Profiling of miRNA expression of HeLa cells infected with Salmonella Typhimurium
ABSTRACT: We identified miRNAs differentially regulated upon Salmonella infection by comparative deep-sequencing analysis of cDNA libraries prepared from the small RNA population (10–29 nt) of HeLa cells infected with Salmonella (20 hpi) and mock-treated cells. Considering that at a MOI of 25 Salmonella is internalized in only 10-15% of the HeLa cells, we separated the fraction of cells which had internalized Salmonella (Salmonella+) from the bystander fraction (Salmonella-) by fluorescence-activated cell sorting (FACS), and extended the analysis of miRNA changes to these samples. Interestingly, we observed that Salmonella infection induces a significant decrease in the expression of all the detected members of the miR-15 family miRNA profiles of HeLa cells infected with Salmonella Typhimurium were generated by deep sequencing, using Illumina HiSeq2000.
Project description:We evaluated the transcriptome changes induced by infection with Salmonella (20 hpi, MOI 100). Transcriptmic profiles of HeLa cells infected with Salmonella Typhimurium were generated by deep sequencing, using Illumina HiSeq 2000.
Project description:The α-proteobacterium Sinorhizobium fredii NGR234 has an exceptionally wide host range as it forms nitrogen-fixing nodules with more legumes than any other known microsymbiont. Within its 6.9 Mbp genome it encodes two N-acyl-homoserine-lactone synthase genes (i.e. traI and ngrI) involved in the biosynthesis of two distinct autoinducer I-type molecules. Here we report on the construction of a NGR234-ΔtraI and a NGR234-ΔngrI mutant and the genome wide RNA-seq-based transcriptome analysis in the parent strain and in the two constructed mutants. A high-resolution RNA-seq analysis of early stationary phase cultures in the background of NGR234-ΔtraI suggested that up to 316 genes (4.9% of all predicted genes) were differentially expressed in the mutant vs. the parent strain. Similarly, in the background of NGR234-ΔngrI 466 differentially regulated genes (7.3% of all predicted genes) were identified. A considerable overlap in the gene expression pattern was uncovered between both mutants resulting in a common set of 186 genes which were almost identically regulated. Co-regulated genes that were linked to the ngrI- and the traI-dependent autoinducer regulatory circuits included 53 flagella biosynthesis genes and genes linked to EPS succinoglycan biosynthesis. Among the genes and ORFs that were differentially regulated in NGR234-ΔtraI were those linked to replication of the pNGR234a symbiotic plasmid and cytochrome c-related genes. In the NGR234-ΔngrI mutant biotin and pyrroloquinoline quinone (PQQ) biosynthesis genes were differentially expressed as well as the entire cluster of 21 genes linked to assembly of the NGR234 type three secretion system II (T3SS-II). We also discovered that genes responsible for octopine catabolism in NGR234 are strongly repressed in the presence of high levels of N-acyl-homoserine-lactones (AHLs). Surprisingly, only few truly symbiosis-related genes were identified that appeared to be quorum sensing regulated. Together with nodulation assays, the RNAseq-based findings suggested that QS-dependent gene regulation appears to be of higher relevance during non-symbiotic growth rather than for life within root nodules. In total 12 samples were analyzed (six different treatments with two independent samples), treatment A: NGR234 wild type strain in stationary phase, treatment B: NGR234 traI-mutant strain in stationary phase, treatment C: NGR234 ngrI-mutant strain in stationary phase, treatment D: NGR234 wild type strain in exponential phase, treatment E: NGR234 wild type supplemented with 0.05 µM AHLs in exponential phase, treatment F: NGR234 wild type supplemented with 50 µM AHLs in exponential phase
Project description:A comparative RNA-seq approach between R. sphaeroides 2.4.1 wild type and an RNase J deletion strain 2.4.1∆rnj revealed the accumulation of different mRNA fragments in the mutant. The structural characteristics of these RNA fragments suggest that RNase J is responsible for the decay of degradation intermediates that cannot serve as substrates for the 3´-to-5´ exoribonucleases. Total RNA from exponentially grown cultures (OD660 0,4) of 2.4.1 wild type and 2.4.1∆rnj has been isolated in triplicates using the hot phenol method and prepared for sequencing on a Illumina Genome Analyzer IIx machine.
Project description:In this study transcriptional start sites (TSS) of E. coli were determined for several growth conditions To detect the complement of transcripts expressed from E. coli, we collected two independent biological replicates (B1 and B2 samples) from MG1655 wild type strain grown to exponential (OD 600 ~0.4) or stationary phase (OD 600 ~2.0) in LB medium (samples LB 0.4 and LB 2.0, respectively) as well as grown to exponential phase (OD 600 ~0.4) in M63 minimal glucose medium (sample M63 0.4). For all six samples, total RNA was extracted and subjected to differential RNA-seq (dRNA-seq) library preparation for primary transcriptome analysis as described previously (Sharma et al., 2010). Specifically, prior to cDNA library construction half of each RNA sample was treated with 5’ terminator exonuclease (+TEX samples), which degrades RNAs containing a 5’-mono-phosphate (5’-P) and, thus, enriching enriches for primary transcripts containing 5’-tri-phosphates (5’-PPP). The other half of each sample was left untreated (-TEX samples) and thus contains both primary transcripts (5’-PPP) and processed RNAs (5’-P).
Project description:We report the transcriptome profiles (RNA seq) of three different co-isolates of S.aureus that have been identified and isolated in both laboratory and infective scenarios. The transcriptome profiles were generated by deep sequencing and transcript levels assessed. For this the raw reads underwent quality-trimming (using the FastX suite), polyA-clipping, size filtering, mapping to the reference genome, coverage calculation, gene wise expression quantification followed by differential gene expression analysis (all done by the tool "READemption" (Förstner et al., unpublished) using "segemehl" (Hoffmann et al., 2009) and "DESeq" (Anders et al., 2010). RT-PCR as well as phenotypical assays were further used to validate the data. Differential gene expression between the isolates was observed in 4% (116 of 2774 genes). This had a significant impact on the physiology of each strain. RNA profiles of 3 different subpopulations of S.aureus generated by deep sequencing
Project description:High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) in the Fenton reaction, while depletion of iron limits the availability of iron containing proteins, some of which have important functions in the oxidative stress defense. Vice versa increased ROS levels lead to damage of proteins with iron sulfur centers. Thus organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge on the molecular mechanisms underlying the coregulation of these responses is still limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the alpha proteobacterium Rhodobacter sphaeroides to iron limitation in presence or absence of oxygen. While some genes respond to iron limitation exclusively or much stronger in presence of oxygen, other genes show much stronger response in anaerobic conditions. Remarkably few genes show even opposite response to iron depletion in presence or absence of iron. RNA samples collected from anaerobically or microaerobically grown cultures in presence or absence of iron were analyzed by RNA_sequencing
Project description:The small RNAs associated with protein Hfq constitute one of the largest classes of post-transcriptional regulators known to date. Most previously investigated members of this class are encoded by conserved free-standing genes. Here, deep sequencing of Hfq-bound transcripts from multiple stages of growth of Salmonella Typhimurium revealed a plethora of new small RNA species from within mRNA loci, including DapZ which overlaps with the 3’ region of the biosynthetic gene, dapB. Synthesis of the DapZ small RNA is independent of DapB protein synthesis, and controlled by HilD, the master regulator of Salmonella invasion genes. DapZ carries a short G/U-rich domain similar to that of the globally acting GcvB small RNA, and uses GcvB-like seed pairing to repress translation of the major ABC transporters, DppA and OppA. This exemplifies double functional output from an mRNA locus by the production of both a protein and an Hfq-dependent trans-acting RNA. Our atlas of Hfq targets suggests that the 3’ regions of mRNA genes constitute a rich reservoir to feed the Hfq network with new regulatory small RNAs. Hfq-associated RNAs were systemically analyzed in Salmonella at 7 different growth stages in standard labortory condition (LB)
Project description:To have a global picture of the targets of the miR-15 family, we assessed transcriptome changes, by deep-sequencing, of HeLa cells transfected with 3 members of the miR-15 family (miR-15a, miR-16 or miR-503) or a control miRNA (cel-miR-231). We observed a very extensive overlap between the genes down-regulated by these 3 miRNAs, as expected for miRNAs belonging to the same family. transcriptmic profiles of HeLa cells treated miR-15a, miR-16, miR-503 and control-miR were generated by deep sequencing, using Illumina HiSeq2000.
Project description:Three biological replicates of H. volcanii grown under optimal conditions to mid-exponential growth phase were used to determine the primary transcriptome and map 5’-ends of transcripts. In total, 4,749 potential transcriptional start sites (TSS) were detected. A position weight matrix was derived for promoter prediction, showing that 64% of the TSS were preceded by stringent or relaxed basal promoters. 1,851 TSS belonged to protein-coding genes, showing that less than half (46%) of the 4040 protein-coding genes are expressed under optimal growth conditions. 72% of all protein-coding transcripts were leaderless, underscoring that this is the default pathway for translation initiation in haloarchaea. The 5’-UTRs of transcripts with leaders had a widely varying length distribution without any optimum. 2,898 of the TSS belong to potential non-coding RNAs, representing an unexpectedly high fraction (61%) among all transcripts. 2792 of the non-coding TSS had not been described before and were thus novel (59% of all TSS). A large fraction of the potential novel non-coding transcripts are cis-antisense RNAs (1,244 aTSS). There was a strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs, suggesting that negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts correspond to internal transcripts overlapping with mRNAs (1,153 iTSS) and intergenic small RNA (sRNA) candidates (395 TSS). Three biological replicates were performed with slight differences in library preparation. In each case, part of the sample was treated with terminator 5'-phosphate-dependent exonuclease (+TEX), while part of the sample remained untreated (-TEX). Therefore, in total, six samples were analysed by high-throughput sequencing.
Project description:Background: Prokaryotes have relatively small genomes, densely-packed and apparently dominated by protein-encoding sequences. However, data now generated by high throughput RNA sequencing (RNA-seq) reveal surprisingly more-complex transcriptomes with many previously unrecognized and unanticipated non-coding small and antisense transcripts. To date, such studies have investigated primarily Bacteria. Here, we report the transcripts present in Thermococcus kodakarensis, a model hyperthermophilic Archaeon, synthesized under different growth and metabolic conditions. Results: cDNA libraries, generated from RNA preparations isolated from cells growing in media with sulfur or pyruvate, with sulfur to stationary phase, and growing with pyruvate but with sulfur added 20 min before RNA isolation, have been deep-sequenced. The results identify >2,700 sites of transcription initiation, establish a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements in T. kodakarensis. Primary transcription start sites (TSS) are identified upstream of 1,254 annotated genes, including ~78 % of those predicted by promoter locations, and an additional 644 primary TSS and their promoters have been identified within genes. Most of the mRNAs have a 5'-untranslated region (5'-UTR) between 10 and 50 nt long (median length = 16 nt), ~20 % have 5'-UTRs from 50 to 300 nt long, ~14 % are leaderless with 5'-UTRs ≤8 nt, and ~50% contain a consensus ribosome binding sequence. The results also identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA. The data confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 snoRNAs predicted to be encoded in the T. kodakarensis genome. Two transcripts, putatively identified as riboswitches, were present in RNA preparations isolated from growing but not from stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the data obtained also provide a semi-quantitative overview of global operon expression. They document substantial differences in gene expression under different physiological conditions and are consistent with previous observations of substrate-dependent specific gene expression. Many previously unrecognized and unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile. Conclusion: We have identified >2,700 TSS that include almost all of the primary sites of transcription initiation upstream of annotated genes, and also many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the results reveal the presence of many non-coding RNAs and predict extensive RNA-based regulation in T. kodakarensis. cDNA libraries were generated and sequenced from RNA isolated from T. kodakarensis cells growing exponentially (Sexp) and to stationary phase (Sstat) in ASW-YT medium with sulfur, growing exponentially in ASW-YT with pyruvate (Pexp), and from cells growing exponentially in pyruvate but 20 min after sulfur addition (PS). The cDNAs were generated after first incubating the RNA preparations with terminator exonuclease (TEX). TEX does not degrade primary transcripts with a 5'-triphosphate (Sharma et al., 2010) but does digest RNAs generated by transcript processing that have a 5'-monophosphate. As a control and to fully document all transcripts, a cDNA library (C) was also generated and sequenced from an aliquot of an RNA preparation isolated from the cells growing exponentially with sulfur that was not exposed to TEX digestion.