In intimate mutualisms between hosts and symbionts, selection can act repeatedly over the development times of the interacting individuals. Although much is now known about the overall ecological conditions that favor the evolution of mutualism, a current challenge is to understand how natural selection acts on the number and kinds of partners to shape the evolution and stability of these interactions. Using the obligate fig-fig wasp mutualism, our experiments showed that the proportion of figs developed to maturity increased quickly to 1.0 as the number of foundresses increased, regardless of whether the foundresses carried pollen. Selection against pollen-free wasps did not occur at this early stage in fig development. Within figs that developed, the proportion of galls producing adult wasps remained high as the number of pollen-carrying foundresses increases. In contrast, the proportion of galls producing adult wasps decreased as the number of pollen-free foundresses increased. Viable seed production increased as the number or proportion of pollen-carrying foundresses increased, but the average number of wasp offspring per pollen-carrying foundress was highest when she was the sole foundress. These results show that figs and their pollinator wasps differ in how fitness effects are distributed throughout the development of the interaction and depend on the number and proportion of pollen-carrying foundresses contributing to the interaction. These results suggest that temporal fluctuations in the local number and proportion of pollen-carrying wasps available to enter figs are likely to have strong but different effects on the figs and the wasps.

RNA-binding proteins (RBPs) play pivotal roles in directing RNA fate and function. Yet the current annotation of RBPs is largely limited to proteins carrying known RNA-binding domains. To systematically reveal dynamic RNA–protein interactions, we surveyed the human proteome by a protein array-based approach and identified 671 proteins with RNA-binding activity. Among these proteins, 525 lack annotated RNA-binding domains and are enriched in transcriptional and epigenetic regulators, metabolic enzymes, and small GTPases. Using an improved CLIP (crosslinking and immunoprecipitation) method, we performed genome-wide target profiling of isocitrate dehydrogenase 1 (IDH1), a novel RBP. IDH1 binds to thousands of RNA transcripts with enriched functions in transcription and chromatin regulation, cell cycle and RNA processing. Purified IDH1, but not an oncogenic mutant, binds directly to GA- or AU-rich RNA that are also enriched in IDH1 CLIP targets. Our study provides useful resources of unconventional RBPs and IDH1-bound transcriptome, and convincingly illustrates, for the first time, the in vivo and in vitro RNA targets and binding preferences of IDH1, revealing an unanticipated complexity of RNA regulation in diverse cellular processes.

Early embryogenesis in vertebrates is initially directed by maternal RNAs and proteins, yet the mechanisms controlling this program remain largely unknown. Recent transcriptome-wide studies on RNA structure have revealed its pervasive and crucial roles in RNA processing and functions. Here we established the first global map of four nucleotide-based mRNA structures during zebrafish early embryogenesis. Strikingly, we observed RNA structurally variable regions are enriched in the 3’ UTR, and contain cis-regulatory elements important for maternal-to-zygotic transition (MZT). We found that the RNA binding protein Elavl1a stabilized maternal mRNAs by binding to the cis-elements. Conversely, RNA structure formation suppressed Elavl1a’s binding leading to the decay of its maternal targets. Overall, our findings revealed a broad and fundamental role of RNA structure-based regulation in vertebrate early embryogenesis.

Preimplantation genetic testing (PGT) requires an invasive biopsy to obtain embryonic material for genetic analysis. The availability of a less invasive procedure would increase the overall efficacy of PGT. The aim of the study was to explore the potential of blastocoele fluid (BF) as an alternative source of embryonic DNA for PGT. Collection of BF was performed by aspiration with a fine needle prior to vitrification. BF DNA was subjected to whole-genome amplification (WGA) and analyzed by high-resolution next-generation sequencing (NGS). A high-quality WGA product was obtained from 8 of 11 (72.7 %) samples. Comparison of matching BF and blastomere samples showed that the genomic representation of sequencing reads was consistently similar with respect to density and regional coverage across the 24 chromosomes. A genome-wide survey of the sample sequencing data also indicated that BF was highly representative of known single gene sequences, and this observation was validated by PCR analyses of ten randomly selected genes, with an overall efficiency of 84 %. This study provides further evidence that BF is a promising alternative source of DNA for PGT.

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

5-Methylcytosine (m

5-Methylcytosine (m5C) is a posttranscriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. Many known or novel m5C sites have been validated by using advanced high-throughput techniques combined with next-generation sequencing (NGS), especially RNA bisulfite sequencing (RNA-BisSeq). Here we introduce an optimized RNA-BisSeq method by using ACT random hexamers to prime the reverse transcription of bisulfite-treated RNA samples to detect the m5C sites.

Epigenetic modifications include the chemical modifications on DNA, RNA and proteins characterized by altered gene expression and function without any changes in the gene sequence. In addition to well-established DNA and protein epigenetic modifications, the reversible RNA methylation has led the third wave of studies in the epigenetic field. RNA has more than 100 chemical modifications, among which methylation is the major type. The identification of catalyzing enzymes for RNA methylation and the development of high-throughput detection technologies for RNA modification at the transcriptomic level are the prerequisites for revealing the regulatory role of RNA methylation in gene expression and biological functions. In this review, we summarize the recent frontier in RNA methylation-mediated epitranscriptomics from our and other laboratories, with emphasis on the discoveries of RNA modification demethylase , methyltransferase and binding protein as well as the illustration of regulatory roles of RNA methylation modification in hematopoietic stem cell differentiation, spermatogenesis, brain development and other pivotal life processes. These findings have shown that RNA methylation is just as reversible as DNA methylation, and opened up a novel field in RNA methylation-mediated epitranscriptomics, which appends a new layer of epigenetic regulation to the central genetic dogma.

The paper outlines the configuration and the hardware system architecture of the quad-rotor aircraft, describes the overall design of the hardware system of the quad-rotor aircraft, expounds the design method of each subsystem of the hardware system of the quad-rotor aircraft, and finally gets the complete hardware system of the quad-rotor aircraft by joining the design results of subsystems.

In some insect nursery pollination mutualisms, plant hosts impose net costs to uncooperative "cheater" symbionts. These "sanctions" promote mutualism stability but their precise adaptive nature remains unclear. In fig-wasp mutualisms host trees (Ficus spp.) are only pollinated by female agaonid wasps whose larvae only use galled fig flowers as food. In actively pollinated systems, if wasps fail to pollinate, sanctions can result via fig abortion, killing all wasp offspring, or by increased offspring mortality within un-aborted figs. These sanctions result from selective investment to pollinated inflorescences, a mechanism present in almost all angiosperms. To more fully understand how selective investment functions as sanctions requires the measurement of variation in their costs and benefits to both hosts and symbionts. Gynodioecious fig-tree-fig-wasp mutualisms are particularly suitable for this because pollen and wasps are produced only in the figs of "male" trees and seeds only in the figs of "female" trees. Male and female trees thus incur different net costs of pollen absence, and costs of sanctions to pollen-free "cheater" wasps only occur in male trees. We used the actively pollinated host tree Ficus hispida and introduced into male and female figs either 1, 3, 5, 7, or 9 all pollen-laden "cooperative" (P+) or all pollen-free "cheater" (P-) wasps. Abortion in both male and female trees was highest in P- figs, with P- fig abortion higher in females (~90%) than in males (~40%). Fig abortion was negatively associated with foundress number mainly in P+ figs; in P- figs abortion was only weakly associated with the number of "cheater" wasps, especially in female figs. In un-aborted male figs, wasp offspring mortality was higher in P- figs than in P+ figs, and in P- figs correlated positively with foundress (cheater) number. Increased offspring mortality was biased against female wasp offspring and likely resulted from reduced larval nutrition in unpollinated flowers. Variation in selective investment to P- figs thus reflects costs and benefits of pollen absence/presence to hosts, variation that translates directly to net costs to cheater wasps.

Summary Sanctioning or punishing is regarded as one of the most important dynamics in the evolution of cooperation. However, it has not been empirically examined yet whether or not such enforcement selection by sanctioning or punishing and classical theories like kin or reciprocity selection are separate mechanisms contributing to the evolution of cooperation. In addition, it remains largely unclear what factors determine the intensity or effectiveness of sanction. Here, we show that in the obligate, interspecific cooperation between figs and fig wasps, the hosted figs can discriminatively sanction cheating individuals by decreasing the offspring development ratio. Concurrently, the figs can reward the cooperative pollinators with a higher offspring development ratio. This sanction intensity and effectiveness largely depend on how closely the host and symbiont are related either in terms of reciprocity exchange or genetic similarity as measured by the reciprocal of the foundress number. Our results imply that in asymmetric systems, symbionts might be forced to evolve to be cooperative or even altruistic through discriminative sanction against the noncooperative symbiont and reward to the cooperative symbiont by the host (i.e. through a game of ‘carrot and stick’).

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional mechanism that targets aberrant transcripts and regulates the cellular RNA reservoir. Genetic modulation in vertebrates suggests that NMD is critical for cellular and tissue homeostasis, although the underlying mechanism remains elusive. Here, we generate knockout mice lacking Smg6/Est1, a key nuclease in NMD and a telomerase cofactor. While the complete loss of Smg6 causes mouse lethality at the blastocyst stage, inducible deletion of Smg6 is compatible with embryonic stem cell (ESC) proliferation despite the absence of telomere maintenance and functional NMD. Differentiation of Smg6-deficient ESCs is blocked due to sustained expression of pluripotency genes, normally repressed by NMD, and forced down-regulation of one such target, c-Myc, relieves the differentiation block. Smg6-null embryonic fibroblasts are viable as well, but are refractory to cellular reprograming into induced pluripotent stem cells (iPSCs). Finally, depletion of all major NMD factors compromises ESC differentiation, thus identifying NMD as a licensing factor for the switch of cell identity in the process of stem cell differentiation and somatic cell reprograming.

Discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes has excited the field of epigenetics, but information on the genome-wide distribution of 5hmC is limited. Globozoospermia is a rare but severe cause of male infertility. To date, the epigenetic mechanism, especially 5hmC profiles involved in globozoospermia progression, remains largely unknown. Here, utilizing the chemical labeling and biotin-enrichment approach followed by Illumina HiSeq sequencing, we showed that (i) 6664, 9029 and 6318 genes contain 5hmC in normal, abnormal, and globozoospermia sperm, respectively; (ii) some 5hmC-containing genes significantly involves in spermatogenesis, sperm motility and morphology, and gamete generation; (iii) 5hmC is exclusively localized in sperm intron; (iv) approximately 40% imprinted genes have 5hmC modification in sperm genomes, but globozoospermia sperm exhibiting a large portion of imprinted genes lose the 5hmC modification; (v) six imprinted genes showed different 5hmC patterns in abnormal sperm (GDAP1L1, GNAS, KCNK9, LIN28B, RB1, RTL1), and five imprinted genes showed different 5hmC patterns in globozoospermia sperm (KCNK9, LIN28B, RB1, SLC22A18, ZDBF2). These results suggested that differences in genome-wide 5hmC patterns may in part be responsible for the sperm phenotype. All of this may improve our understanding of the basic molecular mechanism underlying sperm biology and the etiology of male infertility.

Recent studies on enzymes regulating dynamic N6-methyl-adenosine (m6A) in RNA together with the findings from m6A-methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq/m6Aseq) have revealed a broad biological role of m6A in RNA processing, development, differentiation, metabolism and fertility. RNA m6A methylation is catalyzed by a multicomponent methyltransferase complex composed of at least three subunits: METTL3, METTL14 and Wilms tumor 1-associated protein (WTAP), in which METTL3 and METTL14 serve as catalytic subunits, while WTAP as regulatory subunit. Dioxygenases FTO and ALKBH5, as the first two known m6A demethylases, catalyze m6A removal. Five m6A-binding proteins are classified into cytoplasmic YT521-B homology (YTH) domain-containing family YTHDF1-3 and nuclear YTHDC1-2. Perturbation of enzymatic activities catalyzing dynamic m6A results in altered expression of thousands of genes and affectsmRNAstability and splicing at the cellular level. Here, we summarize recent discoveries about m6A methyltransferases (writers), demethylases (erasers) and binding proteins (readers), and further discuss the potential impacts of m6A on RNA processing, especially on mRNA splicing.

METTL3 catalyzes the formation of N6-methyl-adenosine (m6A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m6A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m6A modification in spermatogenesis and reproduction in mammals.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

In some mutualisms, cooperation in symbionts is promoted by hosts sanctioning "cheats," who obtain benefits but fail to reciprocate. In fig-wasp mutualisms, agaonid wasps pollinate the trees (Ficus spp.), but are also exploitative by using some flowers as larval food. Ficus can sanction cheats that fail to pollinate by aborting some un-pollinated figs. However, in those un-pollinated figs retained by trees, cheats successfully reproduce. When this occurs, wasp broods are reduced, suggesting sanctions increase offspring mortality within un-pollinated figs. We investigated sanction mechanisms of abortion and larval mortality against wasp cheats in the monoecious Ficus racemosa by introducing into figs 1, 3, 5, 7, or 9 female wasps (foundresses) that were either all pollen-laden (P+) or all pollen-free (P-). The abortion rates of P- figs were highest (-60%) when single foundresses were present. Abortion declined with increased foundresses and ceased with seven or more wasps present, irrespective of pollination. In un-aborted figs, wasp fitness (mean offspring per foundress) declined as foundress number increased, especially in P- figs. Reduced broods in P- figs resulted from increased larval mortality of female offspring as foundress number increased, resulting in more male-biased sex ratios. Overall sanctions estimated from both abortion rates and reduced offspring production strengthened as the number of cheats increased. In a second experiment, we decoupled pollination from wasp oviposition by introducing one pollen-free foundress, followed 24 h later by seven pollen-laden ovipositor-excised wasps. Compared with P+ and P- single-foundress figs, delayed pollination resulted in intermediate larval mortality and wasp fitness, which concurred with patterns of female offspring production. We conclude that fig abortion reflects both pollinator numbers and pollen presence. Sanctions within P- figs initiate soon after oviposition and discriminate against female offspring, thus reducing the benefits to cheats from adaptively biasing their offspring sex ratios. We suggest that costs to cheats via these discriminative sanctions are likely to promote stability in this mutualism.

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.