Project description:Background: Microorganisms are the major cause of food spoilage during storage, processing and distribution. Pseudomonas fluorescens is a typical spoilage bacterium that contributes to a large extent to the spoilage process of proteinaceous food. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Our previous work revealed that RpoS contributed to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. Results: RNA-seq transcriptomics analysis combined with quantitative proteomics analysis basing on multiplexed isobaric tandem mass tag (TMT) labeling was performed for the P. fluorescens wild-type strain UK4 and its derivative carrying a rpoS mutation. A total of 375 differentially expressed genes (DEGs) and 212 differentially expressed proteins (DEPs) were identified in these two backgrounds. The DGEs were further verified by qRT-PCR tests, and the genes directly regulated by RpoS were confirmed by 5’-RACE-PCR sequencing. The combining transcriptome and proteome analysis revealed a role of this regulator in several cellular processes, including polysaccharide metabolism, intracellular secretion and extracellular structures, cell well biogenesis, stress responses, ammonia and biogenic amine production, which may contribute to biofilm formation, stress resistance and spoilage activities of P. fluorescens. Moreover, in this work we indeed observed that RpoS contributed to the production of the macrocolony biofilm’s matrix.
Project description:As a newly identified mRNA modification, the regulation of ac4C remains largely unexplored. RNA-binding proteins (RBPs) that specifically binds to ac4C modification and mediate downstream cellular activities (readers) have not been reported yet. We synthesized acetylated and non-acetylated RNA probes by in vitro transcription. The sequences of the probes were segments of FUS and 18s rRNA, which contain ac4C sites as reported. A biotin-RNA pulldown assay and mass spectrometry were performed with HEK 293T cell lysates.
Project description:Background: The lack of obvious symptoms of early gastric cancer (GC) as well as the absence of sensitive and specific biomarkers results in poor clinical outcomes. Tubulin is currently emerging as important regulators of the microtubule cytoskeleton and thus have a strong potential to be implicated in a number of disorders, however, its mechanism of action in gastric cancer is still unclear. Tubulin alpha-1C(TUBA1C) is a subtype of α-tubulin, high TUBA1C expression has been shown to be closely related to a poor prognosis in in various cancers,this study, for the first time, revealed the mechanism of TUBA1C promotes malignant progression of gastric cancer in vitro and in vivo. Methods: The expression of lncRNA EGFR-AS1 was detected in human GC cell lines by qRT–PCR. Mass spectrometry experiments following RNA pulldown assays found that EGFR-AS1 directly binds to TUBA1C, the CCK8, EdU, transwell, wound-healing, cell cycle assays and animal experiments were conducted to investigate the function of TUBA1C in GC. Combined with bioinformatics analyses, reveal interaction between Ki-67, E2F1, PCNA and TUBA1C by western blot. Rescue experiments furtherly demonstrated the relationship of EGFR-AS1and TUBA1C. Results: TUBA1C was proved to be a direct target of EGFR-AS1, TUBA1C promotes gastric cancer proliferation, migration and invasion by accelerating the progression of the cell cycle from the G1 phase to the S phase and activating the expression of oncogenes: Ki-67,E2F1 and PCNA. Conclusions: TUBA1C is a new potential target of LncRNA EGFR-AS1 promotes gastric cancer progression and could be a novel biomarker and therapeutic target for GC.
Project description:In a previous study, we found that H2S alleviates salinity stress in cucumber by maintaining the Na+/K+ balance and by regulating H2S metabolism and the oxidative stress response. However, little is known about the molecular mechanisms behind H2S-regulated salt-stress tolerance in cucumber. Here, an integrated transcriptomic and proteomic analysis based on RNA-seq and 2-DE was used to investigate the global mechanism underlying H2S-regulated salt-stress tolerance. In total, 11 761 differentially expressed genes (DEGs) and 61 differentially expressed proteins (DEPs) were identified. Analysis of the pathways associated with the DEGs showed that salt stress enriched expression of genes in primary and energy metabolism, such as photosynthesis, carbon metabolism and biosynthesis of amino acids. Application of H2S significantly decreased these DEGs but enriched DEGs related to plant-pathogen interaction, sulfur-containing metabolism, cell defense and signal transduction pathways. Notably, changes related to sulfur-containing metabolism and cell defense were also observed through proteome analysis, such as Cysteine synthase 1, Glutathione S-transferase U25-like, Protein disulfide-isomerase and Peroxidase 2. We present the first global analysis of the mechanism underlying H2S regulation of salt-stress tolerance in cucumber through tracking changes in the expression of specific proteins and genes.
Project description:Adults heterozygous for the hi2217 retroviral insertion within the HAI locus (Mathias et al 2007 JCS) were in-crossed for RNA from hi2217 mutants and corresponding WT siblings. Adults heterozygous for the hi1520 retroviral insertion within the Clint1 locus (Dodd et al, 2009) were in-crossed for RNA from hi1520 mutants and corresponding WT siblings. Both hi2217 and hi1520 are mutants with epidermal defects and showing symptoms of chronic inflammation. In this study we aimed to compare the RNA expression changes between Wild-type siblings (a non-inflammatory status) and homozygous mutants (a chronic inflammatory status) for each mutant background (hi2217 and hi1520). Pools of 30-50 zebrafish embryos of WT siblings and hi2217 or hi1520 mutants were collected for RNA isolation. RNA from the hi2217 mutant embryos or hi1520 mutant embryos (test samples)was labelled with Cy5 and hybridized against Cy3-labelled RNA from the corresponding WT sibling embryos (reference samples) . These experiments were performed in biological triplicate.
Project description:Thiol-based redox regulation is a crucial post-translational mechanism to acclimate plants to changing light availability. Here, we conduct a biotin-switch-based redox proteomics study to systematically investigate dynamics of the thiol-redox network in response to temporal changes in light availability and across genotypes lacking parts of the NTRC/thioredoxin (Trx) systems in the chloroplast. Temporal dynamics revealed light leading to marked decreases in the oxidation states of 75 chloroplast proteins mainly involved in photosynthesis during the first 10 min, followed by their partial re-oxidation after 2-6 hours into the photoperiod. This involved f, m and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys-Prx and Trx y2 showed an opposing pattern, being more oxidized in the light, compared to the dark. In Arabidopsis trxf1f2, trxm1m2 or ntrc mutants, most protein candidates showed increased oxidation states, compared to the wild type, suggesting their light-dependent dynamics to be related to the NTRC/Trx network. In this context, deficiencies in f- and m-type Trxs were found to have different impacts on the thiol-redox proteome depending on the light environment, being higher in constant and fluctuating light, respectively, while NTRC deficiency having a strong influence in all light conditions. Results indicate the plant redox proteome to be subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. This involves f-type Trxs and NTRC to play a role in constant light conditions, while both m-type Trxs and NTRC being important to balance changes in protein redox-pattern during dynamic alterations in fluctuating light intensities.
Project description:The nuclear export of HBV RNAs allows the virus to synthesize its proteins through the translation machinery and replicate its genome through reverse transcription in the cytoplasm. However, the molecular mechanisms underlying this important process remain largely obscure. To illustrate this process, we took advantage of an unbiased HBV RNA-host protein interaction screen using a quantitative proteomics approach and identified embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) as a viral RNA binding partner. RNA scope and subcellular mRNA assays indicated that genetic and pharmaceutic inhibition of ELAVL1 inhibits HBV RNA nuclear export and suppresses viral replication in cell cultures. The observations of an HBV replication mouse model with ELAVL1 displayed similar results. RNA pulldown and RNA electrophoretic mobility shift assays revealed direct interaction between ELAVL1 and HBV pgRNA and confirmed AU-rich elements as the binding sites through site-directed mutagenesis of pgRNA. RNA-immunoprecipitation revealed that HBV RNAs associate with ELAVL1, which in turn binds to acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) and ANP32B. These interactions subsequently recruit CRM1. A nuclear RNase-targeted siRNA screen uncovered RNA exosome-mediated degradation of retardant HBV RNA after ELAVL1 or CRM1 knockdown. Further investigation revealed that ELAVL1 protects pgRNA from degradation. Notably, HBc deletion had no effect on pgRNA-CRM1 interaction and HBV RNA nuclear export. In summary, our work indicates that ELAVL1 functions both in HBV RNA stability and nucleocytoplasmic transport via the CRM1 nuclear export pathway.
Project description:Light initiates the seedling deetiolation transition by promoting major changes in gene expression mainly regulated by phytochrome (phy) photoreceptors. During the initial dark-to-light transition, phy photoactivation induces rapid changes in gene expression that eventually lead to the photomorphogenic development. Recent reports indicate that this process is achieved by phy-induced degradation of Phy-Interacting bHLH transcription Factors (PIFs) PIF1, PIF3 PIF4 and PIF5, which are partly redundant constitutive repressors of photomorphogenesis that accumulate in darkness. In order to test whether light/phy-regulated gene expression occurs through these PIFs, we have performed whole-genome expression analysis in the pif1pif3pif4pif5 quadruple mutant (pifq). Wild-type and pifq mutant seeds were plated on GM medium without sucrose at room temperature. During this procedure the seeds were routinely exposed to white light (WL) for a total of 1.5 hours after imbibition. Seeds were then stratified for 5 days at 4ºC in darkness, induced to germinate with a 5-min red pulse (Rp) (46 μmol/m2/s) and then incubated in the dark for 3h at 21°C before exposure to a terminal 5-min far red pulse (FRp) (58 μmol/m2/s) to suppress pseudo-dark effects. Seeds were then placed in either dark (D) or constant red light (Rc) (6.7 μmol/ m2/s) at 21°C for 45h (2d-old seedlings). Alternatively, 2d-old dark-grown seedlings were treated with 1h of red light (R1) (7.5 μmol/m2/s). Seed samples were harvested after stratification (5d stratified seeds).
Project description:Extremophilic archaea employ diverse chemical RNA modifications, providing a rich source of new enzymes for biotechnologically valuable RNA manipulations. Our understanding of the modified nucleoside profiles in Archaea, as well as the functions and dynamic regulation of specific RNA modifications is far from complete. Here, we established an extensive profile of nucleoside modifications in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS analysis and rigorous non-coding RNA depletion, identifying - with high confidence - at least four previously unannotated modifications in archaeal mRNAs. Nucleoside quantification analysis conducted on total, large, small, and mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus kodakarensis revealed a series of modifications whose abundance is dynamically responsive to growth temperatures, implying that specific RNA modifications are fitness relevant under specific growth conditions. To predict the RNA-modifying enzymes most likely to generate the new and dynamic RNA modifications, we leveraged a bioinformatics analysis of open-access databases to annotate likely functional domains of archaeal proteins. Putative enzyme activities were confirmed in vitro and in vivo by assessing the presence of the target RNA modification in genetic deletion strains of T. kodakarensis. Our approach led to the discovery of a methyltransferase-encoded gene responsible for m7G modification in the P-loop of 23S rRNA peptidyl transferase center. This novel finding validates an effective platform for discovering RNA-modifying enzymes through LC-MS/MS analysis that will accelerate efforts of the community towards uncovering the complex and dynamic roles of RNA modifications.
Project description:In this study, we make used of mRNA-seq and its ability to reliably quantify isoforms, integrating this data with ribosome profiling and LC-MS/MS, to assign ribosome footprints and peptides at the isoform level. We leverage the principle that most cell types, and even tissues, predominantly express a single principal isoform to set isoform-level mRNA-seq quantifications as priors to guide and improve allocation of footprints or peptides to isoforms. Through tightly integrated mRNAseq, ribosome footprinting and/or LC-MS/MS proteomics we demonstrate that a principal isoform can be identified in over 80% of gene products in homogenous HEK293 cell culture and over 70% of proteins detected in complex human brain tissue. Defining isoforms in experiments with matched RNA-seq and translatomic/proteomic data increases the functional relevance of such datasets and will further broaden our understanding of multi-level control of gene expression. In this PRIDE submission you will find the raw files for the HEK293 cell proteomics. Files for the human brain proteomics can be found at PXD005445. We have also uploaded a zip file that contains the input files for our HEK293 cell analysis, and the isoform level output files – there is a separate folder within the zip files for these. The data used to create the manuscript figures is in the Rdata file. Code for assigning peptides and footprints to isoforms can be found on Github here: https://github.com/rkitchen/EMpire