Gene expression analysis of enSLiT KO cells against Salmonella infection
ABSTRACT: Eukaryotic genomes are pervasively transcribed, resulting in the production of many unstable nuclear long noncoding RNAs such as nuclear Short-Lived noncoding Transcripts (nSLiTs). However, the biological significance and turnover mechanism of nSLiTs are largely unknown. We used microarrays to detail the global program of gene expression that is regulated by enSLiT upon Salmonella infection Overall design: To assess the role of enSLiT against Salmonella infection, we knockout enSLiT07573 by using CRISPR/Cas9. KO was performed replicate for each transcripts. We comparied Salmonella infected/not infected HeLa cells and KO cells. All data was obtained biological triplicate.
INSTRUMENT(S): Agilent-072363 SurePrint G3 Human GE v3 8x60K Microarray 039494 [Probe Name Version]
Project description:Eukaryotic genomes are pervasively transcribed, resulting in the production of many unstable nuclear long noncoding RNAs such as nuclear Short-Lived noncoding Transcripts (nSLiTs). However, the biological significance and turnover mechanism of nSLiTs are largely unknown. We used microarrays to detail the global program of gene expression that is regulated by LinSLiT08211 upon Salmonella infection Overall design: To assess the role of LinSLiT against Salmonella infection, we knockout LinSLiT08211 by using CRISPR/Cas9. KO was performed replicate for each transcripts. We comparied Salmonella infected/not infected HeLa cells and KO cells. All data was obtained biological triplicate.
Project description:Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon <i>Salmonella</i> infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to <i>Salmonella</i> infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.
Project description:The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3' end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3' end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets.
Project description:Purpose: To understand the role of SUMOylation of c-Fos in the differential regulation of target genes and altered cellular pathways upon STm infection. To address this, we performed a RNA-seq experiment with stable over expressing WT-FOS or SUMO-def-FOS in f10 c-FOS-Knock out MEFs upon STm infection. Methods: Three biological replicates of c-FOS-KO-WT-FOS, c-FOS-KO-WT-FOS with salmonella infection, c-FOS-KO-SUMO-def-FOS and c-FOS-KO-SUMO-def-FOS with salmonella infection, cells were collected and total RNA was extracted according to kit's protocol. The total RNA was used to generate a cDNA library using Quantseq 3' mRNA kit. Results: Transcriptional profiling revealed that genes involved in immune response, proliferation, metastasis etc. are differentially regulated in salmonella infected c-FOS-KO-SUMO-def-FOS MEFs compare to salmonella infected c-FOS-KO-WT-FOS MEFs. Conclusions: Our study revealed the extensive transcriptomics analysis from c-FOS-KO-WT-FOS and c-FOS-KO-SUMO-def-FOS MEFs upon salmonella infection. We found that SUMOylation of c-Fos provides selectivity that causes differential regulation of target genes which are involved in immune response, proliferation etc. pathways of host. Together, our findings illuminate an important regulatory role played by SUMOylated c-Fos upon STm infection. Overall design: RNA-seq
Project description:To identify bacterial transcripts that may be associated with type I IFN production in Salmonella enterica subsp typhimurium (SL1344) infected macrophages we transformed macrophages with an ISRE-GFP reporter construct and sorted separate populations of GFP positive and GFP negative infected macrophages. We then did whole transcriptome profiling, collecting both host and bacterial transcripts, for differential expression analysis Analysis of ISRE positive, negative, and mixed populations at two time points (unexposed and 24hours) in duplicate (biological replicates). A sample consisting of Salmonella prior to infection was also included
Project description:FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3'UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs.IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked "third domain" of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.
Project description:Frog virus 3 (FV3) and other ranaviruses are responsible for die-offs involving wild, farmed, and captive amphibians, fish, and reptiles. To ascertain which elements of the immune system respond to infection, we explored transcriptional responses following infection of fathead minnow cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18 kDa immediate early gene (18K). At 8h post infection we observed marked upregulation of multiple transcripts encoding proteins affecting innate and acquired immunity. Sequences expressed 4-fold or higher in wt-infected cells included transcripts encoding interferon (IFN), IFN regulatory factors (IRFs), IFN stimulated genes (ISGs) such as Mx and MHC class I, and interleukins IL-1?, IL-8, IL-17C and IL-12. Cells infected with the 18K KO mutant (?18K) showed qualitative differences and lower levels of induction. Collectively, these results indicate that ranavirus infection induced expression of multiple cellular genes affecting both innate and acquired immunity.
Project description:Genome-wide studies have identified abundant small, noncoding RNAs, including small nuclear RNAs, small nucleolar RNAs (snoRNAs), cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs), that are transcribed by RNA polymerase II (pol II) and terminated by an Nrd1-dependent pathway. Here, we show that the prolyl isomerase Ess1 is required for Nrd1-dependent termination of noncoding RNAs. Ess1 binds the carboxy-terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of approximately 10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, stable unannotated transcripts, and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase. We also provide evidence for a competition between Nrd1 and Pcf11 for CTD binding that is regulated by Ess1. These data indicate that a prolyl isomerase is required for specifying the "CTD code."
Project description:Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.