Biology subjects

Lai, Y.

Publications and source records attributed to Lai, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Kindlin-2 preserves integrity of the articular cartilage to protect against osteoarthritis

Osteoarthritis (OA) is an aging-related degenerative joint disease, which has no cure partly due to limited understanding of its pathological mechanism(s). Here we report that the focal adhesion protein Kindlin-2, but not Kindlin-1 or -3, is highly expressed in articular chondrocytes of the hyaline cartilage, which is dramatically decreased in the degenerated articular cartilage of aged mice and patients with OA. Inducible deletion of Kindlin-2 in chondrocytes at adult stage leads to spontaneous OA and much severe OA lesions in the mice receiving the surgery of destabilization of the medial meniscus. Mechanistically, Kindlin-2 deficiency promotes mitochondrial oxidative stress and activates Stat3 in articular chondrocytes, leading to Runx2-mediated chondrocyte hypertrophic differentiation and catabolism. In vivo, systemic pharmacological blockade of Stat3 activation or genetic ablation of Stat3 in chondrocytes reverses aberrant accumulation of Runx2 and ECM-degrading enzymes and limits OA deteriorations caused by Kindlin-2 deficiency. Furthermore, genetic inactivation of Runx2 in chondrocytes reverses structural changes and OA lesions caused by Kindlin-2 deletion without down-regulating p-Stat3 in articular chondrocytes. Of translational significance, intraarticular injection of Kindlin-2-expressing adeno-associated virus decelerates progression of aging- and instability-induced knee joint OA in mice. Collectively, we identify a novel pathway comprising of Kindlin-2, Stat3 and Runx2 in articular chondrocytes responsible for maintaining integrity of the articular cartilage and define a potential therapeutic target for OA.

pathology↗

bioRxiv2021

Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism

SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC50 of 0.086 g mL-1 (DL4) and 0.385 g mL-1 (DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50 of 0.414 g mL-1 for Alpha, 1.060 g mL-1 for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.

biochemistry↗

bioRxiv2021

Structure of mycobacterial cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates

Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of M. tuberculosis cytochrome bcc alone (2.68 [A] resolution) and in complex with clinical drug candidates Q203 (2.67 [A] resolution) and TB47 (2.93 [A] resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol binding pockets. Q203 and TB47 are bound to the quinol-binding site. Hydrogen bonds are formed between the inhibitor and the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution structures provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad spectrum drugs to treat mycobacterial infections.

microbiology↗

bioRxiv2021

piRNA pathway is essential for generating functional oocytes in golden hamster

Piwi-interacting RNAs (piRNAs) are small RNAs predominantly expressed in germ cells that are critical for gametogenesis in various species. However, PIWI-deficient female mice are fertile and mouse oocytes express a panel of small RNAs that do not appear widely representative of mammals, and piRNA function in the oogenesis of other mammals has therefore remained elusive. Recent studies revealed the small RNA and PIWI transcriptional profiles in golden hamster oocytes more closely resemble that of humans than mice. Herein, we generated PIWIL1-, PLD6- and MOV10L1-deficient golden hamsters and found that all female mutants were sterile, with embryos arrested at the two-cell stage. In PIWIL1 mutant oocytes, we observed transposon accumulation and broad transcriptomic dysregulation, while zygotic gene activation was impaired in early embryos. Intriguingly, PIWIL1-piRNAs exhibited a unique, preferential silencing of endogenous retroviruses (ERVs), whereas silencing LINE1s depended on both PIWIL1- and PIWIL3-piRNAs. Moreover, we showed that piRNAs participate in the degradation of maternal mRNAs in MII oocytes and embryos via partially complementary targets. Together, our findings demonstrate that piRNAs are indispensable for generating functional oocytes in golden hamster, and show the informative value of this model for functional and mechanistic investigations of piRNAs, especially those related to female infertility.

developmental biology↗

bioRxiv2021

Large field of view-spatially resolved transcriptomics at nanoscale resolution

Spatially resolved transcriptomic technologies are promising tools to study cell fate decisions in a physical microenvironment, which is fundamental for enhancing our knowledge of mammalian development. However, the imbalance between resolution, transcript capture and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation mammalian embryos. Here, we combined DNA nanoball (DNB) patterned arrays and tissue RNA capture to create SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq). This approach allows transcriptomic profiling of large histological sections with high resolution and sensitivity. We have applied Stereo-seq to study the kinetics and directionality of transcriptional variation in a time course of mouse organogenesis. We used this information to gain insight into the molecular basis of regional specification, neuronal migration and differentiation in the developing brain. Furthermore, we mapped the expression of a panel of developmental disease-related loci on our global transcriptomic maps to define the spatiotemporal windows of tissue vulnerability. Our panoramic atlas constitutes an essential resource to investigate longstanding questions concerning normal and abnormal mammalian development.

cell biology↗

bioRxiv2021

Increased theta/alpha synchrony in the habenula-prefrontal network with negative emotional stimuli in human patients

Lateral habenula is believed to encode negative motivational stimuli and plays key roles in the pathophysiology of psychiatric disorders. However, how habenula activities are modulated during the perception and processing of emotional information is still poorly understood. We recorded local field potentials from bilateral habenula areas with simultaneous cortical magnetoencephalography in nine patients with psychiatric disorders during an emotional picture viewing task. Oscillatory activity in the theta/alpha band (5-10 Hz) within the habenula and prefrontal cortical regions, as well as the coupling between these structures, are increased during the perception and processing of negative emotional stimuli compared to positive emotional stimuli. The evoked increase in theta/alpha band synchronization in the frontal cortex-habenula network correlated with the emotional valence not the arousal score of the stimuli. These results provide direct evidence for increased theta/alpha synchrony within the habenula area and prefrontal cortex-habenula network in the perception of negative emotion in human participants.

neuroscience↗

bioRxiv2020

Assessing Study Reproducibility through MMRI: A Novel Approach for Large-scale High-throughput Association Studies

Magnetic Resonance Imaging (MRI) technology has been increasingly used in large-scale association studies. Reproducibility of statistically significant findings generated by MRI-based association studies, especially structural MRI (sMRI) and functional MRI (fMRI), has been recently heavily debated. However, there is still a lack of overall reproducibility assessment for MRI-based association studies. It is also crucial to elucidate the relationship between overall reproducibility and sample size in an experimental design. In this study, we proposed an overall reproducibility index for large-scale high-throughput MRI-based association studies. We performed the overall reproducibility assessments for several recent large sMRI/fMRI databases and observed satisfactory overall reproducibility. Furthermore, we performed the sample size evaluation for the purpose of achieving a desirable overall reproducibility. Additionally, we evaluated the overall reproducibility of GMV changes for UKB vs. PPMI and UKB vs. HCP. We demonstrated that both sample size and some experimental factors play important roles in the overall reproducibility for different experiments. In summary, a systematic assessment of overall reproducibility is fundamental and crucial in the current large-scale high-throughput MRI-based research.

neuroscience↗

bioRxiv2020

Restoration of β-globin expression with optimally designed lentiviral vector for β-thalassemia treatment in Chinese patients

{beta}-thalassemia is one of the most prevalent genetic diseases worldwide. The current treatment for {beta}-thalassemia is allogeneic hematopoietic stem cell transplantation (HSCT), which is limited due to lack of matched donors. Gene therapy has been developed as an alternative therapeutic option for transfusion-ependent {beta}-thalassemia (TDT). However, successful gene therapy for {beta}-thalassemia patients in China has not been reported. Here we present the results of preclinical studies of an optimally designed LV named LentiHBBT87Q in hematopoietic stem cells (HSCs) derived from Chinese TDT patients. LentiHBBT87Q was selected from a series of LVs with optimized backbone and de novo cloning strategy. It contains an exogenous T87Q {beta}-globin gene (HBBT87Q) driven by a specific reconstituted locus control region (rLCR) and efficiently express HBB mRNA and HBB protein in erythroblasts derived from cord blood (CB) HSCs. To facilitate clinical transformation, we manufactured clinical grade LentiHBBT87Q (cLentiHBBT87Q) and optimized its transduction procedure. Importantly, transduction of cLentiHBBT87Qrestored expression of HBB monomer and adult hemoglobin (HbA) tetramer to relatively normal level in erythroblasts from bone marrow (BM) HSCs of Chinese TDT patients, that carry the most common mutation types and cover various genotypes, including {beta}0/{beta}0. Furthermore, viral integration sites (VIS) of cLentiHBBT87Q were similar to other LVs safely used in previous clinical trials and the associated risk of tumorigenesis was not observed in cLentiHBBT87Q transduced HSCs through comprehensive analysis. Taken together, we have engineered the cLentiHBBT87Q that can restore {beta}-globin expression in the HSCs-derived erythroblasts of Chinese TDT patients with minimal risk on tumorigenesis, providing a favorable starting point for future clinical application.

cell biology↗

bioRxiv2020

RND3/RhoE deficiency in preeclamptic women impairs placental mitochondrial function via regulation of the PPARgamma-UCP2 pathway

Preeclampsia (PE) is a life-threatening disease of pregnant women associated with severe hypertension, proteinuria, or multi-organ injuries. Mitochondrial-mediated placental oxidative stress plays a key role in the pathogenesis of PE. However, the underlying mechanism remains to be revealed. Here, we identify Rnd3, a small Rho GTPase, regulating placental mitochondrial reactive oxygen species (ROS). We showed that Rnd3 is down-regulated in primary trophoblasts isolated from PE patients. Loss of Rnd3 in trophoblasts resulted in excessive ROS generation, cell apoptosis, mitochondrial injury, and proton leakage from the respiratory chain. Moreover, Rnd3 overexpression partially rescues the mitochondrial defects and oxidative stress in human PE primary trophoblasts. Rnd3 physically interacts with the peroxisome proliferators-activated receptor {gamma} (PPAR{gamma}) and promotes the PPAR{gamma}-mitochondrial uncoupling protein 2 (UCP2) cascade. Forced expression of PPAR{gamma} rescues deficiency of Rnd3-mediated mitochondrial dysfunction. We conclude that Rnd3 acts as a novel protective factor in placental mitochondria through PPAR{gamma}-UCP2 signaling and highlight that downregulation of Rnd3 is a potential factor involved in PE pathogenesis.

pathology↗

bioRxiv2020

Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, [~]15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 g mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

biochemistry↗

bioRxiv2020

Single-cell regulatory landscape and disease vulnerability map of adult Macaque cortex

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell chromatin accessibility (single-cell ATAC) and transcriptomic data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult cynomolgus monkey brain, and integrated this dataset with Stereo-seq (Spatio-Temporal Enhanced REsolution Omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

neuroscience↗

bioRxiv2020

High-throughput CRISPR screens to dissect macrophage-Shigella interactions

Shigellosis causes most diarrheal deaths worldwide, particularly affecting children. Shigella invades and replicates in the epithelium of the large intestine, eliciting inflammation and tissue destruction. To understand how Shigella rewires macrophages prior to epithelium invasion, we performed genome-wide and focused secondary CRISPR knockout and CRISPR interference (CRISPRi) screens in Shigella flexneri-infected human monocytic THP-1 cells. Knockdown of the Toll-like receptor 1/2 signaling pathway significantly reduced pro-inflammatory cytokine and chemokine production, enhanced host cell survival, and controlled intracellular pathogen growth. Knockdown of the enzymatic component of the mitochondrial pyruvate dehydrogenase complex enhanced THP-1 cell survival. Small molecule inhibitors blocking key components of these pathways had similar effects; these were validated with human monocyte-derived macrophages, which closely mimic the in vivo physiological state of cells post-infection. High-throughput CRISPR screens can elucidate how S. flexneri triggers inflammation and redirects host pyruvate catabolism for energy acquisition before killing macrophages, pointing to new shigellosis therapies.

microbiology↗

bioRxiv2020

Single-cell atlas of a non-human primate reveals new pathogenic mechanisms of COVID-19

Stopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans. Bullet pointsO_LIWe generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19. C_LIO_LIACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2. C_LIO_LIACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities. C_LIO_LIWe unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry. C_LI

cell biology↗

bioRxiv2020

Illuminating host-mycobacterial interactions with functional genomic screening to inhibit mycobacterial pathogenesis

Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used CRISPR/Cas9-mediated high-throughput genetic screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Guerin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens can elucidate highly complex host-pathogen interactions and serve to identify HDTs with the potential to improve TB treatment.

microbiology↗

bioRxiv2019

GmPTF1 Modifies Root Architecture Responses to Phosphate Starvation in Soybean

Though root architecture modifications may be critically important for improving phosphorus (P) efficiency in crops, the regulatory mechanisms triggering these changes remain unclear. In this study, we demonstrate that genotypic variation in GmEXPB2 expression is strongly correlated with root elongation and P acquisition efficiency, and enhancing its transcription significantly improves soybean yield in the field. Promoter deletion analysis was performed using six 5 truncation fragments (P1-P6) of GmEXPB2 fused with the GUS reporter gene in transgenic hairy roots, which revealed that the P1 segment containing 3 E-box elements significantly enhances induction of gene expression in response to phosphate (Pi) starvation. Further experimentation demonstrated that GmPTF1, a bHLH transcription factor, is the regulatory factor responsible for the induction of GmEXPB2 expression in response to Pi starvation. In short, Pi starvation induced expression of GmPTF1, with the GmPTF1 product not only directly binding the E-box motif in the P1 region of the GmEXPB2 promoter, but also activating GUS expression in a dosage dependent manner. Further work with soybean transgenic composite plants showed that, altering GmPTF1 expression significantly impacted GmEXPB2 transcription, and thereby affected root growth, biomass and P uptake. Taken together, this work identifies a novel regulatory factor, GmPTF1, involved in changing soybean root architecture through regulation the expression of GmEXPB2. These findings contribute to understanding the molecular basis of root architecture modifications in response to P deficiency, and, in the process, suggest candidate genes and a promoter region to target for improving soybean yield through molecular breeding of P efficiency. One Sentence SummaryThe bHLH transcription factor GmPTF1 regulates the expression of {beta}-expansin gene GmEXPB2 to modify root architecture, and thus promote phosphate acquisition, and biomass in soybean.

physiology↗

bioRxiv2019

A portable and cost-effective microfluidic system for massively parallel single-cell transcriptome profiling

Single-cell technologies are becoming increasingly widespread and have been revolutionizing our understanding of cell identity, state, diversity and function. However, current platforms can be slow to apply to large-scale studies and resource-limited clinical arenas due to a variety of reasons including cost, infrastructure, sample quality and requirements. Here we report DNBelab C4 (C4), a negative pressure orchestrated, portable and cost-effective device that enables high-throughput single-cell transcriptional profiling. C4 system can efficiently allow discrimination of species-specific cells at high resolution and dissect tissue heterogeneity in different organs, such as murine lung and cerebral cortex. Finally, we show that the C4 system is comparable to existing platforms but has huge benefits in cost and portability and, as such, it will be of great interest for the wider scientific community.

genomics↗

bioRxiv2019

Tyrosyl-DNA phosphodiesterase 1 and topoisomerase I activities as predictive indicators for Glioblastoma susceptibility to genotoxic agents

BackgroundGlioblastoma (GBM) patients have an estimated survival of [~]15 months with treatment, and the standard of care only modestly enhances patient survival. Identifying biomarkers representing vulnerabilities may allow for selection of efficacious chemotherapy options to address personalized variations in GBM tumors. Irinotecan, currently in clinical trials for GBM, targets topoisomerase I (TOP1) by forming a ternary DNA-TOP1 cleavage complex (TOP1cc) inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that may reduce the effectiveness of irinotecan.\n\nMethodsWe treated GBM cell lines with increasing concentrations of irinotecan and compared the IC50 values. TOP1 and TDP1 protein levels from each cell type as well as GBM patient tumors were determined by Western blot analysis, while activity levels were ascertained by specific enzymatic assays. Cellular TDP1 was elevated by ectopic expression of wild-type or mutant TDP1.\n\nResultsAfter comparing cellular susceptibility to TDP1 and TOP1 concentrations and activities, we found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation coefficient R = 0.92) with IC50 values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by ectopic expression of wild-type TDP1 increased in irinotecan IC50, while expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan.\n\nConclusionsTDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability to irinotecan, allowing for selection of individual patients for irinotecan treatment based on risk-benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to improve GBM patient responsiveness to genotoxic chemotherapies.\n\nKey PointsO_LITDP1/TOP1 activity ratio correlates with irinotecan sensitivity in GBM cell lines.\nC_LIO_LITDP1 and TOP1 protein levels are not reliable predictors for irinotecan activity.\nC_LIO_LITDP1 inhibition is a plausible approach to improve irinotecan effectiveness in GBM.\nC_LI\n\nImportance of the StudyThe current standard of care (surgery, radiation, and chemotherapy) for GBM patients modestly enhances survival beyond [~]15 months. Thus, there is a great need for effective therapies and biomarkers that address personalized variations in GBM tumors to improve treatment outcome. Topoisomerase I (TOP1) is the target of irinotecan. The repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) is known to excise irinotecan-induced TOP1-DNA cleavage complexes (TOP1ccs). Consequently, this study examines the relationship between TOP1 and TDP1 expression and activities in GBM cells and their correlation with irinotecan sensitivity. The results reveal that the TDP1/TOP1 activity ratio predicts irinotecan vulnerability in GBM cell lines. TDP1/TOP1 activity ratio was found to vary among GBM patient tumors. This potential predictive indicator may permit selection of patients responsive to irinotecan based on the capacity to repair TOP1cc. Moreover, inhibitors of TDP1 may represent a promising approach to enhance irinotecan efficacy in GBM patients.

cancer biology↗

bioRxiv2019

Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA

Chemical modification of transcripts with 5 caps occurs in all organisms. Here we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of the cap epitranscriptome in any organism. The method was piloted with 21 canonical caps - m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG - and 5 \"metabolite\" caps - NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus, Escherichia coli, yeast, mice, and humans, we discovered four new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and surprisingly high proportions of caps lacking 2-O-methylation, such as m7Gpppm6A in mammals and m7GpppA in dengue virus, and we did not detect cap m1A/m1Am in humans. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps. The mystery around cap m1A/m1Am analysis remains unresolved.

systems biology↗