Single-Cell Transcriptomic Atlas of Primate Ovarian Aging.

Molecular mechanisms of ovarian aging and female age-related fertility decline remain unclear. We surveyed the single-cell transcriptomic landscape of ovaries from young and aged non-human primates (NHPs) and identified seven ovarian cell types with distinct gene-expression signatures, including oocyte and six types of ovarian somatic cells. In-depth dissection of gene-expression dynamics of oocytes revealed four subtypes at sequential and stepwise developmental stages. Further analysis of cell-type-specific aging-associated transcriptional changes uncovered the disturbance of antioxidant signaling specific to early-stage oocytes and granulosa cells, indicative of oxidative damage as a crucial factor in ovarian functional decline with age. Additionally, inactivated antioxidative pathways, increased reactive oxygen species, and apoptosis were observed in granulosa cells from aged women. This study provides a comprehensive understanding of the cell-type-specific mechanisms underlying primate ovarian aging at single-cell resolution, revealing new diagnostic biomarkers and potential therapeutic targets for age-related human ovarian disorders.

On-enzyme refolding permits small RNA and tRNA surveillance by the CCA-adding enzyme.

Transcription in eukaryotes produces a number of long noncoding RNAs (lncRNAs). Two of these, MALAT1 and Menß, generate a tRNA-like small RNA in addition to the mature lncRNA. The stability of these tRNA-like small RNAs and bona fide tRNAs is monitored by the CCA-adding enzyme. Whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Here, we characterize how these two scenarios are distinguished. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates.

Transcriptome profiling in swine macrophages infected with African swine fever virus at single-cell resolution.

African swine fever virus (ASFV) is the causative agent of African swine fever, a highly contagious and usually fatal disease in pigs. The pathogenesis of ASFV infection has not been clearly elucidated. Here, we used single-cell RNA-sequencing technology to survey the transcriptomic landscape of ASFV-infected primary porcine alveolar macrophages. The temporal dynamic analysis of viral genes revealed increased expression of viral transmembrane genes. Molecular characteristics in the ASFV-exposed cells exhibited the activation of antiviral signaling pathways with increased expression levels of interferon-stimulated genes and inflammatory- and cytokine-related genes. By comparing infected cells with unexposed cells, we showed that the unfolded protein response (UPR) pathway was activated in low viral load cells, while the expression level of UPR-related genes in high viral load cells was less than that in unexposed cells. Cells infected with various viral loads showed signature transcriptomic changes at the median progression of infection. Within the infected cells, differential expression analysis and coregulated virus­host analysis both demonstrated that ASFV promoted metabolic pathways but inhibited interferon and UPR signaling, implying the regulation pathway of viral replication in host cells. Furthermore, our results revealed that the cell apoptosis pathway was activated upon ASFV infection. Mechanistically, the production of tumor necrosis factor alpha (TNF-α) induced by ASFV infection is necessary for cell apoptosis, highlighting the importance of TNF-α in ASFV pathogenesis. Collectively, the data provide insights into the comprehensive host responses and complex virus­host interactions during ASFV infection, which may instruct future research on antiviral strategies.

Experimental Realization of Self-Contained Quantum Refrigeration.

A fundamental challenge in quantum thermodynamics is the exploration of inherent dimensional constraints in thermodynamic machines. In the context of two-level systems, the most compact refrigerator necessitates the involvement of three entities, operating under self-contained conditions that preclude the use of external work sources. Here, we build such a smallest refrigerator using a nuclear spin system, where three distinct two-level carbon-13 nuclei in the same molecule are involved to facilitate the refrigeration process. The self-contained feature enables it to operate without relying on net external work, and the unique mechanism sets this refrigerator apart from its classical counterparts. We evaluate its performance under varying conditions and systematically scrutinize the cooling constraints across a spectrum of scenarios, which sheds light on the interplay between quantum information and thermodynamics.

Integrated transcriptomics and epigenomics reveal chamber-specific and species-specific characteristics of human and mouse hearts.

DNA methylation, chromatin accessibility, and gene expression represent different levels information in biological process, but a comprehensive multiomics analysis of the mammalian heart is lacking. Here, we applied nucleosome occupancy and methylome sequencing, which detected DNA methylation and chromatin accessibility simultaneously, as well as RNA-seq, for multiomics analysis of the 4 chambers of adult and fetal human hearts, and adult mouse hearts. Our results showed conserved region-specific patterns in the mammalian heart at transcriptome and DNA methylation level. Adult and fetal human hearts showed distinct features in DNA methylome, chromatin accessibility, and transcriptome. Novel long noncoding RNAs were identified in the human heart, and the gene expression profiles of major cardiovascular diseases associated genes were displayed. Furthermore, cross-species comparisons revealed human-specific and mouse-specific differentially expressed genes between the atria and ventricles. We also reported the relationship among multiomics and found there was a bell-shaped relationship between gene-body methylation and expression in the human heart. In general, our study provided comprehensive spatiotemporal and evolutionary insights into the regulation of gene expression in the heart.

Establishment of bovine expanded potential stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression.

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and ß-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Does public health education improve migrant workers' health status in China?-evidence from China Migrants Dynamic Survey.

This study investigates the effect of public health education (PHE) on migrant workers' health status in China, using the data collected from the China Migrants Dynamic Survey project. The analysis employs a probit model, whose results suggest that, in general, PHE has a statistically significant and positive impact on migrant workers' self-rated health status and exerts a negative impact on their incidence rate of daily diseases. We also utilize the conditional mixed process method to address the potential endogenous issue. Further analyses reveal that there are significant differences in the impacts of different modes of PHE on migrant workers' health status, among which the mode of health knowledge lectures plays the most prominent role. Nonetheless, an additional analysis indicates that in addition to PHE, other public health services, such as the establishment of health records, also have a significant effect on the promotion of migrant workers' health status. A disaggregated analysis reveals that this impact is heterogeneous among different generations, genders as well as those with different income levels. The findings shed light on the importance of promoting equal access to public health services.

Single-cell RNA-seq identifies a reversible mesodermal activation in abnormally specified epithelia of p63 EEC syndrome.

Mutations in transcription factor p63 are associated with developmental disorders that manifest defects in stratified epithelia including the epidermis. The underlying cellular and molecular mechanism is however not yet understood. We established an epidermal commitment model using human induced pluripotent stem cells (iPSCs) and characterized differentiation defects of iPSCs derived from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients carrying p63 mutations. Transcriptome analyses revealed stepwise cell fate transitions during epidermal commitment: Specification from multipotent simple epithelium to basal stratified epithelia and ultimately to the mature epidermal fate. Differentiation defects of EEC iPSCs caused by p63 mutations occurred during the specification switch from the simple epithelium to the basal-stratified epithelial fate. Single-cell transcriptome and pseudotime analyses of cell states identified mesodermal activation that was associated with the deviated commitment route of EEC iPSCs. Integrated analyses of differentially regulated genes and p63-dependent dynamic genomic enhancers during epidermal commitment suggest that p63 directly controls epidermal gene activation at the specification switch and has an indirect effect on mesodermal gene repression. Importantly, inhibitors of mesodermal induction enhanced epidermal commitment of EEC iPSCs. Our findings demonstrate that p63 is required for specification of stratified epithelia, and that epidermal commitment defects caused by p63 mutations can be reversed by repressing mesodermal induction. This study provides insights into disease mechanisms underlying stratified epithelial defects caused by p63 mutations and suggests potential therapeutic strategies for the disease.

Exploring prognostic indicators in the pathological images of hepatocellular carcinoma based on deep learning.

OBJECTIVE: Tumour pathology contains rich information, including tissue structure and cell morphology, that reflects disease progression and patient survival. However, phenotypic information is subtle and complex, making the discovery of prognostic indicators from pathological images challenging. DESIGN: An interpretable, weakly supervised deep learning framework incorporating prior knowledge was proposed to analyse hepatocellular carcinoma (HCC) and explore new prognostic phenotypes on pathological whole-slide images (WSIs) from the Zhongshan cohort of 1125 HCC patients (2451 WSIs) and TCGA cohort of 320 HCC patients (320 WSIs). A 'tumour risk score (TRS)' was established to evaluate patient outcomes, and then risk activation mapping (RAM) was applied to visualise the pathological phenotypes of TRS. The multi-omics data of The Cancer Genome Atlas(TCGA) HCC were used to assess the potential pathogenesis underlying TRS. RESULTS: Survival analysis revealed that TRS was an independent prognosticator in both the Zhongshan cohort (p<0.0001) and TCGA cohort (p=0.0003). The predictive ability of TRS was superior to and independent of clinical staging systems, and TRS could evenly stratify patients into up to five groups with significantly different prognoses. Notably, sinusoidal capillarisation, prominent nucleoli and karyotheca, the nucleus/cytoplasm ratio and infiltrating inflammatory cells were identified as the main underlying features of TRS. The multi-omics data of TCGA HCC hint at the relevance of TRS to tumour immune infiltration and genetic alterations such as the FAT3 and RYR2 mutations. CONCLUSION: Our deep learning framework is an effective and labour-saving method for decoding pathological images, providing a valuable means for HCC risk stratification and precise patient treatment.

The transcription factor AtGLK1 acts upstream of MYBL2 to genetically regulate sucrose-induced anthocyanin biosynthesis in Arabidopsis.

BACKGROUND: The regulation of anthocyanin biosynthesis by various factors including sugars, light and abiotic stresses is mediated by numerous regulatory factors acting at the transcriptional level. Here experimental evidence was provided in order to demonstrate that the nuclear GARP transcription factor AtGLK1 plays an important role in regulating sucrose-induced anthocyanin biosynthesis in Arabidopsis. RESULTS: The results obtained using real-time quantitative PCR and GUS staining assays revealed that AtGLK1 was mainly expressed in the green tissues of Arabidopsis seedlings and could be induced by sucrose. The loss-of-function glk1 glk2 double mutant has lower anthocyanin levels than the glk2 single mutant, although it has been determined that loss of AtGLK1 alone does not affect anthocyanin accumulation. Overexpression of AtGLK1 enhances the accumulation of anthocyanin in transgenic Arabidopsis seedlings accompanied by increased expression of anthocyanin biosynthetic and regulatory genes. Moreover, we found that AtGLK1 also participates in plastid-signaling mediated anthocyanin accumulations. Genetic, physiological, and molecular biological approaches demonstrated that AtGLK1 acts upstream of MYBL2, which is a key negative regulator of anthocyanin biosynthesis, to genetically regulate sucrose-induced anthocyanin biosynthesis. CONCLUSION: Our results indicated that AtGLK1 positively regulates sucrose-induced anthocyanin biosynthesis in Arabidopsis via MYBL2.

Identifying metabolites by integrating metabolome databases with mass spectrometry cheminformatics.

Novel metabolites distinct from canonical pathways can be identified through the integration of three cheminformatics tools: BinVestigate, which queries the BinBase gas chromatography-mass spectrometry (GC-MS) metabolome database to match unknowns with biological metadata across over 110,000 samples; MS-DIAL 2.0, a software tool for chromatographic deconvolution of high-resolution GC-MS or liquid chromatography-mass spectrometry (LC-MS); and MS-FINDER 2.0, a structure-elucidation program that uses a combination of 14 metabolome databases in addition to an enzyme promiscuity library. We showcase our workflow by annotating N-methyl-uridine monophosphate (UMP), lysomonogalactosyl-monopalmitin, N-methylalanine, and two propofol derivatives.

Nitration of Chitin Monomer: From Glucosamine to Energetic Compound.

The nitration of chitin monomer in a mixture of nitric acid and acetic anhydride was conducted and a highly nitrated (3R,4R,6R)-3-acetamido-6-((nitrooxy)methyl)tetrahydro-2H-pyran-2,4,5-triyl trinitrate (1) was obtained. Its structure was fully characterized using infrared spectroscopy, NMR spectroscopy, elemental analysis, and X-ray diffraction. Compound 1 possesses good density (ρ: 1.721 g·cm-3) and has comparable detonation performance (Vd: 7717 m·s-1; P: 25.6 GPa) to that of nitrocellulose (NC: Vd: 7456 m·s-1; P: 23 GPa; Isp = 239 s) and microcrystalline nitrocellulose (MCNC; Vd: 7683 m·s-1; P: 25 GPa; Isp = 250 s). However, Compound 1 has much lower impact sensitivity (IS: 15 J) than the regular nitrocellulose (NC; IS: 3.2 J) and MCNC (IS: 2.8 J). Compound 1 was calculated to exhibit a good specific impulse (Isp: 240 s), which is comparable with NC (Isp: 239 s) and MCNC (Isp: 250 s). By replacing the nitrocellulose with Compound 1 in typical propellants JA2, M30, and M9, the specific impulse was improved by up to 4 s. These promising properties indicate that Compound 1 has a significant potential as an energetic component in solid propellants.

Efficient COI barcoding using high throughput single-end 400 bp sequencing.

BACKGROUND: Over the last decade, the rapid development of high-throughput sequencing platforms has accelerated species description and assisted morphological classification through DNA barcoding. However, the current high-throughput DNA barcoding methods cannot obtain full-length barcode sequences due to read length limitations (e.g. a maximum read length of 300 bp for the Illumina's MiSeq system), or are hindered by a relatively high cost or low sequencing output (e.g. a maximum number of eight million reads per cell for the PacBio's SEQUEL II system). RESULTS: Pooled cytochrome c oxidase subunit I (COI) barcodes from individual specimens were sequenced on the MGISEQ-2000 platform using the single-end 400 bp (SE400) module. We present a bioinformatic pipeline, HIFI-SE, that takes reads generated from the 5' and 3' ends of the COI barcode region and assembles them into full-length barcodes. HIFI-SE is written in Python and includes four function modules of filter, assign, assembly and taxonomy. We applied the HIFI-SE to a set of 845 samples (30 marine invertebrates, 815 insects) and delivered a total of 747 fully assembled COI barcodes as well as 70 Wolbachia and fungi symbionts. Compared to their corresponding Sanger sequences (72 sequences available), nearly all samples (71/72) were correctly and accurately assembled, including 46 samples that had a similarity score of 100% and 25 of ca. 99%. CONCLUSIONS: The HIFI-SE pipeline represents an efficient way to produce standard full-length barcodes, while the reasonable cost and high sensitivity of our method can contribute considerably more DNA barcodes under the same budget. Our method thereby advances DNA-based species identification from diverse ecosystems and increases the number of relevant applications.

Single-cell RNA-seq analysis reveals the progression of human osteoarthritis.

OBJECTIVES: Understanding the molecular mechanisms underlying human cartilage degeneration and regeneration is helpful for improving therapeutic strategies for treating osteoarthritis (OA). Here, we report the molecular programmes and lineage progression patterns controlling human OA pathogenesis using single-cell RNA sequencing (scRNA-seq). METHODS: We performed unbiased transcriptome-wide scRNA-seq analysis, computational analysis and histological assays on 1464 chondrocytes from 10 patients with OA undergoing knee arthroplasty surgery. We investigated the relationship between transcriptional programmes of the OA landscape and clinical outcome using severity index and correspondence analysis. RESULTS: We identified seven molecularly defined populations of chondrocytes in the human OA cartilage, including three novel phenotypes with distinct functions. We presented gene expression profiles at different OA stages at single-cell resolution. We found a potential transition among proliferative chondrocytes, prehypertrophic chondrocytes and hypertrophic chondrocytes (HTCs) and defined a new subdivision within HTCs. We revealed novel markers for cartilage progenitor cells (CPCs) and demonstrated a relationship between CPCs and fibrocartilage chondrocytes using computational analysis. Notably, we derived predictive targets with respect to clinical outcomes and clarified the role of different cell types for the early diagnosis and treatment of OA. CONCLUSIONS: Our results provide new insights into chondrocyte taxonomy and present potential clues for effective and functional manipulation of human OA cartilage regeneration that could lead to improved health.

Detection of Colorectal Cancer in Circulating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing.

BACKGROUND: Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cell-free DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and other cancer types. METHODS: We applied the methylated CpG tandem amplification and sequencing (MCTA-Seq) method, with a fully methylated molecules algorithm, to plasma samples from patients with CRC (n = 147) and controls (n = 136), as well as cancer and adjacent noncancerous tissue samples (n = 66). We also comparatively analyzed plasma samples from patients with hepatocellular carcinoma (HCC; n = 36). RESULTS: Dozens of DNA hypermethylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5, CHST11, APBB1IP, and TJP2) genes were identified for effectively detecting CRC in cfDNA. A panel of 80 markers discriminated early-stage CRC patients and controls with a clinical sensitivity of 74% and clinical specificity of 90%. Patients with early-stage CRC and HCC could be discriminated at clinical sensitivities of approximately 70% by another panel of 128 markers. CONCLUSIONS: MCTA-Seq is a promising method for the noninvasive detection of CRC.

DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA.

Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2΄-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2΄-deoxyadenosines in DNA/RNA hybrids.

Dynamic chromatin accessibility modeled by Markov process of randomly-moving molecules in the 3D genome.

Chromatin three-dimensional (3D) structure plays critical roles in gene expression regulation by influencing locus interactions and accessibility of chromatin regions. Here we propose a Markov process model to derive a chromosomal equilibrium distribution of randomly-moving molecules as a functional consequence of spatially organized genome 3D structures. The model calculates steady-state distributions (SSD) from Hi-C data as quantitative measures of each chromatin region's dynamic accessibility for transcription factors and histone modification enzymes. Different from other Hi-C derived features such as compartment A/B and interaction hubs, or traditional methods measuring chromatin accessibility such as DNase-seq and FAIRE-seq, SSD considers both chromatin-chromatin and protein-chromatin interactions. Through our model, we find that SSD could capture the chromosomal equilibrium distributions of activation histone modifications and transcription factors. Compared with compartment A/B, SSD has higher correlations with the binding of these histone modifications and transcription factors. In addition, we find that genes located in high SSD regions tend to be expressed at higher level. Furthermore, we track the change of genome organization during stem cell differentiation, and propose a two-stage model to explain the dynamic change of SSD and gene expression during differentiation, where chromatin organization genes first gain chromatin accessibility and are expressed before lineage-specific genes do. We conclude that SSD is a novel and better measure of dynamic chromatin activity and accessibility.

Prevailing genotype distribution and characteristics of human respiratory syncytial virus in northeastern China.

Although human respiratory syncytial virus (RSV) is one of the most common viruses inducing respiratory tract infections in young children and the elderly, the genotype distribution and characteristics of RSV in northeastern China have not been investigated. Here, we identified 25 RSV-A and 8 RSV-B strains from 80 samples of patients with respiratory infections between February 2015 and May 2015. All 25 RSV-A viruses were classified as the ON1 genotype, which rapidly spread and became the dominant genotype in the world since being identified in Ontario (Canada) in December 2010. All eight RSV-B viruses belonged to the BA genotype with a 60-nucleotide duplication, seven of which formed two new genotypes, BA-CCA and BA-CCB. The remaining RSV-B virus clustered with one of the Hangzhou strains belonging to genotype BA11. Construction of a phylogenetic tree and amino acid substitution analysis showed that Changchun ON1 viruses exclusively constituted Lineages 3, 5 and 6, and contained several unique and newly identified amino acid substitutions, including E224G, R244K, L289I, Y297H, and L298P. Selective pressure was also evaluated, and various N and O-glycosylation sites were predicted. This study provides the first genetic analysis of RSV in northeastern China and may facilitate a better understanding of the evolution of this virus locally and globally. J. Med. Virol. 89:222-233, 2017. © 2016 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.

Synthesis and evaluation of an alkyne-modified ATP analog for enzymatic incorporation into RNA.

Alkyne-modified nucleoside analogs are useful for nucleic acid localization as well as functional and structural studies because of their ability to participate in copper-catalyzed azide/alkyne cycloaddition (CuAAC) reactions. Here we describe the synthesis of the triphosphate of 7-ethynyl-8-aza-7-deazaadenosine (7-EAATP) and the enzymatic incorporation of 7-EAA into RNA. The free nucleoside of 7-EAA is taken up by HeLa cells and incorporated into cellular RNA by endogenous RNA polymerases. In addition, 7-EAATP is a substrate for both T7 RNA polymerase and poly (A) polymerase from Escherichia coli in vitro, albeit at lower efficiencies than with ATP. This work adds to the toolbox of nucleoside analogs useful for RNA labeling.