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.)

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N 6 -methyladenosine (m 6 A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m 6 A methylation of circRNAs. Changes in m 6 A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m 6 A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m 6 A modification patterns. The majority of circRNAs that showed post-stroke differential m 6 A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m 6 A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m 6 A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Biological processes in intertidal species follow tidal rhythms that enhance survival and fitness. Whereas tidal effects on behaviour and metabolic rates have been widely studied, impacts on other key process such as protein synthesis are still poorly understood. To date, no studies have examined the effect of immersion/emersion and tidal cycles on protein synthesis rates (k s ). Patella vulgata is an intertidal limpet present in North-Eastern Atlantic rocky shores from high to low shore. Previously reported P. vulgata respiration and heart rate measurements suggest aerobic metabolism is maintained during emersion and growth rates increase from high to low shore, but whether these patterns are reflected in k s is currently unclear. Here, we measured for the first time in any intertidal organism, k s , RNA to protein ratios and RNA translational efficiency (k RNA ) in P. vulgata over a full tidal cycle, at three different shore heights. k s increased during emersion (p < 0.001) and was significantly higher in low shore animals compared to the other shore heights (p < 0.001), additionally k s was negatively correlated to body mass (p = 0.002). RNA to protein ratios remained unchanged over the tidal cycle (p = 0.659) and did not vary with shore height (p = 0.591). k RNA was significantly higher during emersion and was also higher in low shore limpets (p < 0.001). This study demonstrates that P. vulgata increases k s during emersion, an important adaptation in a species that spends a considerable amount of its lifecycle emersed. Intertidal species are highly exposed to increasing air temperatures, making knowledge of physiological responses during emersion critical in understanding and forecasting climate warming impacts., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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).)

One of the significant challenges during the purification and characterization of antimicrobial peptides (AMPs) from Bacillus sp. is the interference of unutilized peptides from complex medium components during analytical procedures. In this study, a semi-synthetic medium was devised to overcome this challenge. Using a genetic algorithm, the production medium of AMP is optimized. The parent organism, Bacillus licheniformis MCC2514, produces AMP in very small quantities. This AMP is known to inhibit RNA biosynthesis. The findings revealed that lactose, NH 4 Cl and NaNO 3 were crucial medium constituents for enhanced AMP synthesis. The potency of the AMP produced was studied using bacterium, Kocuria rhizophila ATCC 9341. The AMP produced from the optimized medium was eightfold higher than that produced from the unoptimized medium. Furthermore, activity was increased by 1.5-fold when cultivation conditions were standardized using the optimized medium. Later, AMP was produced in a 5 L bioreactor under controlled conditions, which led to similar results as those of shake-flask production. The mode of action of optimally produced AMP was confirmed to be inhibition of RNA biosynthesis. Here, we demonstrate that improved production of AMP is possible with the developed semi-synthetic medium recipe and could help further AMP production in an industrial setup., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.

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).)

Main conclusion: Transcriptome analysis was carried out for wheat seedlings and spikes from hybrid Jingmai 8 and both inbred lines to unravel mechanisms underlying heterosis.Heterosis, known as one of the most successful strategies for increasing crop yield, has been widely exploited in plant breeding systems. Despite its great importance, the molecular mechanism underlying heterosis remains elusive. In the present study, RNA sequencing (RNA-seq) was performed on the seedling and spike tissues of the wheat (Triticum aestivum) hybrid Jingmai 8 (JM8) and its homozygous parents to unravel the underlying mechanisms of wheat heterosis. In total, 1686 and 2334 genes were identified as differentially expressed genes (DEGs) between the hybrid and the two inbred lines in seedling and spike tissues, respectively. Gene Ontology analysis revealed that DEGs from seedling tissues were significantly enriched in processes involved in photosynthesis and carbon fixation, and the majority of these DEGs expressed at a higher level in JM8 compared to both inbred lines. In addition, cell wall biogenesis and protein biosynthesis-related pathways were also significantly represented. These results confirmed that a combination of different pathways could contribute to heterosis. The DEGs between the hybrid and the two inbred progenitors from the spike tissues were significantly enriched in biological processes related to transcription, RNA biosynthesis and molecular function categories related to transcription factor activities. Furthermore, transcription factors such as NAC, ERF, and TIF-IIA were highly expressed in the hybrid JM8. These results may provide valuable insights into the molecular mechanisms underlying wheat heterosis. [ABSTRACT FROM AUTHOR]

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non-Hfq-dependent sRNA scaffold that uses a modifiable 20 nucleotide antisense binding region to target mRNAs selectively and influence protein expression. The system was developed for regulation of a fluorescent reporter in vivo using Escherichia coli, but the system was found to be more responsive and produced statistically significant results when applied to protein synthesis using in vitro cell-free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high-energy stem loop structure and the 3' end of mRNA yielded protein expression knock-downs that approached 70%. Notably, targeting a low-energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in protein expression (p < 0.05). These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in a CFS. It is possible the designs developed here can be applied to improve/regulate expression of other proteins in a CFS., (© 2023 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

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.)

We have developed a simple NMR-based method to determine the turnover of nucleotides and incorporation into RNA by stable isotope resolved metabolomics (SIRM) in A549 lung cancer cells. This method requires no chemical degradation of the nucleotides or chromatography. During cell growth, the free ribonucleotide pool is rapidly replaced by de novo synthesized nucleotides. Using [U-C]-glucose and [U-C,N]-glutamine as tracers, we showed that virtually all of the carbons in the nucleotide riboses were derived from glucose, whereas glutamine was preferentially utilized over glucose for pyrimidine ring biosynthesis, via the synthesis of Asp through the Krebs cycle. Incorporation of the glutamine amido nitrogen into the N3 and N9 positions of the purine rings was also demonstrated by proton-detected N NMR. The incorporation of C from glucose into total RNA was measured and shown to be a major sink for the nucleotides during cell proliferation. This method was applied to determine the metabolic action of an anti-cancer selenium agent (methylseleninic acid or MSA) on A549 cells. We found that MSA inhibited nucleotide turnover and incorporation into RNA, implicating an important role of nucleotide metabolism in the toxic action of MSA on cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular mRNAs that contain partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. MiRNA was originally discovered in the intergenic regions of the Caenorhabditis elegans genome as native RNA fragments that modulate a wide range of genetic regulatory pathways during animal development. However, neither RNA promoter nor polymerase responsible for miRNA biogenesis was determined. Recent findings of intron-derived miRNA in C. elegans, mouse, and human have inevitably led to an alternative pathway for miRNA biogenesis, which relies on the coupled interaction of Pol-II-mediated pre-mRNA transcription and intron excision, occurring in certain nuclear regions proximal to genomic perichromatin fibrils. [Copyright &y& Elsevier]

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.)

The extent of the relationship between hormone dependence and cytostatica sensitivity of the tumors was examined for primary breast cancers under standardized in vitro conditions. The effects of estradiol and adriamycin on the RNA biosynthesis in the tumor cells were compared on the basis of the H-uridine incorporation in a short-term test system. Estradiol particularly influenced the RNA metabolism of higly differentiated carcinomas, whereas adriamycin exhibited the greatest inhibitory effect on the RNA synthesis in the tumor cells of undifferentiated carcinomas. An exact, unequivocal separation of the breast cancers investigated into hormone-sensitive, cytostatica-resistant and hormone-independent, cytostatica-sensitive tumors, however, was not possible under the in vitro conditions chosen. [ABSTRACT FROM AUTHOR]

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.)

Once it was believed that ribosomal RNA encodes proteins, and GTP hydrolysis supplies the energy for protein synthesis. Everything has changed, when Alexander Spirin joined the science. It turned out that proteins are encoded by a completely different RNA, and GTP hydrolysis only accelerates the process already provided with energy. It was Spirin who first put forward the idea of a Brownian ratchet and explained how and why molecular machines could arise in the RNA world.

Recent studies underscore RNA modifications as a novel mechanism to coordinate expression and function of different genes. While modifications on the sugar or base moieties of tRNA are well known, their roles in mRNA regulation are only starting to emerge. Interestingly, some modifications are present in both tRNA and mRNA, and here we discuss the functional significance of these common features. We describe key modifications that are present in both RNA types, elaborate on proteins that interact with them, and indicate recent works that identify roles in communicating tRNA processes and mRNA regulation. We propose that as tools are developed, the shortlist of features that are common between types of RNA will greatly expand and proteins that interact with them will be identified. In conclusion, the presence of the same modification in both RNA types provides an intersect between tRNA processes and mRNA regulation and implies a novel mechanism for connecting diverse cellular processes.

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.)

A mechanism of nucleoside triphosphorylation would have been critical in an evolving "RNA world" to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. Previous reports have demonstrated that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under prebiotically-relevant conditions, but their reaction rates were unknown and the influence of reaction conditions not well-characterized. Here we established a sensitive assay that allowed for the determination of second-order rate constants for all four rNTPs, ranging from 1.7×10 -6 to 6.5×10 -6  M -1  s -1 . The ATP reaction shows a linear dependence on pH and Mg 2+ , and an enthalpy of activation of 88±4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication., (© 2021 Wiley-VCH GmbH.)

Liposomes, which are vesicles surrounded by lipid membranes, can be used as biochemical reactors by encapsulating various reactions. Accordingly, they are useful for studying cellular functions under controlled conditions that mimic the environment within a cell. However, one of the shortcomings of liposomes as biochemical reactors is the difficulty of introducing or removing proteins due to the impermeability of the membrane. In this study, we established a method for exchanging proteins in liposomes by forming reversible pores in the membrane. We used the toxic protein streptolysin O (SLO); this forms pores in membranes made of phospholipids containing cholesterol that can be closed by the addition of calcium ions. After optimizing the experimental procedure and lipid composition, we observed the exchange of fluorescent proteins (transferrin Alexa Fluor 488 and 647) in 9.9 % of liposomes. We also introduced T7 RNA polymerase, a 98-kDa enzyme, and observed RNA synthesis in ∼8 % of liposomes. Our findings establish a new method for controlling the internal protein composition of liposomes, thereby increasing their utility as bioreactors., (© 2021 Wiley-VCH GmbH.)