RNA-binding proteins (RBPs) play important roles in the regulation of gene expression through a variety of post-transcriptional mechanisms. The p53-induced RBP Wig-1 (Zmat3) binds RNA through its zinc finger domains and enhances stability of p53 and N-Myc mRNAs and decreases stability of FAS mRNA. To identify novel Wig-1-bound RNAs, we performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells. We identified 286 Wig-1-bound mRNAs common between ...[more]
Project description:RBP2 is downstream of pRB pathway; We used gene expression profiling experiments to investigate if gene expression changes in cells with RBP2 knockdown significantly overlap with gene expression changes in cells overexpressing pRB, consistent with the data that knockdown of RBP2 phenocopies reintroduction of pRB in SAOS-2 (RB-/-) cells Experiment Overall Design: Knockdown of RBP2 by siRNA to RBP2 (RBP2siRNA) in SAOS-2 (RB-/-) cells in comparison to overexpression of pRB in the SOAS-2 (RB-/-) cells and scrambled version of the RBP2 siRNA (RBP2scsiRNA)
Project description:The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-Activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL11 mRNA, inducing autocrine of IL11 and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for anti-metastatic therapies. To identify mRNA species bound by lncRNA-ATB, we performed an RIP to pull down endogenous mRNAs associated with the lncRNA-ATB and sequenced the retrieved RNA.
Project description:Here we delineate the roles of RNA binding for WDR5 function on chromatin state and murine embryonic stem cell (ESC) maintenance. Examination of global transcriptional changes and mapping genome-wide transcription factors occupancy at distinct time points during the transdifferentiation process
Project description:Recent estimates of the human proteome suggest that there are ~20,000 protein-coding genes, the protein products of which contain >145,000 phosphorylation sites. Unfortunately, in-depth examination of the human phosphoproteome has far outpaced the ability to annotate the kinases that mediate these post-translational modifications. To obtain actionable information about phosphorylation-driven signaling cascades, it is essential to identify the kinases responsible for phosphorylating sites that differ across disease states. To fill in these knowledge gaps, we have developed an unbiased, chemoproteomic approach for identifying high confidence kinase-substrate interactions with phosphosite specificity. Using this assay, we have uncovered the role of cyclin-dependent kinase 4 (CDK4), a clinically validated kinase important for cell cycle progression, in regulating cap-dependent translation via phosphorylation of the tumor suppressor 4E-BP1.
Project description:The p53-family member p73 functions in various cellular signaling pathways and can have tumor suppressor properties. Several isoforms of p73 exist that differ considerably in their function. Whereas the functions of the N-terminal isoforms (TA and ΔNp73) and their opposing pro- and anti-apoptotic roles became evident, the functional differences of the distinct C-terminal spliceforms of TAp73 have remained unclear. Here, we characterized the genomic binding sites for TAp73α and TAp73β and identified a specific p73 consensus binding-motif. Furthermore, an AP1 motif is strongly enriched close to binding sites for TAp73α. These AP1 motif-containing target genes are selectively upregulated by TAp73α, while their mRNA expression is repressed upon TAp73β induction. Recruitment of c-Jun to the respective AP1 sites was impaired upon TAp73β expression in part due to downregulation of c-Jun. We show that several of these AP1-site containing TAp73α-induced genes reduce on apoptosis-induction suggesting an underlying molecular mechanism for the observed functional differences between TAp73α and TAp73β. ChIP-seq and RNA-seq profiles of TAp73alpha, TAp73beta and p53 stably transfected in human osteosarcoma Saos cells
Project description:The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3’ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. CLIP-seq for DGCR8 and Drosha, RIP-seq for DGCR8, sequencing of AGO2-assocated miRNA
Project description:Comparative analyses of Mex67 and Npl3 binding to mRNA at normal growth condition (25°C) and upon shift to heat stress (30 min, 42°C). Comparative analyses of mRNA binding of Mex67, Npl3 and no tag control at normal growth condition (25°C) and upon heat stress (30 min, 42°C) via RNA Co-IP experiments. Respective co-purified mRNAs were subjected to cDNA Microarray chips.
Project description:This SuperSeries is composed of the following subset Series: GSE23669: Changes in polysome loading as a consequence of RHA downregulation [Agilent] GSE23688: RNA immunoprecipitation to identify RHA-binding transcripts in HEK293 cells Refer to individual Series
Project description:Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from four experiments with samples obtained in four independent RNA immunoprecipitations identified 375 transcripts that co-immunoprecipitate with FLAG RHA. Since N-terminal FLAG tagged RHA specifically co-immunoprecipitates PCE-containing mRNAs HEK293 cells were transfected with a CMV-FLAG-RHA construct. Cytoplasmic lysates were immunoprecipitated with anti-FLAG beads. RNA was extracted from the immunoprecipitate and used to probe a human Agilent expression arrays. An immunoprecipitation with cells transfected with empty FLAG plasmid was used as negative control.