Project description:NIH-3T3 cells were pretreated for 15 min with either DMSO (mock) or cycloheximide followed by addition of either mock, 100 U/ml IFNalpha or 100 U/ml IFNgamma for 1h. During the last 30 min, 500 µM 4-thiouridine was added to cell culture medium. Total cellular RNA was isolated using Trizol reagent and nascent RNA was purified as described (Dölken et al. RNA 2008) . Three replicates of nascent RNA were analyzed by Affymetrix Mouse Gene ST 1.0 arrays Primary (translation independent) from secondary (translation dependent) IFN-mediated differential gene expression were studied in NIH-3T3 fibroblasts by studying studying differential gene expression in presence and absence of Cycloheximide (CHX). In addition, the effect of 75 min CHX during the last 30 min of treatment was studied in nascent RNA.
Project description:Numerous microRNAs and their target mRNAs are co-expressed across diverse cell types. However, it is unknown whether they are regulated in a cellular context-independent or -dependent manner. Here, we explored transcriptome-wide targeting and gene regulation by miR-155, whose activation-induced expression plays important roles in innate and adaptive immunity. Through mapping of miR-155 targets using differential Argonaute iCLIP, mRNA quantification with RNA-Seq, and 3’UTR usage analysis using polyadenylation (polyA)-Seq in activated miR-155-sufficient and deficient macrophages, dendritic cells, T and B lymphocytes, we identified numerous targets differentially bound by miR-155. While alternative cleavage and polyadenylation (ApA) contributed to differential miR-155 binding to some transcripts, in a majority of cases identical 3’UTR isoforms were differentially regulated across cell types, suggesting ApA-independent and cellular context-dependent miR-155-mediated gene regulation reminiscent of sequence-specific transcription factors. Our study provides comprehensive maps of miR-155 regulatory RNA networks and offers a valuable resource for dissecting context-dependent and -independent miRNA-mediated gene regulation in key cell types of the adaptive and innate immune systems.
Project description:Numerous microRNAs and their target mRNAs are co-expressed across diverse cell types. However, it is unknown whether they are regulated in a cellular context-independent or -dependent manner. Here, we explored transcriptome-wide targeting and gene regulation by miR-155, whose activation-induced expression plays important roles in innate and adaptive immunity. Through mapping of miR-155 targets using differential Argonaute iCLIP, mRNA quantification with RNA-Seq, and 3’UTR usage analysis using polyadenylation (polyA)-Seq in activated miR-155-sufficient and deficient macrophages, dendritic cells, T and B lymphocytes, we identified numerous targets differentially bound by miR-155. While alternative cleavage and polyadenylation (ApA) contributed to differential miR-155 binding to some transcripts, in a majority of cases identical 3’UTR isoforms were differentially regulated across cell types, suggesting ApA-independent and cellular context-dependent miR-155-mediated gene regulation reminiscent of sequence-specific transcription factors. Our study provides comprehensive maps of miR-155 regulatory RNA networks and offers a valuable resource for dissecting context-dependent and -independent miRNA-mediated gene regulation in key cell types of the adaptive and innate immune systems.
Project description:Numerous microRNAs and their target mRNAs are co-expressed across diverse cell types. However, it is unknown whether they are regulated in a cellular context-independent or -dependent manner. Here, we explored transcriptome-wide targeting and gene regulation by miR-155, whose activation-induced expression plays important roles in innate and adaptive immunity. Through mapping of miR-155 targets using differential Argonaute iCLIP, mRNA quantification with RNA-Seq, and 3’UTR usage analysis using polyadenylation (polyA)-Seq in activated miR-155-sufficient and deficient macrophages, dendritic cells, T and B lymphocytes, we identified numerous targets differentially bound by miR-155. While alternative cleavage and polyadenylation (ApA) contributed to differential miR-155 binding to some transcripts, in a majority of cases identical 3’UTR isoforms were differentially regulated across cell types, suggesting ApA-independent and cellular context-dependent miR-155-mediated gene regulation reminiscent of sequence-specific transcription factors. Our study provides comprehensive maps of miR-155 regulatory RNA networks and offers a valuable resource for dissecting context-dependent and -independent miRNA-mediated gene regulation in key cell types of the adaptive and innate immune systems.