Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells.

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.

Altered nucleocytoplasmic export of adenosine-rich circRNAs by PABPC1 contributes to neuronal function.

Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.

Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment.

Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying CsfcrRNA complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the CsfcrRNA complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.

Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

Creation of memory-memory entanglement in a metropolitan quantum network.

Towards realizing the future quantum internet1,2, a pivotal milestone entails the transition from two-node proof-of-principle experiments conducted in laboratories to comprehensive multi-node set-ups on large scales. Here we report the creation of memory-memory entanglement in a multi-node quantum network over a metropolitan area. We use three independent memory nodes, each of which is equipped with an atomic ensemble quantum memory3 that has telecom conversion, together with a photonic server where detection of a single photon heralds the success of entanglement generation. The memory nodes are maximally separated apart for 12.5 kilometres. We actively stabilize the phase variance owing to fibre links and control lasers. We demonstrate concurrent entanglement generation between any two memory nodes. The memory lifetime is longer than the round-trip communication time. Our work provides a metropolitan-scale testbed for the evaluation and exploration of multi-node quantum network protocols and starts a stage of quantum internet research.

Cytoplasmic SIRT6-mediated ACSL5 deacetylation impedes nonalcoholic fatty liver disease by facilitating hepatic fatty acid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive hepatic lipid accumulation, which can progress to nonalcoholic steatohepatitis (NASH). Histone deacetylase Sirtuin 6 (SIRT6) regulates NAFLD by regulating metabolism-related gene expression, but an extrachromosomal role for SIRT6 in NAFLD development remains elusive. We investigated whether SIRT6 functions on NAFLD in the cytoplasm. We found that SIRT6 binds saturated fatty acids, especially palmitic acid. This binding leads to its nuclear export, where it deacetylates long-chain acyl-CoA synthase 5 (ACSL5), thereby facilitating fatty acid oxidation. High-fat diet-induced NAFLD is suppressed by ACSL5 hepatic overexpression but is exacerbated by its depletion. As confirmation, overexpression of a deacetylated ACSL5 mimic attenuated NAFLD in Sirt6 liver-specific knockout mice. Moreover, NASH-hepatic tissues from both patients and diet-fed mice exhibited significantly reduced cytoplasmic SIRT6 levels and increased ACSL5 acetylation. The SIRT6/ACSL5 signaling pathway has a critical role in NAFLD progression and might constitute an avenue for therapeutic intervention.

Programming and inheritance of parental DNA methylomes in mammals.

The reprogramming of parental methylomes is essential for embryonic development. In mammals, paternal 5-methylcytosines (5mCs) have been proposed to be actively converted to oxidized bases. These paternal oxidized bases and maternal 5mCs are believed to be passively diluted by cell divisions. By generating single-base resolution, allele-specific DNA methylomes from mouse gametes, early embryos, and primordial germ cell (PGC), as well as single-base-resolution maps of oxidized cytosine bases for early embryos, we report the existence of 5hmC and 5fC in both maternal and paternal genomes and find that 5mC or its oxidized derivatives, at the majority of demethylated CpGs, are converted to unmodified cytosines independent of passive dilution from gametes to four-cell embryos. Therefore, we conclude that paternal methylome and at least a significant proportion of maternal methylome go through active demethylation during embryonic development. Additionally, all the known imprinting control regions (ICRs) were classified into germ-line or somatic ICRs.

Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane.

The entry of SARS-CoV-2 into host cells depends on the refolding of the virus-encoded spike protein from a prefusion conformation, which is metastable after cleavage, to a lower-energy stable postfusion conformation1,2. This transition overcomes kinetic barriers for fusion of viral and target cell membranes3,4. Here we report a cryogenic electron microscopy (cryo-EM) structure of the intact postfusion spike in a lipid bilayer that represents the single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membrane-interacting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos.

5-methylcytosine is a major epigenetic modification that is sometimes called "the fifth nucleotide." However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo's methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos.

An integrated space-to-ground quantum communication network over 4,600 kilometres.

Quantum key distribution (QKD)1,2 has the potential to enable secure communication and information transfer3. In the laboratory, the feasibility of point-to-point QKD is evident from the early proof-of-concept demonstration in the laboratory over 32 centimetres4; this distance was later extended to the 100-kilometre scale5,6 with decoy-state QKD and more recently to the 500-kilometre scale7-10 with measurement-device-independent QKD. Several small-scale QKD networks have also been tested outside the laboratory11-14. However, a global QKD network requires a practically (not just theoretically) secure and reliable QKD network that can be used by a large number of users distributed over a wide area15. Quantum repeaters16,17 could in principle provide a viable option for such a global network, but they cannot be deployed using current technology18. Here we demonstrate an integrated space-to-ground quantum communication network that combines a large-scale fibre network of more than 700 fibre QKD links and two high-speed satellite-to-ground free-space QKD links. Using a trusted relay structure, the fibre network on the ground covers more than 2,000 kilometres, provides practical security against the imperfections of realistic devices, and maintains long-term reliability and stability. The satellite-to-ground QKD achieves an average secret-key rate of 47.8 kilobits per second for a typical satellite pass-more than 40 times higher than achieved previously. Moreover, its channel loss is comparable to that between a geostationary satellite and the ground, making the construction of more versatile and ultralong quantum links via geosynchronous satellites feasible. Finally, by integrating the fibre and free-space QKD links, the QKD network is extended to a remote node more than 2,600 kilometres away, enabling any user in the network to communicate with any other, up to a total distance of 4,600 kilometres.

Anti-Epstein-Barr Virus BNLF2b for Mass Screening for Nasopharyngeal Cancer.

BACKGROUND: Population screening of asymptomatic persons with Epstein-Barr virus (EBV) DNA or antibodies has improved the diagnosis of nasopharyngeal carcinoma and survival among affected persons. However, the positive predictive value of current screening strategies is unsatisfactory even in areas where nasopharyngeal carcinoma is endemic. METHODS: We designed a peptide library representing highly ranked B-cell epitopes of EBV coding sequences to identify novel serologic biomarkers for nasopharyngeal carcinoma. After a retrospective case-control study, the performance of the novel biomarker anti-BNLF2b total antibody (P85-Ab) was validated through a large-scale prospective screening program and compared with that of the standard two-antibody-based screening method (EBV nuclear antigen 1 [EBNA1]-IgA and EBV-specific viral capsid antigen [VCA]-IgA). RESULTS: P85-Ab was the most promising biomarker for nasopharyngeal carcinoma screening, with high sensitivity (94.4%; 95% confidence interval [CI], 86.4 to 97.8) and specificity (99.6%; 95% CI, 97.8 to 99.9) in the retrospective case-control study. Among the 24,852 eligible participants in the prospective cohort, 47 cases of nasopharyngeal carcinoma (38 at an early stage) were identified. P85-Ab showed higher sensitivity than the two-antibody method (97.9% vs. 72.3%; ratio, 1.4 [95% CI, 1.1 to 1.6]), higher specificity (98.3% vs. 97.0%; ratio, 1.01 [95% CI, 1.01 to 1.02]), and a higher positive predictive value (10.0% vs. 4.3%; ratio, 2.3 [95% CI, 1.8 to 2.8]). The combination of P85-Ab and the two-antibody method markedly increased the positive predictive value to 44.6% (95% CI, 33.8 to 55.9), with sensitivity of 70.2% (95% CI, 56.0 to 81.4). CONCLUSIONS: Our results suggest that P85-Ab is a promising novel biomarker for nasopharyngeal carcinoma screening, with higher sensitivity, specificity, and positive predictive value than the standard two-antibody method. (Funded by the National Key Research and Development Program of China and others; ClinicalTrials.gov number, NCT04085900.).

Characterization of network hierarchy reflects cell state specificity in genome organization.

Dynamic chromatin structure acts as the regulator of transcription program in crucial processes including cancer and cell development, but a unified framework for characterizing chromatin structural evolution remains to be established. Here, we performed graph inferences on Hi-C data sets and derived the chromatin contact networks. We discovered significant decreases in information transmission efficiencies in chromatin of colorectal cancer (CRC) and T-cell acute lymphoblastic leukemia (T-ALL) compared to corresponding normal controls through graph statistics. Using network embedding in the Poincaré disk, the hierarchy depths of chromatin from CRC and T-ALL patients were found to be significantly shallower compared to their normal controls. A reverse trend of change in chromatin structure was observed during early embryo development. We found tissue-specific conservation of hierarchy order in chromatin contact networks. Our findings reveal the top-down hierarchy of chromatin organization, which is significantly attenuated in cancer.

Dual-channel hypergraph convolutional network for predicting herb-disease associations.

Herbs applicability in disease treatment has been verified through experiences over thousands of years. The understanding of herb-disease associations (HDAs) is yet far from complete due to the complicated mechanism inherent in multi-target and multi-component (MTMC) botanical therapeutics. Most of the existing prediction models fail to incorporate the MTMC mechanism. To overcome this problem, we propose a novel dual-channel hypergraph convolutional network, namely HGHDA, for HDA prediction. Technically, HGHDA first adopts an autoencoder to project components and target protein onto a low-dimensional latent space so as to obtain their embeddings by preserving similarity characteristics in their original feature spaces. To model the high-order relations between herbs and their components, we design a channel in HGHDA to encode a hypergraph that describes the high-order patterns of herb-component relations via hypergraph convolution. The other channel in HGHDA is also established in the same way to model the high-order relations between diseases and target proteins. The embeddings of drugs and diseases are then aggregated through our dual-channel network to obtain the prediction results with a scoring function. To evaluate the performance of HGHDA, a series of extensive experiments have been conducted on two benchmark datasets, and the results demonstrate the superiority of HGHDA over the state-of-the-art algorithms proposed for HDA prediction. Besides, our case study on Chuan Xiong and Astragalus membranaceus is a strong indicator to verify the effectiveness of HGHDA, as seven and eight out of the top 10 diseases predicted by HGHDA for Chuan-Xiong and Astragalus-membranaceus, respectively, have been reported in literature.

Functional importance and divergence of plant apurinic/apyrimidinic endonucleases in somatic and meiotic DNA repair.

Apurinic/apyrimidinic (AP) sites are one of the most abundant DNA lesions and are mainly repaired by AP endonucleases (APEs). While most eukaryotic genomes encode two APEs, plants usually possess three APEs, namely APE1L, APE2, and ARP. To date, the biological relevance and functional divergence of plant APEs are unclear. Here, we show that the three plant APEs have ancient origins, with the APE1L clade being plant-specific. In Arabidopsis thaliana, simultaneously mutating APE1L and APE2, but not ARP alone or in combination with either APE1L or APE2, results in clear developmental defects linked to genotoxic stress. Genetic analyses indicated that the three plant APEs have different substrate preferences in vivo. ARP is mainly responsible for AP site repair, while APE1L and APE2 prefer to repair 3'-blocked single-stranded DNA breaks. We further determined that APEs play an important role in DNA repair and the maintenance of genomic integrity in meiotic cells. The ape1l ape2 double mutant exhibited a greatly enhanced frequency of sporulation 1 (SPO11-1)-dependent and SPO11-1-independent double-stranded DNA breaks. The DNA damage response (DDR) was activated in ape1l ape2 to trigger pollen abortion. Our findings suggest functional divergence of plant APEs and reveal important roles of plant APEs during vegetative and reproductive development.

Target ssDNA activates the NADase activity of prokaryotic SPARTA immune system.

Argonaute proteins (Agos), which use small RNAs or DNAs as guides to recognize complementary nucleic acid targets, mediate RNA silencing in eukaryotes. In prokaryotes, Agos are involved in immunity: the short prokaryotic Ago/TIR-APAZ (SPARTA) immune system triggers cell death by degrading NAD+ in response to invading plasmids, but its molecular mechanisms remain unknown. Here we used cryo-electron microscopy to determine the structures of inactive monomeric and active tetrameric Crenotalea thermophila SPARTA complexes, revealing mechanisms underlying SPARTA assembly, RNA-guided recognition of target single-stranded DNA (ssDNA) and subsequent SPARTA tetramerization, as well as tetramerization-dependent NADase activation. The small RNA guides Ago to recognize its ssDNA target, inducing SPARTA tetramerization via both Ago- and TIR-mediated interactions and resulting in a two-stranded, parallel, head-to-tail TIR rearrangement primed for NAD+ hydrolysis. Our findings thus identify the molecular basis for target ssDNA-mediated SPARTA activation, which will facilitate the development of SPARTA-based biotechnological tools.

Glycolysis-Mediated Activation of v-ATPase by Nicotinamide Mononucleotide Ameliorates Lipid-Induced Cardiomyopathy by Repressing the CD36-TLR4 Axis.

BACKGROUND: Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a pivotal role for the proton-pump vacuolar-type H+-ATPase (v-ATPase), which acidifies endosomes, and for lipid-transporter CD36, which is stored in acidified endosomes. During lipid overexposure, an increased influx of lipids into cardiomyocytes is sensed by v-ATPase, which then disassembles, causing endosomal de-acidification and expulsion of stored CD36 from the endosomes toward the sarcolemma. Once at the sarcolemma, CD36 not only increases lipid uptake but also interacts with inflammatory receptor TLR4 (Toll-like receptor 4), together resulting in lipid-induced insulin resistance, inflammation, fibrosis, and cardiac dysfunction. Strategies inducing v-ATPase reassembly, that is, to achieve CD36 reinternalization, may correct these maladaptive alterations. For this, we used NAD+ (nicotinamide adenine dinucleotide)-precursor nicotinamide mononucleotide (NMN), inducing v-ATPase reassembly by stimulating glycolytic enzymes to bind to v-ATPase. METHODS: Rats/mice on cardiomyopathy-inducing high-fat diets were supplemented with NMN and for comparison with a cocktail of lysine/leucine/arginine (mTORC1 [mechanistic target of rapamycin complex 1]-mediated v-ATPase reassembly). We used the following methods: RNA sequencing, mRNA/protein expression analysis, immunofluorescence microscopy, (co)immunoprecipitation/proximity ligation assay (v-ATPase assembly), myocellular uptake of [3H]chloroquine (endosomal pH), and [14C]palmitate, targeted lipidomics, and echocardiography. To confirm the involvement of v-ATPase in the beneficial effects of both supplementations, mTORC1/v-ATPase inhibitors (rapamycin/bafilomycin A1) were administered. Additionally, 2 heart-specific v-ATPase-knockout mouse models (subunits V1G1/V0d2) were subjected to these measurements. Mechanisms were confirmed in pharmacologically/genetically manipulated cardiomyocyte models of lipid overload. RESULTS: NMN successfully preserved endosomal acidification during myocardial lipid overload by maintaining v-ATPase activity and subsequently prevented CD36-mediated lipid accumulation, CD36-TLR4 interaction toward inflammation, fibrosis, cardiac dysfunction, and whole-body insulin resistance. Lipidomics revealed C18:1-enriched diacylglycerols as lipid class prominently increased by high-fat diet and subsequently reversed/preserved by lysine/leucine/arginine/NMN treatment. Studies with mTORC1/v-ATPase inhibitors and heart-specific v-ATPase-knockout mice further confirmed the pivotal roles of v-ATPase in these beneficial actions. CONCLUSION: NMN preserves heart function during lipid overload by preventing v-ATPase disassembly.

Unearthing a novel function of SRSF1 in binding and unfolding of RNA G-quadruplexes.

SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but not other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.

Persistent corotation of the large-scale flow of thermal convection and an immersed free body.

Inspired by the superrotation of the Earth's solid core, we investigate the dynamics of a free-rotating body as it interacts with the large-scale circulation (LSC) of the Rayleigh-Bénard thermal convection in a cylindrical container. A surprising and persistent corotation of both the free body and the LSC emerges, breaking the axial symmetry of the system. The corotational speed increases monotonically with the intensity of thermal convection, measured by the Rayleigh number Ra, which is proportional to the temperature difference between the heated bottom and cooled top. The rotational direction occasionally and spontaneously reverses, occurring more frequently at higher Ra. The reversal events follow a Poisson process; it is feasible that flow fluctuations randomly interrupt and reestablish the rotation-sustaining mechanism. This corotation is powered by thermal convection alone and promoted by the addition of a free body, enriching the classical dynamical system.

Methionine biosynthesis enzyme MoMet2 is required for rice blast fungus pathogenicity by promoting virulence gene expression via reducing 5mC modification.

The emergence of fungicide resistance severely threatens crop production by limiting the availability and application of established fungicides. Therefore, it is urgent to identify new fungicidal targets for controlling plant diseases. Here, we characterized the function of a conserved homoserine O-acetyltransferase (HOA) from the rice blast fungus Magnaporthe oryzae that could serve as the candidate antifungal target. Deletion of the MoMET2 and MoCYS2 genes encoding HOAs perturbed the biosynthesis of methionine and S-adenyl methionine, a methyl group donor for epigenetic modifications, and severely attenuated the development and virulence of M. oryzae. The ∆Momet2 mutant is significantly increased in 5-methylcytosine (5mC) modification that represses the expression of genes required for pathogenicity, including MoGLIK and MoCDH-CYT. We further showed that host-induced gene silencing (HIGS) targeting MoMET2 and MoCYS2 effectively controls rice blasts. Our studies revealed the importance of HOA in the development and virulence of M. oryzae, which suggests the potential feasibility of HOA as new targets for novel anti-rice blast measurements.

Fine structure and assembly pattern of a minimal myophage Pam3.

The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.