Time-course and concentration-effect experiments with multiple time points and drug concentrations provide far more valuable information than experiments with just two design-points (treated vs. control), as commonly performed in most microarray studies. Analysis of the data from such complex experiments, however, remains a challenge. Here we present a semi-automated method for fitting time profiles and concentration-effect patterns, simultaneously, to gene expression data. The submodels for time-course included exponential increase and decrease models with parameters such as initial expression level, maximum effect, and rate-constant (or half-time). The submodel for concentration-effect was a 4-parameter Hill model.
Simultaneous modeling of concentration-effect and time-course patterns in gene expression data from microarrays.
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View SamplesPurpose: The goals of this study were to determine whether the spliceosome interacts with non-intronic mRNAs Methods: RNAseq was performed on RNA that immunoprecipitated with the yeast SMD1 protein. Tandem-affinity-purified RNAs were extracted and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). We also performed RNAseq experiments on rRNA depleted total RNA extracted from an exosome mutant (rrp6?), a temperature-sensitive splicing mutant (prp40-1) and a parental strain (BY4741). The rRNA was depleted using the Invitrogen RiboMinus kit, according to manufactureres procedures. The depleted RNA was subsequently treated with Turbo DNAse I (Ambion) and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). Results: The SM RNAseq data identified a number of non-intronic mRNAs that appeard to be bound by the spliceosome. Among these was the BDF2 mRNA, which enocdes for a bromo-domain protein. BDF2 was highly enriched in both SM-IP datasets and was therefore analyzed in more detail. To determine if other non-intronic mRNAs could be regulated by the spliceosome, we analysed the transcriptome in the rrp6?, the prp40-1 and a parental strain. Bioinformatic analysis of these data sets revealed that roughly 1% of the non-intronic mRNAs in yeast could be targeted by the spliceosome. TopHat revealed cannonical splice junctions in roughly 30 non-intronic mRNAs, indicating that these messages are spliced. Conclusions: We demonstrate, for the first time, that the spliceosome can regulate expression of non-intronic mRNAs via one and/or two RNA cleavage events. We refer to this process as Spliceosome Mediated Decay (SMD). Overall design: We report RNAseq data for two SM immunoprecipitation experiments and RNAseq datasets for the parental strain (BY4741), the prp40-1 mutant, and the rrp6? strain.
Spliceosome-mediated decay (SMD) regulates expression of nonintronic genes in budding yeast.
Subject
View SamplesRibosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3’ major domain of the 20S pre-ribosomal RNA. Overall design: Three datasets of yeast ribosomal samples subjected to different chemical modifications; 1M7 dataset contains 8 different modified samples and 2 control samples; NAI dataset contains 3 different modified samples and 2 control samples; DMS dataset contains 1 modified sample and 1 control sample. Each sample consists of at least two replicates.
Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution.
Disease, Cell line, Treatment, Subject
View SamplesSkeletal muscle adapts to resistance exercise (RE) performance acutely and chronically. An important regulatory step of muscle adaptation to RE is gene expression. Microarray analysis can be used as an exploratory method to investigate how genes and gene clusters are modulated acutely and chronically by RE. The purpose of the present study was to investigate the effect of training status in the basal (rested) and pre- to 24h post-RE on the global transcriptome in vastus lateralis muscle biopsies of young men. Muscle biopsies of nine young men who undertook RE training for 10-wks were collected pre and 24h post-RE at the first (W1) and last (W10) weeks of training and analysed using microarray. An unaccustomed RE bout (at W1) up-regulated muscle gene transcripts related to stress (e.g., heat shock proteins), damage and inflammation, structural remodelling, protein turnover and increased translational capacity. Trained muscles (at W10) became more efficient metabolically, as training favoured a more oxidative metabolism, refined response to stress, showed by genes suppression related to RE-induced stress and inflammation, and up-regulated genes indicating greater muscle contractile efficiency and contribution to promote muscle growth and development. These data highlight that chronic repetition of RE increases muscle efficiency and adapt muscles to respond more specifically and accurately to RE-induced stress.
Resistance training in young men induces muscle transcriptome-wide changes associated with muscle structure and metabolism refining the response to exercise-induced stress.
Sex, Specimen part
View SamplesGametes rely heavily on post-transcriptional control mechanisms to regulate their differentiation. In eggs, the storage and selective temporal activation of maternal mRNAs is essential for normal development. In the male, transcription ceases during spermiogenesis necessitating the post-transcriptional regulation of many paternal mRNAs required for spermatid differentiation and spermatozoan function. Messenger RNAs that are being actively translated form polysomes. whereas translationally inactive mRNAs are often sequestered in ribonucleoproteins (RNPs). Here we combine polysome display and microarray analyses of RNP and polysome fractions of testes from prepuberal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds.. Consistent with published reports, many post-meiotic mRNAs known to be translationally delayed shift from the RNPs into the polysomes, confirming the validity of this approach. In addition, based upon the criterion of movement from RNPs to polysomes, we detect another 742 mouse testicular genes showing dramatic shifts between RNPs and polysomes. One sub-group of 35 genes including the known translationally delayed Pgk2, are initially transcribed and translationally repressed in meiotic spermatocytes, and translated post-meiotically. This high-through-put approach defines the changing translation patterns of a large number of genes as male germ cells differentiate and identifies a new group of post-transcriptionally regulated meiotic transcripts for future study.
Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated.
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View SamplesSome aquaporins do not show a pronounced function as water diffusion facilitators but act as small molecule transport facilitators for substances such as urea, glycerol, boron or gases such as CO2 . Transcriptome analysis provided distinguishable, specific profiles for water stress or for conditions of increased or decreased CO2 concentrations
T-DNA insertion in aquaporin gene AtPIP1;2 generates transcription profiles reminiscent of a low CO2 response.
Specimen part
View SamplesTranscriptome analysis may provide means to investigate the underlying genetic causes of shared and divergent phenotypes in different populations and help to identify potential targets of adaptive evolution. Applying RNA sequencing to whole male Drosophila melanogaster from the ancestral tropical African environment and a very recently colonized cold-temperate European environment at both standard laboratory conditions and following a cold shock, we seek to uncover the transcriptional basis of cold adaptation. In both the ancestral and the derived populations, the predominant characteristic of the cold shock response is the swift and massive upregulation of heat shock proteins and other chaperones. Although we find ~30% of the genome to be differentially expressed following a cold shock, only relatively few genes (n=26) are up- or down-regulated in a population-specific way. Intriguingly, 24 of these 26 genes show a greater degree of differential expression in the African population. Likewise, there is an excess of genes with particularly strong cold-induced changes in expression in Africa on a genome-wide scale. The analysis of the transcriptional cold shock response most prominently reveals an upregulation of components of a general stress response, which is conserved over many taxa and triggered by a plethora of stressors. Despite the overall response being fairly similar in both populations, there is a definite excess of genes with a strong cold-induced fold-change in Africa. This is consistent with a detrimental deregulation or an overshooting stress response. Thus, the canalization of European gene expression might be responsible for the increased cold tolerance of European flies. Overall design: mRNA profiles of whole Drosophila melanogaster adult males from a Africa (4 lines) and Europe (4 lines) during a 7h cold shock experiment. Samples include room temperature controls, 3.5h into the cold shock, 15 minutes after recovery and 90 minutes after recovery. 2 biological replicates each.
Canalization of gene expression is a major signature of regulatory cold adaptation in temperate Drosophila melanogaster.
Sex, Subject
View SamplesRationale: MicroRNAs play key roles in hypertrophic stress responses. miR-378(-3p) is a highly abundant, cardiomyocyte-enriched microRNA whose downregulation in pressure-overload has been suggested as detrimental to the heart. Previous studies have utilized systemic anti-miR or microRNA-encoding virus administration, and thus questions regarding the cardiomyocyte-autonomous roles of miR-378 remain. Objective: To examine whether persistent overexpression of miR-378 in cardiomyocytes alters the phenotype of the unstressed heart, whether its overexpression is beneficial or deleterious in the setting of pressure-overload, and to comprehensively identify its cardiomyocyte-specific effects on mRNA regulation. Methods and Results: Cardiac function was compared in young (10-12 week-old) mice overexpressing miR-378 in the heart under the control of the Myh6 promoter (alphaMHC-miR-378 mice), in older (40 week-old) mice and their age-matched wild-type controls. Older alphaMHC-miR-378 mice exhibited decreased fractional shortening and modest chamber dilation with an increase in cardiomyocyte length. When subjected to pressure-overload, cardiomyocyte length was increased in young alphaMHC-miR-378 mice, but fractional shortening declined precipitously over two weeks. Transcriptome profiling of wild-type and alphaMHC-miR-378 hearts in unstressed and pressure-overload conditions revealed dysregulation of several upstream metabolic and mitochondrial genes in alphaMHC-miR-378 hearts, compromising the reprogramming that occurs during early adaptation to pressure overload. Ago2 immunoprecipitation with mRNA sequencing revealed novel miR-378 cardiac mRNA targets including Akt1 and Epac2 and demonstrated the contextual nature of previously described miR-378 targeting events. Conclusions: Long-term upregulation of miR-378 levels in the heart is not innocuous and exacerbates contractile dysfunction in pressure-overload hypertrophy through numerous signaling mechanisms. Overall design: Cardiac polyadenylated RNA (mRNA) or RISC-seq (total RNA-seq of Ago2 immunoprecipitate) profiles were generated from nontransgenic and transgenic mouse hearts of FVB/N background, on Illumina HiSeq 2000 instruments. Male mice 8-12 weeks of age were used in these studies, and subjected to sham surgery or 2 weeks of pressure-overload via transverse aortic constriction (TAC). 3 nontransgenic sham, 3 transgenic sham, 7 nontransgenic TAC, 7 transgenic TAC, each with mRNA-seq and RISC-seq data.
Cardiac Disease Status Dictates Functional mRNA Targeting Profiles of Individual MicroRNAs.
No sample metadata fields
View SamplesmiR-133a-3p is a highly abundant cardiomyocyte-enriched microRNA whose expression is persistently decreased in response to pressure overload (or transverse aortic constriction, TAC) in mice. Overexpression of miR-133a in cardiomyocytes of mouse hearts in vivo (under the control of the Myh6 promoter) decreases pressure overload-induced apoptosis and fibrosis. In previous studies using microarray platforms, we detected numerous mRNAs whose transcript levels were altered by either or both of miR-133a overexpression and pressure overload. The data set presented here builds upon our previous study in these mice by examining mRNA-RISC associations (using Ago2-immunoprecipitated RNA) and global mRNA abundances via RNA-sequencing procedures, and tests the hypothesis that mRNAs targeted by overexpressed miR-133a are dissimilar between sham and TAC contexts. Overall design: Cardiac polyadenylated RNA (mRNA) profiles were generated from nontransgenic and transgenic mouse hearts of FVB/N background, on Illumina HiSeq 2000 instruments. Male mice 8-12 weeks of age were used in these studies, and subjected to sham surgery or 1 week of pressure-overload via transverse aortic constriction (TAC). 3 nontransgenic sham, 7 transgenic sham, 5 nontransgenic TAC, 4 transgenic TAC, each with mRNA-seq and RISC-seq (mRNA-seq of Ago2 immunoprecipitate) data.
Cardiac Disease Status Dictates Functional mRNA Targeting Profiles of Individual MicroRNAs.
No sample metadata fields
View SamplesThe in-vitro analysis of the hypomethylation of the imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS).
Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation.
Specimen part, Disease
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