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Influenza A virus (IAV) lacks the enzyme for adding 5' caps to its RNAs and snatches the 5' ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched nor the effect of cap-snatching on host processes is completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNAs from infected cells or relied on annotated host genes to define the snatched host RNAs, and thus lack details on many noncoding host RNAs including snRNAs, snoRNAs, and promoter-associated capped small (cs)RNAs, which are made by "paused" Pol II during transcription initiation. In this study, we used a nonbiased technique, CapSeq, to identify host and viral-capped RNAs including nonpolyadenylated RNAs in the same samples, and investigated the substrate-product correlation between the host RNAs and the viral RNAs. We demonstrated that noncoding host RNAs, particularly U1 and U2, are the preferred cap-snatching source over mRNAs or pre-mRNAs. We also found that csRNAs are highly snatched by IAV. Because the functions of csRNAs remain mostly unknown, especially in somatic cells, our finding reveals that csRNAs at least play roles in the process of IAV infection. Our findings support a model where nascent RNAs including csRNAs are the preferred targets for cap-snatching by IAV and raise questions about how IAV might use snatching preferences to modulate host-mRNA splicing and transcription.
It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.
Bacterial RNA polymerase is able to initiate transcription with adenosine-containing cofactor NAD+, which was proposed to result in a portion of cellular RNAs being 'capped' at the 5' end with NAD+, reminiscent of eukaryotic cap. Here we show that, apart from NAD+, another adenosine-containing cofactor FAD and highly abundant uridine-containing cell wall precursors, UDP-Glucose and UDP-N-acetylglucosamine are efficiently used to initiate transcription in vitro. We show that the affinity to NAD+ and UDP-containing factors during initiation is much lower than their cellular concentrations, and that initiation with them stimulates promoter escape. Efficiency of initiation with NAD+, but not with UDP-containing factors, is affected by amino acids of the Rifampicin-binding pocket, suggesting altered RNA capping in Rifampicin-resistant strains. However, relative affinity to NAD+ does not depend on the -1 base of the template strand, as was suggested earlier. We show that incorporation of mature cell wall precursor, UDP-MurNAc-pentapeptide, is inhibited by region 3.2 of σ subunit, possibly preventing targeting of RNA to the membrane. Overall, our in vitro results propose a wide repertoire of potential bacterial RNA capping molecules, and provide mechanistic insights into their incorporation.
Chemical modification of transcripts with 5' caps occurs in all organisms. Here, we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of an organism's cap epitranscriptome. The method was piloted with 21 canonical caps-m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG-and 5 'metabolite' caps-NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus, Escherichia coli, yeast, mouse tissues, and human cells, we discovered new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and high proportions of caps lacking 2'-O-methylation (m7Gpppm6A in mammals, m7GpppA in dengue virus). While substantial Dimroth-induced loss of m1A and m1Am arose with specific RNA processing conditions, human lymphoblast cells showed no detectable m1A or m1Am in caps. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps.
We show that about one-third of the RNAs produced in vitro by viral RNA polymerases in the presence of m7GpppG dinucleotides have unusual 5' caps. In these RNAs, the initiating dinucleotide is incorporated in an orientation opposite to that expected so that the 7-methyl guanine (m7G) nucleotide is adjacent to the body of the RNA, making a "reverse" cap. The doubly methylated dinucleotide, m7GpppGm, containing a 2' O-methylated guanine (Gm) is incorporated only in the reverse orientation. Precursors of U1 snRNAs containing reverse caps are recognized by antibodies specific for the m7G cap structure. When injected into Xenopus laevis oocyte nuclei, reverse-capped pre-U1 RNAs are exported considerably more slowly than normal. Furthermore, U1 RNAs with reverse caps exhibit a striking defect in nuclear import that can be attributed to the failure of reverse caps to be hypermethylated to m2,2,7G caps. Thus, the presence of reverse-capped RNAs in RNA preparations may affect conclusions about the efficiency and extent of certain m7G cap-dependent processes.
Accurate identification of NAD-capped RNAs is essential for delineating their generation and biological function. Previous transcriptome-wide methods used to classify NAD-capped RNAs in eukaryotes contain inherent limitations that have hindered the accurate identification of NAD caps from eukaryotic RNAs. In this study, we introduce two orthogonal methods to identify NAD-capped RNAs more precisely. The first, NADcapPro, uses copper-free click chemistry and the second is an intramolecular ligation-based RNA circularization, circNC. Together, these methods resolve the limitations of previous methods and allowed us to discover unforeseen features of NAD-capped RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD-RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m7G-capped RNAs can be different, and 3) NAD caps can be added subsequent to transcription initiation. Moreover, we uncovered a dichotomy of NAD-RNAs in translation where they are detected with mitochondrial ribosomes but minimally on cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.
RNA 5' cap structures comprising the metabolic effector nicotinamide adenine dinucleotide (NAD) have been identified in diverse organisms. Here we report a simple, two-step procedure to detect and quantitate NAD-capped RNA, termed "NAD-capQ." By use of NAD-capQ we quantitate NAD-capped RNA levels in Escherichia coli, Saccharomyces cerevisiae, and human cells, and we measure increases in NAD-capped RNA levels in cells from all three organisms harboring disruptions in their respective "deNADding" enzymes. We further show that NAD-capped RNA levels in human cells respond to changes in cellular NAD concentrations, indicating that NAD capping provides a mechanism for human cells to directly sense and respond to alterations in NAD metabolism. Our findings establish NAD-capQ as a versatile, rapid, and accessible methodology to detect and quantitate 5'-NAD caps on endogenous RNA in any organism.
Algae are important primary colonizers of snow and glacial ice, but hitherto little is known about their ecology on Iceland's glaciers and ice caps. Due do the close proximity of active volcanoes delivering large amounts of ash and dust, they are special ecosystems. This study provides the first investigation of the presence and diversity of microbial communities on all major Icelandic glaciers and ice caps over a 3 year period. Using high-throughput sequencing of the small subunit ribosomal RNA genes (16S and 18S), we assessed the snow community structure and complemented these analyses with a comprehensive suite of physical-, geo-, and biochemical characterizations of the aqueous and solid components contained in snow and ice samples. Our data reveal that a limited number of snow algal taxa (Chloromonas polyptera, Raphidonema sempervirens and two uncultured Chlamydomonadaceae) support a rich community comprising of other micro-eukaryotes, bacteria and archaea. Proteobacteria and Bacteroidetes were the dominant bacterial phyla. Archaea were also detected in sites where snow algae dominated and they mainly belong to the Nitrososphaerales, which are known as important ammonia oxidizers. Multivariate analyses indicated no relationships between nutrient data and microbial community structure. However, the aqueous geochemical simulations suggest that the microbial communities were not nutrient limited because of the equilibrium of snow with the nutrient-rich and fast dissolving volcanic ash. Increasing algal secondary carotenoid contents in the last stages of the melt seasons have previously been associated with a decrease in surface albedo, which in turn could potentially have an impact on the melt rates of Icelandic glaciers.
Canonical sexual reproduction among basidiomycete fungi involves the fusion of two haploid individuals of different mating types, resulting in a heterokaryotic mycelial body made up of genetically different nuclei. Using population genomics data and experiments, we discover mushrooms of the invasive and deadly Amanita phalloides can also be homokaryotic; evidence of sexual reproduction by single, unmated individuals. In California, genotypes of homokaryotic mushrooms are also found in heterokaryotic mushrooms, implying nuclei of homokaryotic mycelia are also involved in outcrossing. We find death cap mating is controlled by a single mating type locus, but the development of homokaryotic mushrooms appears to bypass mating type gene control. Ultimately, sporulation is enabled by nuclei able to reproduce alone as well as with others, and nuclei competent for both unisexuality and bisexuality have persisted in invaded habitats for at least 17 but potentially as long as 30 years. The diverse reproductive strategies of invasive death caps are likely facilitating its rapid spread, suggesting a profound similarity between plant, animal and fungal invasions.
We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5'caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins-Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.
In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species. Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution. Here, we show that telomeres on chromosome arms carrying subtelomeric heterochromatic caps in the chimpanzee, which are completely absent in humans, replicate later than telomeres on chromosome arms without caps. In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres. Strikingly, telomere-containing RNA accumulates extrachromosomally in gorilla mitotic cells, suggesting that at least some aspects of telomere-containing RNA biogenesis have diverged in gorilla, perhaps in concert with the evolution of heterochromatic caps in this species.
Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome (CAPS), and to compare the CAPS proteins to human and murine platelet proteomic data. Washed platelets were isolated from healthy dogs, and stimulated with saline (control) or gamma-thrombin (releasate). Proteins were separated by SDS-page, trypsin-digested and analyzed by liquid chromatography and tandem mass spectrometry (MS). CAPS proteins were defined as those with a MS1-abundance ratio of two or more for releasate vs. unstimulated saline control. A total of 1,918 proteins were identified, with 908 proteins common to all dogs and 693 characterized as CAPS proteins. CAPS proteins were similar to human and murine platelet secretomes and were highly represented in hemostatic pathways. Differences unique to CAPS included replacement of platelet factor 4 with other cleavage products of platelet basic protein (e.g. interleukin-8), novel proteins (e.g. C-C motif chemokine 14), and proteins in relatively high (e.g. protease nexin-1) or low (e.g. von Willebrand factor) abundance. This study establishes the first in-depth platelet releasate proteome from healthy dogs with a reference database of 693 CAPS proteins. Similarities between CAPS and the human secretome confirm the utility of dogs as translational models of human disease, but we also identify differences unique to canine platelets. Our findings provide a resource for further investigations into disease-related CAPS profiles, and for comparative pathway analyses of platelet activation among species.
miRNAs are potential regulators of carotid artery stenosis and concordant vulnerable atherosclerotic plaques. Hence, we analyzed miRNA expression in laser captured micro-dissected fibrous caps of either ruptured or stable plaques (n = 10 each), discovering that miR-21 was significantly downregulated in unstable lesions. To functionally evaluate miR-21 in plaque vulnerability, miR-21 and miR-21/apolipoprotein-E double-deficient mice (Apoe-/-miR-21-/-) were assessed. miR-21-/- mice lacked sufficient smooth muscle cell proliferation in response to carotid ligation injury. When exposing Apoe-/-miR-21-/- mice to an inducible plaque rupture model, they presented with more atherothrombotic events (93%) compared with miR-21+/+Apoe-/- mice (57%). We discovered that smooth muscle cell fate in experimentally induced advanced lesions is steered via a REST-miR-21-REST feedback signaling pathway. Furthermore, Apoe-/-miR-21-/- mice presented with more pronounced atherosclerotic lesions, greater foam cell formation, and substantially higher levels of arterial macrophage infiltration. Local delivery of a miR-21 mimic using ultrasound-targeted microbubbles into carotid plaques rescued the vulnerable plaque rupture phenotype. In the present study, we identify miR-21 as a key modulator of pathologic processes in advanced atherosclerosis. Targeted, lesion site-specific overexpression of miR-21 can stabilize vulnerable plaques.
Novel features of coenzyme A (CoA) and its precursor, 3'-dephospho-CoA (dpCoA), recently became evident. dpCoA was found to attach to 5'-ends of small ribonucleic acids (dpCoA-RNAs) in two bacterial species (Escherichia coli and Streptomyces venezuelae). Furthermore, CoA serves, in addition to its well-established coenzymatic roles, as a ubiquitous posttranslational protein modification ('CoAlation'), thought to prevent the irreversible oxidation of cysteines. Here, we first identified and quantified dpCoA-RNAs in the small RNA fraction of the human pathogen Staphylococcus aureus, using a newly developed enzymatic assay. We found that the amount of dpCoA caps was similar to that of the other two bacteria. We furthermore tested the hypothesis that, in the environment of a cell, the free thiol of the dpCoA-RNAs, as well as other sulfur-containing RNA modifications, may be oxidized by disulfide bond formation, e.g., with CoA. While we could not find evidence for such an 'RNA CoAlation', we observed that CoA disulfide reductase, the enzyme responsible for reducing CoA homodisulfides in S. aureus, did efficiently reduce several synthetic dpCoA-RNA disulfides to dpCoA-RNAs in vitro. This activity may imply a role in reversing RNA CoAlation.
In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3'-FADP or CoA and fission yeast Rai1 with 3'-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.
The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5'-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3'-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.
Domestic dogs represent a translational animal model to study naturally occurring human disease. Proteomics has emerged as a promising tool for characterizing human platelet pathophysiology; thus a detailed characterization of the core canine activated platelet secretome (CAPS) will enhance utilization of the canine model. The objectives of this study were development of a robust, high throughput, label-free approach for proteomic identification and quantification of the canine platelet (i) thrombin releasate proteins, and (ii) the protein subgroup that constitutes CAPS.
In addition to their roles in protecting nerves and increasing conduction velocity, peripheral glia plays key functions in blood vessel development by secreting molecules governing arteries alignment and maturation with nerves. Here, we show in mice that a specific, nerve-attached cell population, derived from boundary caps (BCs), constitutes a major source of mural cells for the developing skin vasculature. Using Cre-based reporter cell tracing and single-cell transcriptomics, we show that BC derivatives migrate into the skin along the nerves, detach from them, and differentiate into pericytes and vascular smooth muscle cells. Genetic ablation of this population affects the organization of the skin vascular network. Our results reveal the heterogeneity and extended potential of the BC population in mice, which gives rise to mural cells, in addition to previously described neurons, Schwann cells, and melanocytes. Finally, our results suggest that mural specification of BC derivatives takes place before their migration along nerves to the mouse skin.
Bacterial blotch is a group of economically important diseases affecting the cultivation of common button mushroom, Agaricus bisporus. Despite being studied for more than a century, the identity and nomenclature of blotch-causing Pseudomonas species is still unclear. This study aims to molecularly characterize the phylogenetic and phenotypic diversity of blotch pathogens in Western Europe.
Among SNP markers that become increasingly valuable in molecular breeding of crop plants are the CAPS and dCAPS markers derived from the genes of interest. To date, the number of such gene-based markers is small in polyploid crop plants such as allotetraploid cotton that has A- and D-sub-genomes. The objective of this study was to develop and map new CAPS and dCAPS markers for cotton developmental-regulatory genes that are important in plant breeding programs.
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