Global and single-nucleotide resolution detection of 7-methylguanosine in RNA.

RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.

Identification of a myofibroblast differentiation program during neonatal lung development.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFBs) and a stable but poorly described population of lipid-rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFBs). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single-cell RNA sequencing and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a MyoFB differentiation program that is distinct from other mesenchymal cell types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

DMRT1 regulates human germline commitment.

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages.

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

N7-methylguanosine methylation of tRNAs regulates survival to stress in cancer.

Tumour progression and therapy tolerance are highly regulated and complex processes largely dependent on the plasticity of cancer cells and their capacity to respond to stress. The higher plasticity of cancer cells highlights the need for identifying targetable molecular pathways that challenge cancer cell survival. Here, we show that N7-guanosine methylation (m7G) of tRNAs, mediated by METTL1, regulates survival to stress conditions in cancer cells. Mechanistically, we find that m7G in tRNAs protects them from stress-induced cleavage and processing into 5' tRNA fragments. Our analyses reveal that the loss of tRNA m7G methylation activates stress response pathways, sensitising cancer cells to stress. Furthermore, we find that the loss of METTL1 reduces tumour growth and increases cytotoxic stress in vivo. Our study uncovers the role of m7G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to sensitise cancer cells to chemotherapy.

NOP16 is a histone mimetic that regulates Histone H3K27 methylation and gene repression.

Post-translational modifications of histone tails alter chromatin accessibility to regulate gene expression. Some viruses exploit the importance of histone modifications by expressing histone mimetic proteins that contain histone-like sequences to sequester complexes that recognize modified histones. Here we identify an evolutionarily conserved and ubiquitously expressed, endogenous mammalian protein Nucleolar protein 16 (NOP16) that functions as a H3K27 mimic. NOP16 binds to EED in the H3K27 trimethylation PRC2 complex and to the H3K27 demethylase JMJD3. NOP16 knockout selectively globally increases H3K27me3, a heterochromatin mark, without altering methylation of H3K4, H3K9, or H3K36 or acetylation of H3K27. NOP16 is overexpressed and linked to poor prognosis in breast cancer. Depletion of NOP16 in breast cancer cell lines causes cell cycle arrest, decreases cell proliferation and selectively decreases expression of E2F target genes and of genes involved in cell cycle, growth and apoptosis. Conversely, ectopic NOP16 expression in triple negative breast cancer cell lines increases cell proliferation, cell migration and invasivity in vitro and tumor growth in vivo , while NOP16 knockout or knockdown has the opposite effect. Thus, NOP16 is a histone mimic that competes with Histone H3 for H3K27 methylation and demethylation. When it is overexpressed in cancer, it derepresses genes that promote cell cycle progression to augment breast cancer growth.

METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer.

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.

Epigenetic resetting in the human germ line entails histone modification remodeling.

Epigenetic resetting in the mammalian germ line entails acute DNA demethylation, which lays the foundation for gametogenesis, totipotency, and embryonic development. We characterize the epigenome of hypomethylated human primordial germ cells (hPGCs) to reveal mechanisms preventing the widespread derepression of genes and transposable elements (TEs). Along with the loss of DNA methylation, we show that hPGCs exhibit a profound reduction of repressive histone modifications resulting in diminished heterochromatic signatures at most genes and TEs and the acquisition of a neutral or paused epigenetic state without transcriptional activation. Efficient maintenance of a heterochromatic state is limited to a subset of genomic loci, such as evolutionarily young TEs and some developmental genes, which require H3K9me3 and H3K27me3, respectively, for efficient transcriptional repression. Accordingly, transcriptional repression in hPGCs presents an exemplary balanced system relying on local maintenance of heterochromatic features and a lack of inductive cues.

Identification of a myofibroblast differentiation program during neonatal lung development.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFB) and a stable but poorly described population of lipid rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFB). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single cell RNA sequencing, and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a myofibroblast differentiation program that is distinct form other mesenchymal cells types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

Mitochondrial RNA modifications shape metabolic plasticity in metastasis.

Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.

Transcriptomic mapping uncovers Purkinje neuron plasticity driving learning.

Cellular diversification is critical for specialized functions of the brain including learning and memory1. Single-cell RNA sequencing facilitates transcriptomic profiling of distinct major types of neuron2-4, but the divergence of transcriptomic profiles within a neuronal population and their link to function remain poorly understood. Here we isolate nuclei tagged5 in specific cell types followed by single-nucleus RNA sequencing to profile Purkinje neurons and map their responses to motor activity and learning. We find that two major subpopulations of Purkinje neurons, identified by expression of the genes Aldoc and Plcb4, bear distinct transcriptomic features. Plcb4+, but not Aldoc+, Purkinje neurons exhibit robust plasticity of gene expression in mice subjected to sensorimotor and learning experience. In vivo calcium imaging and optogenetic perturbation reveal that Plcb4+ Purkinje neurons have a crucial role in associative learning. Integrating single-nucleus RNA sequencing datasets with weighted gene co-expression network analysis uncovers a learning gene module that includes components of FGFR2 signalling in Plcb4+ Purkinje neurons. Knockout of Fgfr2 in Plcb4+ Purkinje neurons in mice using CRISPR disrupts motor learning. Our findings define how diversification of Purkinje neurons is linked to their responses in motor learning and provide a foundation for understanding their differential vulnerability to neurological disorders.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

Identification of kidney injury released circulating osteopontin as causal agent of respiratory failure.

Tissue injury can drive secondary organ injury; however, mechanisms and mediators are not well understood. To identify interorgan cross-talk mediators, we used acute kidney injury (AKI)-induced acute lung injury (ALI) as a clinically important example. Using kidney and lung single-cell RNA sequencing after AKI in mice followed by ligand-receptor pairing analysis across organs, kidney ligands to lung receptors, we identify kidney-released circulating osteopontin (OPN) as a novel AKI-ALI mediator. OPN release from kidney tubule cells triggered lung endothelial leakage, inflammation, and respiratory failure. Pharmacological or genetic OPN inhibition prevented AKI-ALI. Transplantation of ischemic wt kidneys caused AKI-ALI, but not of ischemic OPN-global knockout kidneys, identifying kidney-released OPN as necessary interorgan signal to cause AKI-ALI. We show that OPN serum levels are elevated in patients with AKI and correlate with kidney injury. Our results demonstrate feasibility of using ligand-receptor analysis across organs to identify interorgan cross-talk mediators and may have important therapeutic implications in human AKI-ALI and multiorgan failure.

Technologies and perspectives for achieving carbon neutrality.

Global development has been heavily reliant on the overexploitation of natural resources since the Industrial Revolution. With the extensive use of fossil fuels, deforestation, and other forms of land-use change, anthropogenic activities have contributed to the ever-increasing concentrations of greenhouse gases (GHGs) in the atmosphere, causing global climate change. In response to the worsening global climate change, achieving carbon neutrality by 2050 is the most pressing task on the planet. To this end, it is of utmost importance and a significant challenge to reform the current production systems to reduce GHG emissions and promote the capture of CO2 from the atmosphere. Herein, we review innovative technologies that offer solutions achieving carbon (C) neutrality and sustainable development, including those for renewable energy production, food system transformation, waste valorization, C sink conservation, and C-negative manufacturing. The wealth of knowledge disseminated in this review could inspire the global community and drive the further development of innovative technologies to mitigate climate change and sustainably support human activities.

Artificial intelligence: A powerful paradigm for scientific research.

Artificial intelligence (AI) coupled with promising machine learning (ML) techniques well known from computer science is broadly affecting many aspects of various fields including science and technology, industry, and even our day-to-day life. The ML techniques have been developed to analyze high-throughput data with a view to obtaining useful insights, categorizing, predicting, and making evidence-based decisions in novel ways, which will promote the growth of novel applications and fuel the sustainable booming of AI. This paper undertakes a comprehensive survey on the development and application of AI in different aspects of fundamental sciences, including information science, mathematics, medical science, materials science, geoscience, life science, physics, and chemistry. The challenges that each discipline of science meets, and the potentials of AI techniques to handle these challenges, are discussed in detail. Moreover, we shed light on new research trends entailing the integration of AI into each scientific discipline. The aim of this paper is to provide a broad research guideline on fundamental sciences with potential infusion of AI, to help motivate researchers to deeply understand the state-of-the-art applications of AI-based fundamental sciences, and thereby to help promote the continuous development of these fundamental sciences.

RN7SK small nuclear RNA controls bidirectional transcription of highly expressed gene pairs in skin.

Pausing of RNA polymerase II (Pol II) close to promoters is a common regulatory step in RNA synthesis, and is coordinated by a ribonucleoprotein complex scaffolded by the noncoding RNA RN7SK. The function of RN7SK-regulated gene transcription in adult tissue homoeostasis is currently unknown. Here, we deplete RN7SK during mouse and human epidermal stem cell differentiation. Unexpectedly, loss of this small nuclear RNA specifically reduces transcription of numerous cell cycle regulators leading to cell cycle exit and differentiation. Mechanistically, we show that RN7SK is required for efficient transcription of highly expressed gene pairs with bidirectional promoters, which in the epidermis co-regulated cell cycle and chromosome organization. The reduction in transcription involves impaired splicing and RNA decay, but occurs in the absence of chromatin remodelling at promoters and putative enhancers. Thus, RN7SK is directly required for efficient Pol II transcription of highly transcribed bidirectional gene pairs, and thereby exerts tissue-specific functions, such as maintaining a cycling cell population in the epidermis.

Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory.

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2 However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human.

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.