ICLIP of endogenous PURA in HeLa cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE193901 | GEO | 2022/12/12
REPOSITORIES: GEO
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Project description:The protein PURA binds to DNA and RNA and has been suggested to be implicated in different cellular functions. Here, we show that PURA predominantly resides in the cytoplasm, where it binds to a large set of transcripts.
2022-12-12 | GSE193902 | GEO
Project description:The protein PURA binds to DNA and RNA and has been suggested to be implicated in different cellular functions. Here, we show that PURA predominantly resides in the cytoplasm, where it binds to a large set of transcripts.
2022-12-12 | GSE193904 | GEO
Project description:The protein PURA binds to DNA and RNA and has been suggested to be implicated in different cellular functions. Here, we show that PURA predominantly resides in the cytoplasm, where it binds to a large set of transcripts.
2022-12-12 | GSE193903 | GEO
Project description:Here, we show that PURA predominantly resides in the cytoplasm, where it binds to a large set of transcripts. Many of these transcripts change abundance in response to PURA depletion, in parts reflected in altered protein expression levels. A closer inspection of the regulated proteins indicates a role of PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity.
2022-12-12 | GSE193900 | GEO
Project description:The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular functions and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6, and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs decrease. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study on PURA provides a blueprint for the comprehensive understanding of the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.
2022-12-19 | PXD030266 | Pride
Project description:Mycena pura 9144 Standard Draft genome sequencing
| PRJNA585713 | ENA
Project description:Our ChIP resuls suggested that coilin association with U3, snRNA and histone genes might be dependent on coilin-RNA interaction. We used iCLIP of coilin-GFP expressed in HeLa and P19 cell lines at endogenous levels to identify coilin RNA targets and investigate RNA-binding specificity. P19 cells expressing GFP fused to a nuclear localization signal (GFP-NLS) was used as a negative control. iCLIP results revealed that coilin binds several classes of ncRNA including snRNAs, U3 snoRNA and scaRNAs. Interestlignly the majority of coilin targets were intronic snoRNAs, suggesting a novel role of CBs in snoRNA biogenesis.
2014-11-06 | GSE58448 | GEO
Project description:Our ChIP resuls suggested that coilin association with U3, snRNA and histone genes might be dependent on coilin-RNA interaction. We used iCLIP of coilin-GFP expressed in HeLa and P19 cell lines at endogenous levels to identify coilin RNA targets and investigate RNA-binding specificity. P19 cells expressing GFP fused to a nuclear localization signal (GFP-NLS) was used as a negative control. iCLIP results revealed that coilin binds several classes of ncRNA including snRNAs, U3 snoRNA and scaRNAs. Interestlignly the majority of coilin targets were intronic snoRNAs, suggesting a novel role of CBs in snoRNA biogenesis. 5 biological replicates from P19 and 2 biological replicates from HeLa cells after UV-crosslinking. Negative control samples prepared from GFP-NLS fusion protein are stored uder accession E-MTAB-747.
2014-11-06 | E-GEOD-58448 | biostudies-arrayexpress
Project description:Following androgen ablation treatment for advanced prostate cancer, almost all men relapse after a period of initial response to therapy, which eventually is life threatening. We have previously found that purine-rich element binding protein, PUR alpha, was significantly repressed in androgen-independent prostate cancer cell lines in comparison to an androgen-dependent line. Moreover, over-expressing PURa in androgen-independent prostate cancer cells attenuated their cell proliferation. The aim of the studies described here was to uncover some of the mechanisms by which over-expression of PURa attenuates cell proliferation. A set of common genes induced by over-expressing PURa both in PC3 and LNCaP cells was analyzed by DNA microarray. The results were then validated utilizing quantitative reverse transcription-PCR. Using a 5.3-kb region of the PSA promoter containing androgen response elements, the participation of PURa in androgen regulated gene expression was determined. Genes involved in stress response and cell differentiation were induced in cells over-expressing PURa. Some of the genes that are targets of androgen regulation are also induced. Most strikingly, ectopic expression of PURa induced transcriptional activity of the 5.3-kb PSA promoter containing androgen response elements, without androgen stimulation. Based upon the consideration that some of the genes involved in cell stress and differentiation are also regulated by androgens our data suggest that PURa shares some common pathway regulated by the androgen receptor. These findings suggest that regulation of PURa expression in prostate cancer cells may serve as a therapeutic target for hormone refractory prostate cancer. Experiment Overall Design: PC3 cells (low in PURa expression) was stably over-expressed PURa. Total RNA of the cells was collected simultaneously with that of PC3 transfected with mock vector. Experiment Overall Design: LNCaP cells (Express PURa more than PC3 cells) was PURa-over-expressed with retrovirus vector (MSCV-PIG) and selected with puromycin for 72hr. Total RNA of the cells was collected simultaneously with that of LNCaP cells infected with mock vector. Experiment Overall Design: One set of RNA derived from PC3 cells and two independent RNA derived from LNCaP cells retrovirus infection were used for DNA microarray. Quality and concentration of total RNA was verified using Agilent Bioanalyzer and Nanodrop Spectrophotometer, respectively. T7 oligo(dT) primer (Sigma-Proligo, Boulder, CO) and 5ug total RNA are combined for first strand cDNA synthesis. Following second strand cDNA synthesis and cDNA cleanup (Phase Lock Gel Light, Eppendorf, Hamburg, Germany) an in vitro transcription reaction was performed overnight using T7 RNA transcript labeling kit provided by Enzo Life Sciences, Inc. (Farmingdale, NY). IVT reactions were cleaned using RNeasy Mini Kit (Qiagen) and concentration determined by Nanodrop Spectrophotometer. 15ug cRNA was fragmented using 5X fragmentation buffer (made in-house). Hybridization cocktail was made in accordance with Affymetrix eukaryotic expression array protocol (Affymetrix, Santa Clara, CA) and combined with fragmented cRNA. 10ug cRNA was loaded onto Human U133Plus 2.0 genome arrays (Affymetrix, Santa Clara, CA) and hybridized overnight for 16 hours. After hybridization, staining and washing of the arrays was performed following the Affymetrix eukaryotic expression array protocol, including staining with streptavidin-phycoerythrin, antibody stain, and a second streptavidin-phycoerythrin stain. After washing and staining, all arrays were scanned with the Affymetrix GeneChip Scanner and data collected with GeneChip Operating System 1.4 (GCOS, Affymetrix, Santa Clara, CA). Experiment Overall Design: The quality of the microarray experiments was assessed with affyPLM and Affy, two Bioconductor packages for statistical analysis of microarray data. To estimate the gene expression signals, data analysis was conducted on the chipsâ CEL file probe signal values at the Affymetrix probe pair (perfect match (PM) probe and mismatch (MM) probe) level, using the statistical algorithm Robust Multiarray Analysis (RMA) expression measure (Irizarry RA et al. 2003) with Affy. This probe level data processing includes a normalization procedure utilizing the quantile normalization method (Bolstad BM et al. 2003) to reduce the obscuring variation between microarrays, which might be introduced during the processes of sample preparation, manufacture, fluorescence labeling, hybridization and/or scanning. Exploratory data analysis (EDA) was performed with the preprocessed data above. Between-treatment and between-replicate variations were examined with the pair-wise MvA plots, in which the base 2 log ratios (M) between two samples are plotted against their averaged base 2 log signals (A). With the signal estimates, Principal Component analysis (PCA) was also performed to assess sample variability. Experiment Overall Design: With the signal intensities obtained above, an empirical Bayes method with the Gamma-Gamma modeling, as implemented in the bioconductor package EBarrays, was used to estimate the posterior probabilities of the differential expression of genes between PURA and Vector only sample conditions (Newton MA et al. 2001, Kendziorski Cm et al. 2003, Newton MA et al. 2004). The criterion of the posterior probability > 0.5, which means the posterior odds favoring change, was used to produce the differentially expressed gene list. Experiment Overall Design: All Bioconductor packages are available at http://www.bioconductor.org and all computation was performed under R environment (http://www.r-project.org).
2009-02-07 | E-GEOD-14464 | biostudies-arrayexpress
Project description:This experiment uses iCLIP to identify the binding pattern of the spliceosomal protein PRPF8 on RNA. The data shows that PRPF8 binds strongly and specifically in the region 12 to 14nt upstream of 5' splice sites (5ss). Due to PRPF8's role in the formation of the catalytically active spliceosome, this data can be used as a readout of 5ss selection. Here, we performed iCLIP on HeLa cells treated with control or EIF4A3 siRNA, with 4 replicate samples per condition and eIF4A3 protein levels reduced ~50% in knockdown. We investigated the role of the exon junction complex (EJC) in suppressing 5ss that are reconstituted at the junction of two canonical exons (RS-5ss) - selection of these splice sites would result in recursive splicing of canonical exons. We plotted the crosslink sites of reads that span an exon-exon junction, seperating reads that span RS-5ss from those that do not. We found that reads that span an RS-5ss are enriched at the 12-14nt window associated with 5ss selection, while reads that span other exon-exon junctions are not enriched. This effect is magnified greatly by knockdown of eIF4A3. The results indicate that RS-5ss can be used by the spliceosome, but that this process is usually repressed by the EJC. This data is evidence of recursive splicing of canonical exons and the role of the EJC in repressing recursive splicing.
2018-10-14 | E-MTAB-7269 | biostudies-arrayexpress