Project description:HEK T293 cells were treated with Anisomycin or the solvent control DMSO. Cells were fractionated and cytoplasmic and nuclear RNA were isolated. pre-mRNA-Seq was performed on stressed and unstressed nuclear RNA samples. mRNA-Seq was performed on stressed and unstressed cytoplasmic RNA samples. In addition, polyadenylation sites were globally mapped by applying the 3'T-fill method on the same stressed and unstressed cytoplasmic RNA samples.
Project description:Although RNA-binding proteins (RBPs) coordinate many key decisions during cell growth and differentiation, the dynamics of RNA–RBP interactions have not been extensively studied on a global basis. We immunoprecipitated endogenous ribonucleoprotein complexes containing HuR and PABP throughout a T-cell activation time course and identified the associated mRNA populations using microarrays. We used Gaussian mixture modeling as a discriminative model, treating RBP association as a discrete variable (target or not target), and as a generative model, treating RBP-association as a continuous variable (probability of association). We report that HuR interacts with different populations of mRNAs during T-cell activation. These populations encode functionally related proteins that are members of the Wnt pathway and proteins mediating T-cell receptor signaling pathways. Moreover, the mRNA targets of HuR were found to overlap with the targets of other posttranscriptional regulatory factors, indicating combinatorial interdependence of posttranscriptional regulatory networks and modules after activation. Applying HuR mRNA dynamics as a quantitative phenotype in the drug-gene-phenotype Connectivity Map, we identified candidate small molecule effectors of HuR and T-cell activation. We show that one of these candidates, resveratrol, exerts T-cell activation-dependent posttranscriptional effects that are rescued by HuR. Thus, we describe a strategy to systematically link an RBP and condition-specific posttranscriptional effects to small molecule drugs. Keywords: Timecourse, RIP, Immunoprecipitation, HuR, ELAVL1, PABP, T cell activation Timecourse experiment: 0hr, 4hr, and 12hr post-activation of Jurkat cells. Four samples collected: HuR-IP, PABP-IP, Neg-IP and Total RNA. 3 Biological replicates per condition and sample that were independently grown and harvested.
Project description:Although RNA-binding proteins (RBPs) coordinate many key decisions during cell growth and differentiation, the dynamics of RNA–RBP interactions have not been extensively studied on a global basis. We immunoprecipitated endogenous ribonucleoprotein complexes containing HuR and PABP throughout a T-cell activation time course and identified the associated mRNA populations using microarrays. We used Gaussian mixture modeling as a discriminative model, treating RBP association as a discrete variable (target or not target), and as a generative model, treating RBP-association as a continuous variable (probability of association). We report that HuR interacts with different populations of mRNAs during T-cell activation. These populations encode functionally related proteins that are members of the Wnt pathway and proteins mediating T-cell receptor signaling pathways. Moreover, the mRNA targets of HuR were found to overlap with the targets of other posttranscriptional regulatory factors, indicating combinatorial interdependence of posttranscriptional regulatory networks and modules after activation. Applying HuR mRNA dynamics as a quantitative phenotype in the drug-gene-phenotype Connectivity Map, we identified candidate small molecule effectors of HuR and T-cell activation. We show that one of these candidates, resveratrol, exerts T-cell activation-dependent posttranscriptional effects that are rescued by HuR. Thus, we describe a strategy to systematically link an RBP and condition-specific posttranscriptional effects to small molecule drugs. Keywords: Timecourse, RIP, Immunoprecipitation, HuR, ELAVL1, PABP, T cell activation
Project description:Primary outcome(s): Relationship with mRNA expression of B7 family molecules in blood of patients with colorectal cancer and clinicopathological factors
Project description:The expression of the antioxidant genes, katA and katB, in response to oxidative stress in Pseudomonas aeruginosa has recently been shown to be controlled at the translational level. Here, we demonstrate that the expression of katA and katB is also modulated by gidA at the posttranscriptional level.
Project description:Control of mRNA half-life is a powerful strategy to adjust individual mRNA levels to various stress conditions, because the mRNA degradation rate controls not only the steady-state mRNA level but also the transition speed of mRNA levels. Here, we analyzed mRNA half-life changes in response to cold stress in Arabidopsis cells using genome-wide analysis, in which mRNA half-life measurements and transcriptome analysis were combined. Half-lives of average transcripts were determined to be elongated under cold conditions. Taking this general shift into account, we identified more than a thousand transcripts that were classified as relatively stabilized or relatively destabilized. The relatively stabilized class was predominantly observed in functional categories that included various regulators involved in transcriptional, post-transcriptional and post-translational processes. On the other hand, the relatively destabilized class was enriched in categories related to stress and hormonal response proteins, supporting the idea that rapid decay of mRNA is advanta- geous for swift responses to stress. In addition, pentatricopeptide repeat, cyclin-like F-box and Myb transcription factor protein families were significantly over-represented in the relatively destabilized class. The global analysis presented here demonstrates not only the importance of mRNA turn-over control in the cold stress response but also several structural characteristics that might be important in the control of mRNA stability. To demonstrate the importance of mRNA stability control in cold stress response, we investigated global changes in mRNA half-lives in response to cold treatment by micaroarray using Arabidopsis suspension cell cultures (T87 cells). Control cells were collected prior to transcriptional inhibitor (cordycepin) treatment (0 h), and 1 and 3 h after the start of cordycepin treatment. For cold-treated cells, 6 h samples were also used for microarray analyses. The experiments were performed with triplicate sets for each time point.
Project description:Control of mRNA half-life is a powerful strategy to adjust individual mRNA levels to various stress conditions, because the mRNA degradation rate controls not only the steady-state mRNA level but also the transition speed of mRNA levels. Here, we analyzed mRNA half-life changes in response to cold stress in Arabidopsis cells using genome-wide analysis, in which mRNA half-life measurements and transcriptome analysis were combined. Half-lives of average transcripts were determined to be elongated under cold conditions. Taking this general shift into account, we identified more than a thousand transcripts that were classified as relatively stabilized or relatively destabilized. The relatively stabilized class was predominantly observed in functional categories that included various regulators involved in transcriptional, post-transcriptional and post-translational processes. On the other hand, the relatively destabilized class was enriched in categories related to stress and hormonal response proteins, supporting the idea that rapid decay of mRNA is advanta- geous for swift responses to stress. In addition, pentatricopeptide repeat, cyclin-like F-box and Myb transcription factor protein families were significantly over-represented in the relatively destabilized class. The global analysis presented here demonstrates not only the importance of mRNA turn-over control in the cold stress response but also several structural characteristics that might be important in the control of mRNA stability.
Project description:Although being essential to respiratory and reproductive tracts multiciliogenesis, TAp73 is dispensable for multiciliogenesis in the ventricles. TAp73 KO is accompanied by dramatic changes in ciliogenic microRNAs miR34bc and miR449 family members, suggestin TAp73 functions partially thorugh posttranscriptional nodes in brain ciliogenesis.