How Is Precursor Messenger RNA Spliced by the Spliceosome?

Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.

Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching.

Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B∗) is pivotal for understanding the branching reaction. In this study, we assembled the B∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B∗ complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5'-splice site (5'SS) in the three B∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5'SS, with the BPS nucleophile positioned 4 Å away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.

Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae.

The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5 Å. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 5' exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly. The DEAH family ATPase/helicase Prp43 binds Syf1 at the periphery of the spliceosome, with its RNA-binding site close to the 3' end of U6 snRNA. The C-terminal domain of Ntr1/Spp382 associates with the GTPase Snu114, and Ntr2 is anchored to Prp8 while interacting with the superhelical domain of Ntr1. These structural features suggest a plausible mechanism for the disassembly of the ILS complex.

Structure of the Post-catalytic Spliceosome from Saccharomyces cerevisiae.

Removal of an intron from a pre-mRNA by the spliceosome results in the ligation of two exons in the post-catalytic spliceosome (known as the P complex). Here, we present a cryo-EM structure of the P complex from Saccharomyces cerevisiae at an average resolution of 3.6 Å. The ligated exon is held in the active site through RNA-RNA contacts. Three bases at the 3' end of the 5' exon remain anchored to loop I of U5 small nuclear RNA, and the conserved AG nucleotides of the 3'-splice site (3'SS) are specifically recognized by the invariant adenine of the branch point sequence, the guanine base at the 5' end of the 5'SS, and an adenine base of U6 snRNA. The 3'SS is stabilized through an interaction with the 1585-loop of Prp8. The P complex structure provides a view on splice junction formation critical for understanding the complete splicing cycle.

Cervical cancer cell-derived Tie1 expression via PI3K/AKT signaling pathway promotes tumor progression.

BACKGROUND: Tie1 orphan receptor has become a focus of research, Tie1 can form a polymer with Tie2, regulate the Ang/Tie2 pathway and play a vital role in pathological angiogenesis and tumor progression, the function of Tie1 has remained uncertain in the progression of cervical cancer (CC). Here, we investigated the functional influences of Tie1 overexpress on CC in vitro and in vivo. METHODS: We used Immunohistochemistry (IHC) analysis to detect the relative expression of Tie1 in CC, and we analyzed its connection with the overall survival (OS) and progression free survival (PFS)of CC patients. To prove the role of Tie1 in cell proliferation and metastatic, Tie1 expression in CC cell lines was upregulated by lentivirus. RESULTS: The high expression of Tie1 in tumor cells of cervical cancer tissues is significantly correlated with FIGO stage, differentiated tumors, tumors with diameters, deep stromal invasion. We found that cell progression was promoted in Tie1-overexpress CC cell lines in vivo and in vitro. Tie1 potentially exerts a commanding influence on the expression of markers associated with epithelial-mesenchymal transition (EMT) and the PI3K/AKT signaling pathway. CONCLUSIONS: Our research indicates that Tie1 is highly connected to CC progression as it may play a role in the EMT process through the PI3K/AKT signaling pathway.

Boron-Nitrogen-Embedded Polycyclic Aromatic Hydrocarbon-Based Controllable Hierarchical Self-Assemblies through Synergistic Cation-π and C-H···π Interactions for Bifunctional Photo- and Electro-Catalysis.

Boron-Nitrogen-embedded polycyclic aromatic hydrocarbons (BN-PAHs) as novel π-conjugated systems have attracted immense attention owing to their superior optoelectronic properties. However, constructing long-range ordered supramolecular assemblies based on BN-PAHs remains conspicuously scarce, primarily attributed to the constraints arising from coordinating multiple noncovalent interactions and the intrinsic characteristics of BN-PAHs, which hinder precise control over delicate self-assembly processes. Herein, we achieve the successful formation of BN-PAH-based controllable hierarchical assemblies through synergistically leveraged cation-π and C-H···π interactions. By carefully adjusting the solvent conditions in two progressive assembly hierarchies, the one-dimensional (1D) supramolecular assemblies with "rigid yet flexible" assembled units are first formed by cation-π interactions, and then they can be gradually fused into two-dimensional (2D) structures under specific C-H···π interactions, thus realizing the precise control of the transformation process from BN-PAH-based 1D primary structures to 2D higher-order assemblies. The resulting 2D-BNSA, characterized by enhanced electrical conductivity and ordered 2D layered structure, provides anchoring and dispersion sites for loading two appropriate nanocatalysts, thus facilitating the efficient photocatalytic CO2 reduction (with a remarkable CH4 evolution rate of 938.7 µmol g-1 h-1) and electrocatalytic acetylene semihydrogenation (reaching a Faradaic efficiency for ethylene up to 98.5%).

In Vivo Imaging MicroRNA with Bright Fluorescent RNA Aptamer Through Target-Mediated Entropy-Driven Toehold Exchange.

MicroRNAs (miRNAs) play vital roles in biological activities, but their in vivo imaging is still challenging due to the low abundance and the lack of efficient fluorescent tools. RNA aptamers with high affinity and low background emerge for bioimaging yet suffering from low brightness. We introduce a rational design based on target-mediated entropy-driven toehold exchange (EDTE) to induce the release of RNA aptamer and subsequently light up corresponding fluorophore, which achieves selective imaging of miRNAs with good stability in both living cells and tumor-bearing mouse. Through tailoring recognition unit of the EDTE probes, highly sensitive imaging of different miRNAs including miRNA-125b and miRNA-21 is achieved, confirming its universal bioimaging applications. In comparison with the reported "one-to-one" model, the EDTE strategy shows a remarkable 4.6-time improvement in signal/noise ratio for intracellular imaging of the same miRNA. Particularly, it realizes sensitive imaging of miRNA in vivo, providing a promising tool in investigating functions and interactions of disease-associated miRNAs.

Air-Stable Na3.5C6O6 as a Sodium Compensation Additive in Cathode of Na-Ion Batteries.

Sodium-ion battery (SIB) is a candidate for the stationary energy storage systems because of the low cost and high abundance of sodium. However, the energy density and lifespan of SIBs suffer severely from the irreversible consumption of the Na-ions for the formation of the solid electrolyte interphase (SEI) layer and other side reactions on the electrodes. Here, Na3.5C6O6 is proposed as an air-stable high-efficiency sacrificial additive in the cathode to compensate for the lost sodium. It is characteristic of low desodiation (oxidation) potential (3.4-3.6 V vs. Na+/Na) and high irreversible desodiation capacity (theoretically 378 mAh g-1). The feasibility of using Na3.5C6O6 as a sodium compensation additive is verified with the improved electrochemical performances of a Na2/3Ni1/3Mn1/3Ti1/3O2ǀǀhard carbon cells and cells using other cathode materials. In addition, the structure of Na3.5C6O6 and its desodiation path are also clarified on the basis of comprehensive physical characterizations and the density functional theory (DFT) calculations. This additive decomposes completely to supply abundant Na ions during the initial charge without leaving any electrochemically inert species in the cathode. Its decomposition product C6O6 enters the carbonate electrolyte without bringing any detectable negative effects. These findings open a new avenue for elevating the energy density and/or prolonging the lifetime of the high-energy-density secondary batteries.

C1q/Tumor Necrosis Factor-Related Protein-9 Enhances Macrophage Cholesterol Efflux and Improves Reverse Cholesterol Transport via AMPK Activation.

Cholesterol efflux from foam cells in atherosclerotic plaques is crucial for reverse cholesterol transport (RCT), an important antiatherogenic event. ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1, are key receptors in the cholesterol efflux pathway. C1q/tumor necrosis factor-related protein-9 (CTRP9) is a newly discovered adipokine and exhibits an atheroprotective activity. However, the role of CTRP9 in RCT still remains unknown. In this work, we investigated the effect of subcutaneous administration of CTRP9 protein on RCT and atherosclerotic lesion formation in ApoE-/- mice fed with a high-fat diet. CTRP9-dependent regulation of cholesterol efflux and ABC transporters in RAW 264.7 foam cells was determined. Our results showed that CTRP9 protein decreased atherosclerotic lesions, increased cholesterol efflux, and upregulated liver ABCA1 and ABCG1 expression in ApoE-/- mice. CTRP9 treatment dose-dependently increased mRNA and protein expression of ABCA1, ABCG1, and LXR-α in RAW 264.7 foam cells. Moreover, the expression and phosphorylation of AMPK was potentiated upon CTRP9 treatment. Notably, CTRP9-induced cholesterol efflux and upregulation of ABCA, ABCG1, and LXR-α were impaired when AMPK was knocked down. AMPK depletion restored cholesterol accumulation in CTRP9-treated RAW 264.7 cells. Taken together, subcutaneous injection is an effective novel delivery route for CTRP9 protein, and exogenous CTRP9 can facilitate cholesterol efflux and promote RCT in an animal model of atherosclerosis. The atheroprotective activity of CTRP9 is mediated through the activation of AMPK signaling.

Transboundary cooperation in Arctic climate change governance under geopolitical tensions.

Political conflicts or geopolitical tensions can create uncertainty in addressing climate change and environmental management in the Arctic. Dissecting how actors interact with each other and form networks is important for understanding ecological and environmental management challenges during geopolitical tensions, as well as promoting better governance. We construct transboundary networks for Arctic climate change governance (ACCG) from 2013 to 2021 based on the Global Database of Events, Language, and Tone (GDELT). Further, we used network descriptive statistical analysis and Temporal Exponential Random Graph Models (TERGM) to explore the structure of ACCG networks and the key factors influencing cooperation formation. The findings suggest that the overall cooperation density of the ACCG is low, and the dominant position of core actors is continuously strengthening. Non-state actors are less likely to be seen as partners and their participation depends largely on cooperation with states. The results also show that actors with similar stances and problem exposure are more likely to cooperate, but those exposed to high latitudes often choose not to cooperate; first-comers are more likely to perceive as cooperating yet they are inclined to establish internal cooperation. Additionally, two geographically proximate actors are more likely to cooperate. This indicates that under geopolitical tensions, the ACCG faces challenges not only due to the limited capacity of non-state actors to perform transboundary functions but also because the cooperation mechanisms are influenced by regional political logic. Accordingly, we further suggest policy recommendations from developing binding international frameworks to guide transboundary cooperation, enhancing cooperation among non-state actors, and ensuring the representativeness and fairness of non-Arctic actors' participation. This research provides insights into transboundary environmental management under political tensions, while also offering new pathways for analysing large-scale environmental governance structures.

Prevention and Potential Treatment Strategies for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a significant viral pathogen that causes respiratory infections in infants, the elderly, and immunocompromised individuals. RSV-related illnesses impose a substantial economic burden worldwide annually. The molecular structure, function, and in vivo interaction mechanisms of RSV have received more comprehensive attention in recent times, and significant progress has been made in developing inhibitors targeting various stages of the RSV replication cycle. These include fusion inhibitors, RSV polymerase inhibitors, and nucleoprotein inhibitors, as well as FDA-approved RSV prophylactic drugs palivizumab and nirsevimab. The research community is hopeful that these developments might provide easier access to knowledge and might spark new ideas for research programs.

Artesunate-loaded thermosensitive chitosan hydrogel promotes osteogenesis of maxillary tooth extraction through regulating T lymphocytes in type 2 diabetic rats.

BACKGROUND: Type 2 diabetes mellitus (T2DM) causes severe bone loss after tooth extraction as a hyperglycemic environment causes aberrant bone homeostasis. Artesunate (ART) is known to possess anti-inflammation and osteogenic properties. However, its osteogenesis property in alveolar bone remains unclear. This study aimed to explore the osteogenic and immunoregulatory effects of artesunate-loaded thermosensitive chitosan hydrogel (ART-loaded TCH) on maxilla tooth extraction in T2DM rats. METHODS: T2DM rats were induced by a high-fat diet and streptozotocin. Different concentrations of ART-loaded TCH were applied in tooth extraction sockets. Bone loss and the expression of osteogenic regulatory factors (OPG, ALP, RANK) were evaluated. The immunoregulatory effects of ART-loaded TCH were observed through detecting the infiltration of T lymphocytes and their cytokines. The underlying mechanisms were explored. RESULTS: Results showed that the 150 mg/ml ART-loaded TCH group significantly ameliorated maxilla bone height and bone mineral density when compared with the T2DM group (p < 0.05). It also improved the expression of OPG, ALP, and RANK. Although the alteration of CD4+ T, CD8+ T, and CD4+:CD8+ T ratio has no significant difference among groups, the release of Th1 and Th2 in the 150 mg/ml ART-loaded TCH group has been significantly regulated than in the T2DM group (p < 0.05). Besides, ART-loaded TCH treatment inhibited the expression of p38 MAPK and ERK1 in T2DM maxilla. CONCLUSIONS: Therefore, the results indicated that 150 mg/ml ART-loaded TCH could be an effective method to prevent bone loss in T2DM tooth extraction rats by modulating the immunoregulation of Th1 and Th2 and the MAPK signaling pathway.

Multi-Functional Formaldehyde-Nitrate Batteries for Wastewater Refining, Electricity Generation, and Production of Ammonia and Formate.

As bulky pollutants in industrial and agricultural wastewater, nitrate and formaldehyde pose serious threats to the human health and ecosystem. Current purification technologies including chemical and bio-/photo-/electro-chemical methods, are generally high-cost, time-consuming, or energy-intensive. Here, we report a novel formaldehyde-nitrate battery by pairing anodic formaldehyde oxidation with cathodic nitrate reduction, which simultaneously enables wastewater purification, electricity generation, and the production of high-value-added ammonia and formate. As a result, the formaldehyde-nitrate battery remarkably exhibits an open-circuit voltage of 0.75 V, a peak power density of 3.38 mW cm-2 and the yield rates of 32.7 mg h-1 cm-2 for ammonia and 889.4 mg h-1 cm-2 for formate. In a large-scale formaldehyde-nitrate battery (25 cm2 ), 99.9 % of nitrate and 99.8 % of formaldehyde are removed from simulated industrial wastewater and the electricity of 2.03 W⋅h per day is generated. Moreover, the design of such a multi-functional battery is universally applicable to the coupling of NO3 - or NO2 - reduction with various aldehyde oxidization, paving a new avenue for wastewater purification and chemical manufacturing.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].

Bifunctional Supertetrahedral Chalcogenolate Cluster-Based Assembly Materials Constructed by a Photoactive Ligand.

The assembly of supertetrahedral chalcogenolate clusters (SCCs) and multifunctional organic linkers could lead to the formation of tunable structures and synergistic properties. Two SCC-based assembled materials (SCCAM-1 and -2) constructed by a triangular chromophore ligand, tris(4-pyridylphenyl)amine, were successfully synthesized and characterized. The SCCAMs demonstrate unusually long-lived afterglow at low temperatures (83 K) and efficient activities for the photocatalytic degradation of organic dye in water.

Research Progress on the Cardiotoxicity of EGFR-TKIs in Non-Small Cell Lung Cancer.

OPINION STATEMENT: With the development of molecular biology and histology techniques, targeted therapy for non-small cell lung cancer (NSCLC) has emerged, which is highly effective and has marginal side effects. Epidermal growth factor receptor (EGFR) was the first driver gene discovered, whose three generations of therapeutic use have its characteristics and benefits in clinical practice. However, cardiovascular complications by EGFR-tyrosine kinase inhibitors (EGFR-TKIs) in preclinical studies have been increasingly reported, including heart failure, cardiomyopathy, and QT prolongation, among others. Cardiotoxicity of targeted drugs significantly affects the therapeutic effect of NSCLC and has become the second leading cause of death in NSCLC. The aim of the present review was to recognize the potential cardiotoxicity of third-generation targeted drugs in the treatment of NSCLC and their associated mechanisms to help clinicians identify and prevent it early in the treatment, minimize the cardiotoxicity of targeted drugs, and improve the therapeutic effect of patients.

Analysis of risk factors for parenteral nutrition-associated cholestasis in preterm infants: a multicenter observational study.

BACKGROUND: It is proposed that the development of parenteral nutrition-associated cholestasis (PNAC) was significantly associated with preterm birth, low birth weight, infection, etc.; however, the etiology and pathogenesis of PNAC are not fully understood. Most of the studies examining PNAC-associated risk factors were single-center studies with relatively small sample sizes. OBJECTIVE: To analyze the risk factors associated with PNAC in preterm infants in China. METHODS: This is a retrospective multicenter observational study. Clinical data on the effect of multiple oil-fat emulsions (soybean oil-medium chain triglycerides-olive oil-fish oil, SMOF) in preterm infants were collected from a prospective multicenter randomized controlled study. A secondary analysis was performed in which preterm infants were divided into the PNAC group and the non-PNAC group based on the PNAC status. RESULTS: A total of 465 cases very preterm infants or very low birth weight infants were included in the study in which 81 cases were assigned to the PNAC group and 384 cases were assigned to the non-PNAC group. The PNAC group had a lower mean gestational age, lower mean birth weight, longer duration of invasive and non-invasive mechanical ventilation, a longer duration oxygen support, and longer hospital stay (P < 0.001 for all). The PNAC group had higher respiratory distress syndrome, hemodynamically significant patent ductus arteriosus, necrotizing enterocolitis (NEC) with stage II or higher, surgically treated NEC, late-onset sepsis, metabolic bone disease, and extrauterine growth retardation (EUGR) compared to the non-PNAC group (P < 0.05 for all). In contrast with the non-PNAC group, the PNAC group received a higher maximum dose of amino acids and fat emulsion, more medium/long-chain fatty emulsion, less SMOF, had a longer duration of parenteral nutrition, lower rates of breastfeeding, higher incidence of feeding intolerance (FI), more accumulated days to achieve total enteral nutrition, less accumulated days of total calories up to standard 110 kcal/kg/day and slower velocity of weight growth (P < 0.05 for all). Logistic regression analysis indicated that the maximum dose of amino acids (OR, 5.352; 95% CI, 2.355 to 12.161), EUGR (OR, 2.396; 95% CI, 1.255 to 4.572), FI (OR, 2.581; 95% CI, 1.395 to 4.775), surgically treated NEC (OR, 11.300; 95% CI, 2.127 ~ 60.035), and longer total hospital stay (OR, 1.030; 95% CI, 1.014 to 1.046) were independent risk factors for the development of PNAC. SMOF (OR, 0.358; 95% CI, 0.193 to 0.663) and breastfeeding (OR, 0.297; 95% CI, 0.157 to 0.559) were protective factors for PNAC. CONCLUSIONS: PNAC can be reduced by optimizing the management of enteral and parenteral nutrition and reducing gastrointestinal comorbidities in preterm infants.

A basic study for the molecular imaging of dual-energy CT in diagnosing anterior cruciate ligament injury of knee joint.

BACKGROUND: Anterior cruciate ligament (ACL) injury is a common disease in clinical practice that seriously affects the daily life of patients. PURPOSE: To explore the molecular imaging basis of "diminution sign on dual-energy colour mapping" for the diagnosis of ACL injury by dual-energy computed tomography (DECT). MATERIAL AND METHODS: The hydroxylysine and hydroxyproline reagents were prepared in different concentrations. The grouping was shown as follows: a simple concentration change group of an amino acid (group 1/2); a mixed solution group with the concentration increasing synchronously (group 3); a mixed solution group with the concentration reverse increasing and decreasing (group 4); and a mixed solution group that fix one amino acid with increasing concentration of the other (group 5/6). The samples were scanned by DECT. The solution CT value and image signal-to-noise ratio were analyzed. RESULTS: In group 1/2, the brightness of the dual-energy color mapping of each test tube solution and the CT value increased with increasing the concentration of amino acid. In group 6, there was no significant change in the brightness and brilliance of the dual-energy color mapping and the CT value. The remaining three groups showed an increase in the brightness and brilliance of the dual-energy color mapping and the CT value, and this increase was positively associated with the hydroxylysine concentration. CONCLUSION: The dual-energy staining of the DECT imaging in "tendon" mode is related to hydroxylysine and hydroxyproline. Moreover, the degree of dual-energy color mapping is positively correlated with the change of CT value.

Resilient and sustainable production of peanut (Arachis hypogaea) in phosphorus-limited environment by using exogenous gamma-aminobutyric acid to sustain photosynthesis.

Globally, many low to medium yielding peanut fields have the potential for further yield improvement. Low phosphorus (P) limitation is one of the significant factors curtailing Arachis hypogaea productivity in many regions. In order to demonstrate the effects of gamma-aminobutyric acid (GABA) on peanuts growing under P deficiency, we used a pot-based experiment to examine the effects of exogenous GABA on alleviating P deficiency-induced physiological changes and growth inhibition in peanuts. The key physiological parameters examined were foliar gas exchange, photochemical efficiency, proton motive force, reactive oxygen species (ROS), and adenosine triphosphate (ATP) synthase activity of peanuts under cultivation with low P (LP, 0.5 mM P) and control conditions. During low P, the cyclic electron flow (CEF) maintained the high proton gradient (∆pH) induced by low ATP synthetic activity. Applying GABA during low P conditions stimulated CEF and reduced the concomitant ROS generation and thereby protecting the foliar photosystem II (PSII) from photoinhibition. Specifically, GABA enhanced the rate of electronic transmission of PSII (ETRII) by pausing the photoprotection mechanisms including non-photochemical quenching (NPQ) and ∆pH regulation. Thus, GABA was shown to be effective in restoring peanut growth when encountering P deficiency. Exogenous GABA alleviated two symptoms (increased root-shoot ratio and photoinhibition) of P-deficient peanuts. This is possibly the first report of using exogenous GABA to restore photosynthesis and growth under low P availability. Therefore, foliar applications of GABA could be a simple, safe and effective approach to overcome low yield imposed by limited P resources (low P in soils or P-fertilizers are unavailable) for sustainable peanut cultivation and especially in low to medium yielding fields.

Molecular Regulatory Network of Anthocyanin Accumulation in Black Radish Skin as Revealed by Transcriptome and Metabonome Analysis.

To understand the coloring mechanism in black radish, the integrated metabolome and transcriptome analyses of root skin from a black recombinant inbred line (RIL 1901) and a white RIL (RIL 1911) were carried out. A total of 172 flavonoids were detected, and the analysis results revealed that there were 12 flavonoid metabolites in radish root skin, including flavonols, flavones, and anthocyanins. The relative concentrations of most flavonoids in RIL 1901 were higher than those in RIL 1911. Meanwhile, the radish root skin also contained 16 types of anthocyanins, 12 of which were cyanidin and its derivatives, and the concentration of cyanidin 3-o-glucoside was very high at different development stages of black radish. Therefore, the accumulation of cyanidin and its derivatives resulted in the black root skin of radish. In addition, a module positively related to anthocyanin accumulation and candidate genes that regulate anthocyanin synthesis was identified by the weighted gene co-expression network analysis (WGCNA). Among them, structural genes (RsCHS, RsCHI, RsDFR, and RsUGT75C1) and transcription factors (TFs) (RsTT8, RsWRKY44L, RsMYB114, and RsMYB308L) may be crucial for the anthocyanin synthesis in the root skin of black radish. The anthocyanin biosynthesis pathway in the root skin of black radish was constructed based on the expression of genes related to flavonoid and anthocyanin biosynthesis pathways (Ko00941 and Ko00942) and the relative expressions of metabolites. In conclusion, this study not only casts new light on the synthesis and accumulation of anthocyanins in the root skin of black radish but also provides a molecular basis for accelerating the cultivation of new black radish varieties.