Search

RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1″ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 'poison' that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2'-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers 1,2 . Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations 3 . However, fundamental questions about the temporal regulation of transcription and enhancer-gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq-a new single-cell nascent RNA sequencing assay that uses click chemistry-and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO-seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO-seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression., (© 2024. The Author(s).)

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship., (© 2024. The Author(s).)

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast 1 and human 2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control., (© 2023. The Author(s).)

T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This in vitro system, while showing high fidelity in many ways, is also well known for producing longer than encoded RNA products, particularly under high-yield reaction conditions. Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation 1,2 . Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways 1-4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters 1 . A proposed model for this specificity is that promoters have sequence-encoded preferences for certain enhancers, for example, mediated by interacting sets of transcription factors or cofactors 2 . This 'biochemical compatibility' model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila 3-9 . However, the degree to which human enhancers and promoters are intrinsically compatible has not yet been systematically measured, and how their activities combine to control RNA expression remains unclear. Here we design a high-throughput reporter assay called enhancer × promoter self-transcribing active regulatory region sequencing (ExP STARR-seq) and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify simple rules for enhancer-promoter compatibility, whereby most enhancers activate all promoters by similar amounts, and intrinsic enhancer and promoter activities multiplicatively combine to determine RNA output (R 2  = 0.82). In addition, two classes of enhancers and promoters show subtle preferential effects. Promoters of housekeeping genes contain built-in activating motifs for factors such as GABPA and YY1, which decrease the responsiveness of promoters to distal enhancers. Promoters of variably expressed genes lack these motifs and show stronger responsiveness to enhancers. Together, this systematic assessment of enhancer-promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In biological systems, the synthesis of nucleic acids, such as DNA and RNA, is catalyzed by enzymes in various aqueous solutions. However, substrate specificity is derived from the chemical properties of the residues, which implies that perturbations of the solution environment may cause changes in the fidelity of the reaction. Here, we investigated non-promoter-based synthesis of RNA using T7 RNA polymerase (T7 RNAP) directed by an RNA template in the presence of polyethylene glycol (PEG) of various molecular weights, which can affect polymerization fidelity by altering the solution properties. We found that the mismatch extensions of RNA propagated downstream polymerization. Furthermore, PEG promoted the polymerization of non-complementary ribonucleoside triphosphates, mainly due to the decrease in the dielectric constant of the solution. These results indicate that the mismatch extension of RNA-dependent RNA polymerization by T7 RNAP is driven by the stacking interaction of bases of the primer end and the incorporated nucleotide triphosphates (NTP) rather than base pairing between them. Thus, proteinaceous RNA polymerase may display different substrate specificity with changes in dielectricity caused by molecular crowding conditions, which can result in increased genetic diversity without proteinaceous modification., (© 2022. The Author(s).)

CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled several innovative methods that rely on simultaneously targeting numerous genetic loci. Such libraries could be used in a vast number of biological systems and in the development of new technologies, but library generation is hindered by the cost, time, and sequence data required for sgRNA library synthesis. Here, we describe a rapid enzymatic method for generating robust, variant-matched libraries from any source of cDNA in under 3 h. This method, which we have named SLALOM, utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports by a strand displacing polymerase. With this method, we constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its ability to expand the reach of CRISPR technology and facilitate methods requiring custom libraries., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inflammatory memory involves the molecular and cellular 'reprogramming' of innate immune cells following exogenous stimuli, leading to non-specific protection against subsequent pathogen exposure. This phenomenon has now also been described in non-hematopoietic cells, such as human fetal and adult endothelial cells. In this study we mapped the cell-specific DNA methylation profile and the transcriptomic remodelling during the establishment of inflammatory memory in two distinct fetal endothelial cell types - a progenitor cell (ECFC) and a differentiated cell (HUVEC) population. We show that both cell types have a core transcriptional response to an initial exposure to a viral-like ligand, Poly(I:C), characterised by interferon responsive genes. There was also an ECFC specific response, marked by the transcription factor ELF1, suggesting a non-canonical viral response pathway in progenitor endothelial cells. Next, we show that both ECFCs and HUVECs establish memory in response to an initial viral exposure, resulting in an altered subsequent response to lipopolysaccharide. While the capacity to train or tolerize the induction of specific sets of genes was similar between the two cell types, the progenitor ECFCs show a higher capacity to establish memory. Among tolerized cellular pathways are those involved in endothelial barrier establishment and leukocyte migration, both important for regulating systemic immune-endothelial cell interactions. These findings suggest that the capacity for inflammatory memory may be a common trait across different endothelial cell types but also indicate that the specific downstream targets may vary by developmental stage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Weiss, Vlahos, Kim, Wijegunasekara, Shanmuganathan, Aitken, Joo, Imran, Shepherd, Craig, Green, Hiden, Novakovic and Saffery.)

Herein, Broccoli/mCherry and an EGFP/mCherry dual-color fluorescent reporting systems have been established to quantify the promoter activity at transcription and translation levels in eukaryotic cells. Based on those systems, four commonly used promoters (CMV and SV40 of Pol II and U6, H1 of Pol III) were accurately evaluated at both the transcriptional and translational levels by combining accurate protein and RNA quantification. Furthermore, we verified that Pol III promoters can induce proteins expression, and Pol II promoter can be applied to express RNA molecules with defined length by combining a self-cleaving ribozyme and an artificial poly(A) tail. The dual-color fluorescence reporting systems described here could play a significant role in evaluating other gene expression regulators for gene therapy., (© 2021 The Author(s).)

Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell-cell contact-dependent manner, however, it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition., (© 2021. The Author(s).)

The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H 2 O 2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition., Competing Interests: The authors declare no competing interest.

High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation or chromatographic methods. We recently demonstrated that in high-yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer-than-desired, (partially) double-stranded impurities. Current purification methods often fail to fully eliminate these impurities, which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. In this work, we introduce a novel in vitro transcription method that generates high yields of encoded RNA without double-stranded impurities, reducing the need for further purification. Transcription is carried out at high-salt conditions to eliminate RNA product rebinding, while promoter DNA and T7 RNA polymerase are cotethered in close proximity on magnetic beads to drive promoter binding and transcription initiation, resulting in an increase in overall yield and purity of only the encoded RNA. A more complete elimination of double-stranded RNA during synthesis will not only reduce overall production costs, but also should ultimately enable therapies and technologies that are currently being hampered by those impurities., Competing Interests: Conflict of interest E. C. and C. T. M. are listed as inventors on a patent application related to the described technologies., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Iron is an essential cofactor for several metabolic processes, including the generation of ATP in mitochondria, which is required for axonal function and regeneration. However, it is not known how mitochondria in long axons, such as those in sciatic nerves, acquire iron in vivo Because of their close proximity to axons, Schwann cells are a likely source of iron for axonal mitochondria in the PNS. Here we demonstrate the critical role of iron in promoting neurite growth in vitro using iron chelation. We also show that Schwann cells express the molecular machinery to release iron, namely, the iron exporter, ferroportin (Fpn) and the ferroxidase ceruloplasmin (Cp). In Cp KO mice, Schwann cells accumulate iron because Fpn requires to partner with Cp to export iron. Axons and Schwann cells also express the iron importer transferrin receptor 1 (TfR1), indicating their ability for iron uptake. In teased nerve fibers, Fpn and TfR1 are predominantly localized at the nodes of Ranvier and Schmidt-Lanterman incisures, axonal sites that are in close contact with Schwann cell cytoplasm. We also show that lack of iron export from Schwann cells in Cp KO mice reduces mitochondrial iron in axons as detected by reduction in mitochondrial ferritin, affects localization of axonal mitochondria at the nodes of Ranvier and Schmidt-Lanterman incisures, and impairs axonal regeneration following sciatic nerve injury. These finding suggest that Schwann cells contribute to the delivery of iron to axonal mitochondria, required for proper nerve repair. SIGNIFICANCE STATEMENT This work addresses how and where mitochondria in long axons in peripheral nerves acquire iron. We show that Schwann cells are a likely source as they express the molecular machinery to import iron (transferrin receptor 1), and to export iron (ferroportin and ceruloplasmin [Cp]) to the axonal compartment at the nodes of Ranvier and Schmidt-Lanterman incisures. Cp KO mice, which cannot export iron from Schwann cells, show reduced iron content in axonal mitochondria, along with increased localization of axonal mitochondria at Schmidt-Lanterman incisures and nodes of Ranvier, and impaired sciatic nerve regeneration. Iron chelation in vitro also drastically reduces neurite growth. These data suggest that Schwann cells are likely to contribute iron to axonal mitochondria needed for axon growth and regeneration., (Copyright © 2021 the authors.)

Methyl CpG binding protein 2 (MeCP2) is the main DNA methyl-binding protein in the brain that binds to 5-methylcytosine and 5-hydroxymethyl cytosine. MECP2 gene mutations are the main origin of Rett Syndrome (RTT), a neurodevelopmental disorder in young females. The disease has no existing cure, however, metabolic drugs such as metformin and statins have recently emerged as potential therapeutic candidates. In addition, induced MECP2-BDNF homeostasis regulation has been suggested as a therapy avenue. Here, we analyzed nascent RNA synthesis versus steady state total cellular RNA to study the transcriptional effects of metformin (an anti-diabetic drug) on MECP2 isoforms ( E1 and E2 ) and BNDF in a human brain cell line. Additionally, we investigated the impact of simvastatin (a cholesterol lowering drug) on transcriptional regulation of MECP2E1/E2-BDNF . Metformin was capable of post-transcriptionally inducing BDNF and/or MECP2E1 , while transcriptionally inhibiting MECP2E2 . In contrast simvastatin significantly inhibited BDNF transcription without significantly impacting MECP2E2 transcripts. Further analysis of ribosomal RNA transcripts confirmed that the drug neither individually nor in combination affected these fundamentally important transcripts. Experimental analysis was completed in conditions of the presence or absence of serum starvation that showed minimal impact for serum deprival, although significant inhibition of steady state MECP2E1 by simvastatin was only detected in non-serum starved cells. Taken together, our results suggest that metformin controls MECP2E1/E2-BDNF transcriptionally and/or post-transcriptionally, and that simvastatin is a potent transcriptional inhibitor of BDNF . The transcriptional effect of these drugs on MECP2E1/E2-BDNF were not additive under these tested conditions, however, either drug may have potential application for related disorders.

Blood circulates throughout the body via the peripheral tissues, contributes to host homeostasis and maintains normal physiological functions, in addition to responding to lesions. Previously, we revealed that gene expression analysis of peripheral blood cells is a useful approach for assessing diseases such as diabetes mellitus and cancer because the altered gene expression profiles of peripheral blood cells can reflect the presence and state of diseases. However, no chronological assessment of whole gene expression profiles has been conducted. In the present study, we collected whole blood RNA from 61 individuals (average age at registration, 50 years) every 4 years for 8 years and analyzed gene expression profiles using a complementary DNA microarray to examine whether these profiles were stable or changed over time. We found that the genes with very stable expression were related mostly to immune system pathways, including antigen cell presentation and interferon-related signaling. Genes whose expression was altered over the 8-year study period were principally involved in cellular machinery pathways, including development, signal transduction, cell cycle, apoptosis, and survival. Thus, this chronological examination study showed that the gene expression profiles of whole blood can reveal unmanifested physiological changes., (© 2021. The Author(s).)

SPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transcription by RNA polymerase II (RNA Pol II) relies on the elongation factors PAF1 complex (PAF), RTF1, and SPT6. Here, we use rapid factor depletion and multi-omics analysis to investigate how these elongation factors influence RNA Pol II elongation activity in human cells. Whereas depletion of PAF subunits PAF1 and CTR9 has little effect on cellular RNA synthesis, depletion of RTF1 or SPT6 strongly compromises RNA Pol II activity, albeit in fundamentally different ways. RTF1 depletion decreases RNA Pol II velocity, whereas SPT6 depletion impairs RNA Pol II progression through nucleosomes. These results show that distinct elongation factors stimulate either RNA Pol II velocity or RNA Pol II progression through chromatin in vivo. Further analysis provides evidence for two distinct barriers to early elongation: the promoter-proximal pause site and the +1 nucleosome. It emerges that the first barrier enables loading of elongation factors that are required to overcome the second and subsequent barriers to transcription., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Single cell/nucleus RNA sequencing (scRNAseq) is emerging as an essential tool to unravel the phenotypic heterogeneity of cells in complex biological systems. While computational methods for scRNAseq cell type clustering have advanced, the ability to integrate datasets to identify common and novel cell types across experiments remains a challenge. Here, we introduce a cluster-to-cluster cell type matching method-FR-Match-that utilizes supervised feature selection for dimensionality reduction and incorporates shared information among cells to determine whether two cell type clusters share the same underlying multivariate gene expression distribution. FR-Match is benchmarked with existing cell-to-cell and cell-to-cluster cell type matching methods using both simulated and real scRNAseq data. FR-Match proved to be a stringent method that produced fewer erroneous matches of distinct cell subtypes and had the unique ability to identify novel cell phenotypes in new datasets. In silico validation demonstrated that the proposed workflow is the only self-contained algorithm that was robust to increasing numbers of true negatives (i.e. non-represented cell types). FR-Match was applied to two human brain scRNAseq datasets sampled from cortical layer 1 and full thickness middle temporal gyrus. When mapping cell types identified in specimens isolated from these overlapping human brain regions, FR-Match precisely recapitulated the laminar characteristics of matched cell type clusters, reflecting their distinct neuroanatomical distributions. An R package and Shiny application are provided at https://github.com/JCVenterInstitute/FRmatch for users to interactively explore and match scRNAseq cell type clusters with complementary visualization tools., (© The Author(s) 2020. Published by Oxford University Press.)

Despite gene expression programs being notoriously complex, RNA abundance is usually assumed as a proxy for transcriptional activity. Recently developed approaches, able to disentangle transcriptional and post-transcriptional regulatory processes, have revealed a more complex scenario. It is now possible to work out how synthesis, processing and degradation kinetic rates collectively determine the abundance of each gene's RNA. It has become clear that the same transcriptional output can correspond to different combinations of the kinetic rates. This underscores the fact that markedly different modes of gene expression regulation exist, each with profound effects on a gene's ability to modulate its own expression. This review describes the development of the experimental and computational approaches, including RNA metabolic labeling and mathematical modeling, that have been disclosing the mechanisms underlying complex transcriptional programs. Current limitations and future perspectives in the field are also discussed., (© The Author(s) 2020. Published by Oxford University Press.)

Coordination of RNA abundance and production rate with cell size has been observed in diverse organisms and cell populations. However, how cells achieve such 'scaling' of transcription with size is unknown. Here we describe a genome-wide siRNA screen to identify regulators of global RNA production rates in HeLa cells. We quantify the single-cell RNA production rate using metabolic pulse-labelling of RNA and subsequent high-content imaging. Our quantitative, single-cell measurements of DNA, nascent RNA, proliferating cell nuclear antigen (PCNA), and total protein, as well as cell morphology and population-context, capture a detailed cellular phenotype. This allows us to account for changes in cell size and cell-cycle distribution (G1/S/G2) in perturbation conditions, which indirectly affect global RNA production. We also take advantage of the subcellular information to distinguish between nascent RNA localised in the nucleolus and nucleoplasm, to approximate ribosomal and non-ribosomal RNA contributions to perturbation phenotypes. Perturbations uncovered through this screen provide a resource for exploring the mechanisms of regulation of global RNA metabolism and its coordination with cellular states.

Cockayne syndrome (CS) is an autosomal recessive genetic disorder characterized by photosensitivity, developmental defects, neurological abnormalities, and premature aging. Mutations in CSA (ERCC8), CSB (ERCC6), XPB, XPD, XPG, XPF (ERCC4) and ERCC1 can give rise to clinical phenotypes resembling classic CS. Using a yeast two-hybrid (Y2H) screening approach, we identified LEO1 (Phe381-Ser568 region) as an interacting protein partner of full-length and C-terminal (Pro1010-Cys1493) CSB in two independent screens. LEO1 is a member of the RNA polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. Supportive of the Y2H results, purified, recombinant LEO1 and CSB directly interact in vitro, and the two proteins exist in a common complex within human cells. In addition, fluorescently tagged LEO1 and CSB are both recruited to localized DNA damage sites in human cells. Cell fractionation experiments revealed a transcription-dependent, coordinated association of LEO1 and CSB to chromatin following either UVC irradiation or cisplatin treatment of HEK293T cells, whereas the response to menadione was distinct, suggesting that this collaboration occurs mainly in the context of bulky transcription-blocking lesions. Consistent with a coordinated interaction in DNA repair, LEO1 knockdown or knockout resulted in reduced CSB recruitment to chromatin, increased sensitivity to UVC light and cisplatin damage, and reduced RNA synthesis recovery and slower excision of cyclobutane pyrimidine dimers following UVC irradiation; the absence of CSB resulted in diminished LEO1 recruitment. Our data indicate a reciprocal communication between CSB and LEO1 in the context of transcription-associated DNA repair and RNA transcription recovery., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

The synthetic pathways of life's building blocks are envisaged to be through a series of complex prebiotic reactions and processes. However, the strategy to compartmentalize and concentrate biopolymers under prebiotic conditions remains elusive. Liquid-liquid phase separation is a mechanism by which membraneless organelles form inside cells, and has been hypothesized as a potential mechanism for prebiotic compartmentalization. Associative phase separation of oppositely charged species has been shown to partition RNA, but the strongly negative charge exhibited by RNA suggests that RNA-polycation interactions could inhibit RNA folding and its functioning inside the coacervates. Here, we present a prebiotically plausible pathway for non-associative phase separation within an evaporating all-aqueous sessile droplet. We quantitatively investigate the kinetic pathway of phase separation triggered by the non-uniform evaporation rate, together with the Marangoni flow-driven hydrodynamics inside the sessile droplet. With the ability to undergo liquid-liquid phase separation, the drying droplets provide a robust mechanism for formation of prebiotic membraneless compartments, as demonstrated by localization and storage of nucleic acids, in vitro transcription, as well as a three-fold enhancement of ribozyme activity. The compartmentalization mechanism illustrated in this model system is feasible on wet organophilic silica-rich surfaces during early molecular evolution.

In a changing environment, small RNAs (sRNAs) play an important role in the post-transcriptional regulation of gene expression and can vary in abundance depending on the conditions experienced by an individual (phenotypic plasticity) and its parents (non-genetic inheritance). Many sRNAs are unusual in that they can be produced in two ways, either using genomic DNA as the template (primary sRNAs) or existing sRNAs as the template (secondary sRNAs). Thus, organisms can evolve rapid plastic responses to their current environment by adjusting the amplification rate of sRNA templates. sRNA levels can also be transmitted transgenerationally by the direct transfer of either sRNAs or the proteins involved in amplification. Theory is needed to describe the selective forces acting on sRNA levels, accounting for the dual nature of sRNAs as regulatory elements and templates for amplification and for the potential to transmit sRNAs and their amplification agents to offspring. Here, we develop a model to study the dynamics of sRNA production and inheritance in a fluctuating environment. We tested the selective advantage of mutants capable of sRNA-mediated phenotypic plasticity within resident populations with fixed levels of sRNA transcription. Even when the resident was allowed to evolve an optimal constant rate of sRNA production, plastic amplification rates capable of responding to environmental conditions were favored. Mechanisms allowing sRNA transcripts or amplification agents to be inherited were favored primarily when parents and offspring face similar environments and when selection acts before the optimal level of sRNA can be reached within the organism. Our study provides a clear set of testable predictions for the evolution of sRNA-related mechanisms of phenotypic plasticity and transgenerational inheritance., Competing Interests: The authors have declared that no competing interests exist.

With the development of single-cell RNA sequencing (scRNA-seq) technology, it has become possible to perform large-scale transcript profiling for tens of thousands of cells in a single experiment. Many analysis pipelines have been developed for data generated from different high-throughput scRNA-seq platforms, bringing a new challenge to users to choose a proper workflow that is efficient, robust and reliable for a specific sequencing platform. Moreover, as the amount of public scRNA-seq data has increased rapidly, integrated analysis of scRNA-seq data from different sources has become increasingly popular. However, it remains unclear whether such integrated analysis would be biassed if the data were processed by different upstream pipelines. In this study, we encapsulated seven existing high-throughput scRNA-seq data processing pipelines with Nextflow, a general integrative workflow management framework, and evaluated their performance in terms of running time, computational resource consumption and data analysis consistency using eight public datasets generated from five different high-throughput scRNA-seq platforms. Our work provides a useful guideline for the selection of scRNA-seq data processing pipelines based on their performance on different real datasets. In addition, these guidelines can serve as a performance evaluation framework for future developments in high-throughput scRNA-seq data processing., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The NanoString RNA counting assay for formalin-fixed paraffin embedded samples is unique in its sensitivity, technical reproducibility and robustness for analysis of clinical and archival samples. While commercial normalization methods are provided by NanoString, they are not optimal for all settings, particularly when samples exhibit strong technical or biological variation or where housekeeping genes have variable performance across the cohort. Here, we develop and evaluate a more comprehensive normalization procedure for NanoString data with steps for quality control, selection of housekeeping targets, normalization and iterative data visualization and biological validation. The approach was evaluated using a large cohort ($N=\kern0.5em 1649$) from the Carolina Breast Cancer Study, two cohorts of moderate sample size ($N=359$ and$130$) and a small published dataset ($N=12$). The iterative process developed here eliminates technical variation (e.g. from different study phases or sites) more reliably than the three other methods, including NanoString's commercial package, without diminishing biological variation, especially in long-term longitudinal multiphase or multisite cohorts. We also find that probe sets validated for nCounter, such as the PAM50 gene signature, are impervious to batch issues. This work emphasizes that systematic quality control, normalization and visualization of NanoString nCounter data are an imperative component of study design that influences results in downstream analyses., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Transcriptional switch (TS) is a widely observed phenomenon caused by changes in the relative expression of transcripts from the same gene, in spatial, temporal or other dimensions. TS has been associated with human diseases, plant development and stress responses. Its investigation is often hampered by a lack of suitable tools allowing comprehensive and flexible TS analysis for high-throughput RNA sequencing (RNA-Seq) data. Here, we present deepTS, a user-friendly web-based implementation that enables a fully interactive, multifunctional identification, visualization and analysis of TS events for large-scale RNA-Seq datasets from pairwise, temporal and population experiments. deepTS offers rich functionality to streamline RNA-Seq-based TS analysis for both model and non-model organisms and for those with or without reference transcriptome. The presented case studies highlight the capabilities of deepTS and demonstrate its potential for the transcriptome-wide TS analysis of pairwise, temporal and population RNA-Seq data. We believe deepTS will help research groups, regardless of their informatics expertise, perform accessible, reproducible and collaborative TS analyses of large-scale RNA-Seq data., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

At the focus of abiotic chemical reactions is the synthesis of ribose. No satisfactory explanation was provided as to the missing link between the prebiotic synthesis of ribose and prebiotic RNA (preRNA). Hydrogen cyanide (HCN) is assumed to have been the principal precursor in the prebiotic formation of aldopentoses in the formose reaction and in the synthesis of ribose. Ribose as the best fitting aldopentose became the exclusive sugar component of RNA. The elevated yield of ribose synthesis at higher temperatures and its protection from decomposition could have driven the polymerization of the ribose-phosphate backbone and the coupling of nucleobases to the backbone. RNA could have come into being without the involvement of nucleotide precursors. The first nucleoside monophosphate is likely to have appeared upon the hydrolysis of preRNA contributed by the presence of reactive 2'-OH moieties in the preRNA chain. As a result of phosphorylation, nucleoside monophosphates became nucleoside triphosphates, substrates for the selective synthesis of genRNA.

Rationale: Noncoding RNAs (ncRNAs) such as microRNAs (miRs or miRNAs) play important roles in the control of cellular processes through posttranscriptional gene regulation. However, ncRNA research is limited to utilizing RNA agents synthesized in vitro . Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs. Methods: Herein, we developed a novel platform for in vivo fermentation production of humanized recombinant ncRNA molecules, namely hBERAs, carrying payload miRNAs or siRNAs. Target hBERAs were purified by anion exchange FPLC method. Functions of hBERA/miRNAs were investigated in human carcinoma cells and antitumor activities were determined in orthotopic osteosarcoma xenograft spontaneous lung metastasis mouse models. Results: Proper human tRNAs were identified to couple with optimal hsa-pre-miR-34a as new fully-humanized ncRNA carriers to accommodate warhead miRNAs or siRNAs. A group of 30 target hBERAs were all heterogeneously overexpressed (each accounting for >40% of total bacterial RNA), which facilitated large-scale production (8-31 mg of individual hBERAs from 1L bacterial culture). Model hBERA/miR-34a-5p and miR-124-3p were selectively processed to warhead miRNAs in human carcinoma cells to modulate target gene expression, enhance apoptosis and inhibit invasiveness. In addition, bioengineered miR-34a-5p and miR-124-3p agents both reduced orthotopic osteosarcoma xenograft tumor growth and spontaneous pulmonary metastases significantly. Conclusion: This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development., Competing Interests: Competing Interests: A.-M.Y., M.-J.T. and P.Y.H. are inventors on US Patent Application No. 62/674,939 filed on May 22, 2018 as well as US Patent No. 10,619,156 issued on April 14, 2020 and European Patent No. 3150980 granted on May 20, 2020 that are related to this work. A.-M.Y. is a founder of AimRNA, Inc. that intends to license the intellectual property., (© The author(s).)

CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA modifications, long considered to be molecular curiosities embellishing just abundant and non-coding RNAs, have now moved into the focus of both academic and applied research. Dedicated research efforts (epitranscriptomics) aim at deciphering the underlying principles by determining RNA modification landscapes and investigating the molecular mechanisms that establish, interpret and modulate the information potential of RNA beyond the combination of four canonical nucleotides. This has resulted in mapping various epitranscriptomes at high resolution and in cataloguing the effects caused by aberrant RNA modification circuitry. While the scope of the obtained insights has been complex and exciting, most of current epitranscriptomics appears to be stuck in the process of producing data, with very few efforts to disentangle cause from consequence when studying a specific RNA modification system. This article discusses various knowledge gaps in this field with the aim to raise one specific question: how are the enzymes regulated that dynamically install and modify RNA modifications? Furthermore, various technologies will be highlighted whose development and use might allow identifying specific and context-dependent regulators of epitranscriptomic mechanisms. Given the complexity of individual epitranscriptomes, determining their regulatory principles will become crucially important, especially when aiming at modifying specific aspects of an epitranscriptome both for experimental and, potentially, therapeutic purposes.

The RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of nucleic acid polymerases. RdRPs are essential in virus life cycle due to their central role in viral genome replication/transcription processes. However, their contribution in host adaption has not been well documented. By solving the RdRP crystal structure of the tick-borne encephalitis virus (TBEV), a tick-borne flavivirus, and comparing the structural and sequence features with mosquito-borne flavivirus RdRPs, we found that a region between RdRP catalytic motifs B and C, namely region B-C, clearly bears host-related diversity. Inter-virus substitutions of region B-C sequence were designed in both TBEV and mosquito-borne Japanese encephalitis virus backbones. While region B-C substitutions only had little or moderate effect on RdRP catalytic activities, virus proliferation was not supported by these substitutions in both virus systems. Importantly, a TBEV replicon-derived viral RNA replication was significantly reduced but not abolished by the substitution, suggesting the involvement of region B-C in viral and/or host processes beyond RdRP catalysis. A systematic structural analysis of region B-C in viral RdRPs further emphasizes its high level of structure and length diversity, providing a basis to further refine its relevance in RNA virus-host interactions in a general context., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genome-wide association studies have identified common variants associated with platelet-related phenotypes, but because these variants are largely intronic or intergenic, their link to platelet biology is unclear. In 290 normal subjects from the GeneSTAR Research Study (110 African Americans [AAs] and 180 European Americans [EAs]), we generated whole-genome sequence data from whole blood and RNA sequence data from extracted nonribosomal RNA from 185 induced pluripotent stem cell-derived megakaryocyte (MK) cell lines (platelet precursor cells) and 290 blood platelet samples from these subjects. Using eigenMT software to select the peak single-nucleotide polymorphism (SNP) for each expressed gene, and meta-analyzing the results of AAs and EAs, we identify (q-value < 0.05) 946 cis-expression quantitative trait loci (eQTLs) in derived MKs and 1830 cis-eQTLs in blood platelets. Among the 57 eQTLs shared between the 2 tissues, the estimated directions of effect are very consistent (98.2% concordance). A high proportion of detected cis-eQTLs (74.9% in MKs and 84.3% in platelets) are unique to MKs and platelets compared with peak-associated SNP-expressed gene pairs of 48 other tissue types that are reported in version V7 of the Genotype-Tissue Expression Project. The locations of our identified eQTLs are significantly enriched for overlap with several annotation tracks highlighting genomic regions with specific functionality in MKs, including MK-specific DNAse hotspots, H3K27-acetylation marks, H3K4-methylation marks, enhancers, and superenhancers. These results offer insights into the regulatory signature of MKs and platelets, with significant overlap in genes expressed, eQTLs detected, and enrichment within known superenhancers relevant to platelet biology.

Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription., Competing Interests: Declaration of interests J.A.S. is a member of the Molecular Cell Advisory Board. The authors have no further competing interests to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: To examine the characteristics and distribution of possible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target cells in the human trophectoderm (TE) and placenta., Methods: Bioinformatics analysis was performed based on published single-cell transcriptomic datasets of early TE and first- and second-trimester human placentae. We conducted the transcriptomic analysis of 4198 early TE cells, 1260 first-trimester placental cells and 189 extravillous trophoblast cells (EVTs) from 24-week placentae (EVT&#95;24W) using the SMART-Seq2 method. In addition, to confirm the bioinformatic results, we performed immunohistochemical staining of three first-trimester, three second-trimester and three third-trimester placentae from nine women recruited prospectively to this study. We evaluated the expression of the SARS-CoV-2-related molecules angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2)., Results: Via bioinformatic analysis, we identified the existence of ACE2 and TMPRSS2 expression in human TE as well as in first- and second-trimester placentae. In the human TE, 54.4% of TE1 cells, 9.0% of cytotrophoblasts (CTBs), 3.2% of EVTs and 29.5% of syncytiotrophoblasts (STBs) were ACE2-positive. In addition, 90.7% of TE1 cells, 31.5% of CTBs, 22.1% of EVTs and 70.8% of STBs were TMPRSS2-positive. In placental cells, 20.4% of CTBs, 44.1% of STBs, 3.4% of EVTs from 8-week placentae (EVT&#95;8W) and 63% of EVT&#95;24W were ACE2-positive, while 1.6% of CTBs, 26.5% of STBs, 1.9% of EVT&#95;8W and 20.1% of EVT&#95;24W were TMPRSS2-positive. Pathway analysis revealed that EVT&#95;24W cells that were positive for both ACE2 and TMPRSS2 (ACE2 + TMPRSS2-positive) were associated with morphogenesis of branching structure, extracellular matrix interaction, oxygen binding and antioxidant activity. The ACE2 + TMPRSS2-positive TE1 cells were correlated with an increased capacity for viral invasion, epithelial-cell proliferation and cell adhesion. Expression of ACE2 and TMPRSS2 was observed on immunohistochemical staining in first-, second- and third-trimester placentae., Conclusions: ACE2- and TMPRSS2-positive cells are present in the human TE and placenta in all three trimesters of pregnancy, which indicates the possibility that SARS-CoV-2 could spread via the placenta and cause intrauterine fetal infection. © 2020 International Society of Ultrasound in Obstetrics and Gynecology., (© 2020 International Society of Ultrasound in Obstetrics and Gynecology.)

Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from in vitro and in vivo experiments support a model where RNAs produced during early steps in transcription initiation stimulate condensate formation, whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process., Competing Interests: Declaration of Interests R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. T.I.L. is a shareholder of Syros Pharmaceuticals and a consultant to Camp4 Therapeutics. A.K.C. and P.A.S. are shareholders and consultants to Dewpoint Therapeutics. A.K.C. is a SAB member of Omega Therapeutics. The Whitehead Institute has filed a patent application based on this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)