Exon-array profiling of Heat-shock stress response in HeLa cell line
ABSTRACT: The heat-shock stress response was studied at the level of exons using Affymetrix Exon-array profiling for both sense and anti-sense transcripts. Sense transcript profiling was done as per the protocol of Affymetrix Exon 1.0 ST array and anti-sense transcript array profiling was done using a modified protocol (Xijin Ge et al., BMC Genomics. 2008 Jan 22;9:27). In short, for profiling antisense transcripts, first cycle cDNA synthesis and IVT step is skipped. This modified protocol starts directly from the second cycle cDNA synthesis step. The labelled target DNA fragments are in reverse orientation of original mRNAs. Thus the hybridization signals will represent transcripts from the same exonic regions but from the opposite DNA strand. Overall design: Study was undertaken with 2 biological replicates each for - a) Heat-shock treated Sense b) Heat-shock treated Anti-sense c) Untreated control Sense d) Untreated control Anti-Sense
INSTRUMENT(S): [HuEx-1_0-st] Affymetrix Human Exon 1.0 ST Array [probe set (exon) version]
Project description:The heat-shock stress response was studied at the level of exons using Affymetrix Exon-array profiling for both sense and anti-sense transcripts. Sense transcript profiling was done as per the protocol of Affymetrix Exon 1.0 ST array and anti-sense transcript array profiling was done using a modified protocol (Xijin Ge et al., BMC Genomics. 2008 Jan 22;9:27). In short, for profiling antisense transcripts, first cycle cDNA synthesis and IVT step is skipped. This modified protocol starts directly from the second cycle cDNA synthesis step. The labelled target DNA fragments are in reverse orientation of original mRNAs. Thus the hybridization signals will represent transcripts from the same exonic regions but from the opposite DNA strand. Study was undertaken with 2 biological replicates each for - a) Heat-shock treated Sense b) Heat-shock treated Anti-sense c) Untreated control Sense d) Untreated control Anti-Sense
Project description:Background: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse transcription in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. Results: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition specific anti-sense transcription levels is higher than that in condition specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse transcription in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse transcription, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. Conclusions: This study provides a comprehensive picture of the role of antisense transcription mediating sense transcription in different conditions across yeast species. We can conclude from our findings that antisense regulation could act like an on-off switch on sense regulation in different conditions. Transcriptomes of two yeast species under mid-log phase, early stationary phase, and after heat shock treatment were generated by Illumina HiSeq 2000 paired-end sequencing
Project description:Transcription profiling of sense and antisense transcripts of 10 tissues each from human, mouse, and rat. This SuperSeries is composed of the following subset Series: GSE41462: Antisense exon profiling across human, mouse, and rat GSE41464: Sense exon profiling across human, mouse, and rat We profiled the sense and antisense transcription level of 10 tissues each from human, mouse, and rat. Only Affymetrix core probesets were used. Two technical replicates per sample. Reference for protocol: Ge, X., Rubinstein, W.S., Jung, Y.C., and Wu, Q. (2008). Genome-wide analysis of antisense transcription with Affymetrix exon array. BMC Genomics 9, 27.
Project description:We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat-shocked worms. We find antisense transcripts are generally increased after heat shock, and a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hypridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified. Overall design: Total of 11 samples: 1 N2 heat shock input RNA input sample with 2 N2 heat shock J2 immunoprecipated samples. The following samples were re-analyzed from GSE61581. 3 tdp1 J2 immunoprecipitated samples and tdp1 total RNA input with 3 N2 J2 immunoprecipitated controls (with N2 input).
Project description:Expression profiling of a total of 7281 transcripts was compared between control and heat shock conditions. Overall design: In the present study, RNA was extracted from three independent cell cultures for each treatment (control and heat shock).
Project description:Exiqon miRNA array contains LNA-modified oligos which minimizes the Tm-range within which probe hybridizes with the transcript in query, thus minimizing the mis-match. Taking advantage of this, we used the miRCURY LNA array to investigate genome wide signatures for miRNA expression in response to heat shock stress. We also did mRNA expression profiling using the same RNA to look for possible miRNA-mRNA interaction in response to heat shock stress. For the differentially expressed miRNAs, target predictions were done using both miRanda and TargetScan. The consensus set was used for subsequent experimental validation. In total, 6 comparative hybridizations of heat-shock treated versus control were profiled using the Exiqon arrays. These consisted of 3 biological replicates, each with its dye-swap pair.
Project description:Polycistronic mRNAs transcribed from operons are resolved via the trans-splicing of a spliced leader (SL) RNA. The SL is also frequently trans-spliced to monocistronic transcripts. Using a modified cap analysis of gene expression (CAGE) protocol we mapped sites of SL trans-splicing genome-wide in the marine chordate Oikopleura dioica and find evidence for proposed functions of SL-trans-splicing. A recent hypothesis postulates that operons facilitate recovery from growth arrested states in metazoans. We examined the expression dynamics of operons across the life-cycle of the animal and during growth arrest recovery. We show that operons do not facilitate recovery from growth arrest in O. dioica. We find that operons are enriched in the germline and that trans-spliced transcripts are predominantly maternal., Interestingly, there is a TOP-like motif in the SL sequence, and trans-splicing in TOP mRNAs, indicating that trans-spliced mRNAs are targets for nutrient-dependent translational control in O. dioica. Total RNA from a number of stages across development were pooled and used in a modified DeepCAGE protocol. A custom designed spliced-leader primer (using the SL exon) was used in the 2nd strand synthesis step.
Project description:Stress response pathways allow cells to rapidly sense and respond to deleterious environmental changes, including those caused by pathophysiological disease states. A previous screen for small molecules capable of activating the human heat shock response identified the triterpenoid celastrol as a potent activator of the heat shock transcription factor HSF1. We show here that celastrol likewise activates the homologous Hsf1 of Saccharomyces cerevisiae. Celastrol induced Hsf1 hyperphosphorylation and concurrently activated a synthetic transcriptional reporter as well as endogenous inducible Hsp70 proteins at the same effective concentration seen in mammalian cells. Moreover, celastrol treatment conferred significant resistance to subsequent lethal heat shock. Transcriptional profiling experiments revealed that in addition to Hsf1, celastrol treatment induced the Yap1-dependent oxidant defense regulon. Oxidative stress-responsive genes were likewise induced in mammalian cells, demonstrating that celastrol simultaneously activates two major cellular stress-mediating pathways. As the induction of cellular stress pathways has implications in the treatment of a variety of human diseases including neurodegenerative diosorders, cardiovascular disease and cancer, celastrol thus represents an attractive therapeutic compound. Keywords: single-dose, single time-point gene induction by natural small molecule celastrol compared to heat shock in wild type (BY4741) Saccahromyces cerevisiae Overall design: Two heat shock experiments and two drug treatment experiments were conducted and analyzed independently. Heat shock arrays are 39 vs. 30 degrees for 30 min, and drug (celastrol) arrays are 10 µM celastrol in DMSO over DMSO alone, same volumes. Expression numbers are duplicated and averaged on each chip, normalized to the median signal of all hybridizations on the chip.
Project description:Transcriptional profiling of various apple (Malus x domestica Borkh) organ systems using probes complementary to both sense and anti-sense transcripts. Eight apple organs/samples. Biological replicates: 2 for each sample, independently grown and harvested.