Project description:We performed a RNA immunoprecipitations experiments using gfp-specific antibodies to precipitate gfp-tagged La proteins from from gfp-La wild type and sumoylation deficient La mutant (K41/200R) cells and found that specific mRNAs are preferentially enriched gfp-La wild type RIPs when compared to sumoylation deficient La mutant (K41/200R) RIPs.
2017-11-22 | GSE102842 | GEO
Project description:La Crosse Virus in vitro and in in vivo antiviral models
Project description:We recently identified lysine L-lactylation (KL-la) on histones that can be labelled by L-lactate, the end-product of glycolysis. KL-la has two structural isomers, namely N--(carboxyethyl) lysine (Kce) and lysine D-lactylation (KD-la), which can also be caused by metabolites associated with glycolysis. It is unknown if perturbations of glycolysis can lead to dysregulation of KD-la and Kce, in addition to KL-la. Further, current methods have a difficulty to distinguish among these isomers in cellular contexts. To investigate these questions, we first generated specific antibodies against each one of these three modifications. These reagents enable us to distinguish these three isomers. We demonstrated that KL-la, but not KD-la and Kce, is dynamically regulated by glycolysis. KD-la and Kce occur mainly when the major glycolytic pathway is blocked downstream or when the glyoxalase system is incomplete. This result was also independently confirmed by orthogonal HPLC-mass spectrometry, showing that KL-la is the predominant isomer of lactylation on cellular histones. Finally, we demonstrated that lactyl-CoA, an intermediate between L-lactate and lactylation, is dynamically regulated by glycolysis and is positively correlated with KL-la. Thus, our study clearly shows that KL-la, but not KD-la and Kce, is the major glycolytic- and the Warburg-effect associated responsive modification in cells.
Project description:SARS-CoV-2 virus mimics host mRNA by capping its viral RNA to promote replication and evade host immune sensing. SARS-CoV-2 NSP14 is the N7-guanosine methyltransferase (N7-MTase) responsible for RNA cap-0 formation. Targeting NSP14 for antiviral drug development is an under-explored but promising strategy. Here we conducted a high-throughput screening on natural products library derived from Chinese herbal medicine to discover Emodin as a SARS-CoV-2 NSP14 inhibitor. Exploring Emodin derivatives, Questin was identified with potent cellular inhibitory activity (EC50=249 nM) against SARS-CoV-2, which inhibits NSP14 in an RNA cap competitive manner, making it one the most potent anti-coronaviral natural products. Mechanistically, besides catalyzing viral RNA capping, NSP14 by itself could remodel host transcriptome such as enriching CREBBP, a key host factor in cellular cyclic AMP response pathway, to promote viral infection. As a result, targeting NSP14 by Questin significantly impairs viral Replication & Translation step and reverses host transcriptome remodeled by NSP14. We next validated Questin as a promising lead with significantly improved toxicity upon acute exposure in zebrafish larvae. Taken together, our study not only demonstrates Questin as a potent drug lead for clinical antiviral application, but also highlights multiple antiviral potentials of NSP14 as therapeutic target.
Project description:In this study we investigated the steady-state growth of Methylotuvimicrobium alcaliphilum 20ZR in media containing calcium (Ca) or lanthanum (La, a REE element). RNA-seq profiling of Methylomicrobium alcaliphilum strain 20ZR in bioreactor on methane. Sample cultures, La-optimum, La-CH4 limited, Ca-optimum and Ca-CH4 limited, were collected and immediately transferred into tubes containing 5 ml of the stop solution (5% water-equilibrated phenol in ethanol). It was found, that cells supplemented with La show a higher growth rate compared to Ca-cultures; however, the efficiency of carbon conversion, estimated as biomass yield, is higher in cells grown with Ca. The study was financially supported by DOE under FOA DE-FOA-0001085 and by NSF-CBET award 1605031
