m6 A promotes planarian regeneration.

Regeneration is the regrowth of damaged tissues or organs, a vital process in response to damages from primitive organisms to higher mammals. Planarian possesses active whole-body regenerative capability owing to its vast reservoir of adult stem cells, neoblasts, providing an ideal model to delineate the underlying mechanisms for regeneration. RNA N6 -methyladenosine (m6 A) modification participates in many biological processes, including stem cell self-renewal and differentiation, in particular the regeneration of haematopoietic stem cells and axons. However, how m6 A controls regeneration at the whole-organism level remains largely unknown. Here, we demonstrate that the depletion of m6 A methyltransferase regulatory subunit wtap abolishes planarian regeneration, potentially through regulating genes related to cell-cell communication and cell cycle. Single-cell RNA-seq (scRNA-seq) analysis unveils that the wtap knockdown induces a unique type of neural progenitor-like cells (NP-like cells), characterized by specific expression of the cell-cell communication ligand grn. Intriguingly, the depletion of m6 A-modified transcripts grn, cdk9 or cdk7 partially rescues the defective regeneration of planarian caused by wtap knockdown. Overall, our study reveals an indispensable role of m6 A modification in regulating whole-organism regeneration.

Single-cell transcriptome profiling of the vaginal wall in women with severe anterior vaginal prolapse.

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.

m6A Regulates Liver Metabolic Disorders and Hepatogenous Diabetes.

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.

RNA structural dynamics regulate early embryogenesis through controlling transcriptome fate and function.

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.

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.

Dynamic methylome of internal mRNA N7-methylguanosine and its regulatory role in translation.

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. N7-methylguanosine (m7G) 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, m7G 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 m7G individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (m7G miCLIP-seq) to specifically detect internal mRNA m7G modification. Using this approach, we revealed that m7G 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 m7G modification is dynamically regulated under both H2O2 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 m7G modification site displays both enriched m7G modification and increased mRNA translation upon H2O2 treatment compared to the m7G site-mutated minigene reporter (G to A). Taken together, our findings unravel the dynamic profiles of internal mRNA m7G methylome and highlight m7G as a novel epitranscriptomic marker with regulatory roles in translation.

RNA 5-Methylcytosine Facilitates the Maternal-to-Zygotic Transition by Preventing Maternal mRNA Decay.

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.

Author Correction: Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma.

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.

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.

Foundress numbers and the timing of selective events during interactions between figs and fig wasps.

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.

Insight into novel RNA-binding activities via large-scale analysis of lncRNA-bound proteome and IDH1-bound transcriptome.

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.

Differential deployment of sanctioning mechanisms by male and female host trees in a gynodioecious fig-wasp mutualism.

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.

5-Methylcytosine Analysis by RNA-BisSeq.

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.

[RNA methylation: regulations and mechanisms].

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.

METTL3-mediated m6A modification is required for cerebellar development.

N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion-induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.

A Balance of Yki/Sd Activator and E2F1/Sd Repressor Complexes Controls Cell Survival and Affects Organ Size.

The Hippo/Yki and RB/E2F pathways both regulate tissue growth by affecting cell proliferation and survival, but interactions between these parallel control systems are poorly defined. In this study, we demonstrate that interaction between Drosophila E2F1 and Sd disrupts Yki/Sd complex formation and thereby suppresses Yki target gene expression. RBF modifies these effects by reducing E2F1/Sd interaction. This regulation has significant effects on apoptosis, organ size, and progenitor cell proliferation. Using a combination of DamID-seq and RNA-seq, we identified a set of Yki targets that play a diversity of roles during development and are suppressed by E2F1. Further, we found that human E2F1 competes with YAP for TEAD1 binding, affecting YAP activity, indicating that this mode of cross-regulation is conserved. In sum, our study uncovers a previously unknown mechanism in which RBF and E2F1 modify Hippo signaling responses to modulate apoptosis, organ growth, and homeostasis.

Mettl3-mediated m6A regulates spermatogonial differentiation and meiosis initiation.

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 promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.

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.