Genome-wide analysis of mRNAs expression in prometaphase
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE51182 | GEO | 2014/07/03
SECONDARY ACCESSION(S): PRJNA221619
REPOSITORIES: GEO
Similar Datasets
Project description:The goal of this experiment was to compare the transcriptome of cells that overexpressed the RNA-binding protein Staufen1 to that of cells that expressed the empty vector as control. Stau1 abundance fluctuates through the cell cycle: it is high in the G2 phase of the cell cycle and rapidly decreases as cells transit through mitosis. The importance of controlling Stau1 levels was underscored by the observation that its overexpression impairs mitosis as well as proliferation of transformed cell lines. As a major post-transcriptional regulator, Stau1 may exert its role(s) through the spatial and/or temporal regulation of its bound mRNAs. Therefore, to gather clues to support this possibility, we determined if Stau1 overexpression significantly modified the transcriptome of transformed cells in prometaphase explaining the observed impairment in mitosis transit and/or cell proliferation. HEK293T cells were transfected with plasmids coding for Stau155-FLAG or the empty vector as control. Cells were synchronized in prometaphase with nocodazole.
2014-07-03 | E-GEOD-51182 | biostudies-arrayexpress
Project description:The goal of this experiment was to identify Staufen1-bound mRNAs in prometaphase cells. Stau1 abundance fluctuates through the cell cycle: it is high in the G2 phase of the cell cycle and rapidly decreases as cells transit through mitosis. The importance of controlling Stau1 levels was underscored by the observation that its overexpression impairs mitosis as well as proliferation of transformed cell lines. As a major post-transcriptional regulator, Stau1 may exert its role(s) through the spatial and/or temporal regulation of its bound mRNAs. Therefore, to gather clues to support this possibility, we identified Stau1-bound mRNAs in prometaphase as a means to identify mRNAs that could be subjected to post-transcriptional modulation when Stau1 is partly degraded in mitosis. HEK293T cells were transfected with plasmids coding for Stau155-FLAG or the empty vector as control. Cells were synchronized in prometaphase with nocodazole. Stau1-bound mRNAs were isolated after immunoprecipitation using anti-FLAG antibody.
2014-07-03 | E-GEOD-51183 | biostudies-arrayexpress
Project description:The goal of this experiment was to identify Staufen1-bound mRNAs in prometaphase cells. Stau1 abundance fluctuates through the cell cycle: it is high in the G2 phase of the cell cycle and rapidly decreases as cells transit through mitosis. The importance of controlling Stau1 levels was underscored by the observation that its overexpression impairs mitosis as well as proliferation of transformed cell lines. As a major post-transcriptional regulator, Stau1 may exert its role(s) through the spatial and/or temporal regulation of its bound mRNAs. Therefore, to gather clues to support this possibility, we identified Stau1-bound mRNAs in prometaphase as a means to identify mRNAs that could be subjected to post-transcriptional modulation when Stau1 is partly degraded in mitosis.
2014-07-03 | GSE51183 | GEO
Project description:Staufen1 (STAU1) is an RNA-binding protein involved in maturation, localization, translation and decay of mRNAs. STAU1 expression is modulated during the cell cycle and decreases during mitosis. In prometaphase, STAU155 binds specific classes of mRNAs that code for proteins implicated in transcription and cell cycle regulation. In this paper, we report that STAU155 co-localizes with microtubules on the mitotic spindle in human colorectal cancer cell line HCT116, and map the molecular determinant required for this association within the N-terminal 88 amino acids (aa 25-37). Interestingly, STAU1 co-purifies with ribosomal proteins and co-localizes with active sites of translation on the mitotic spindle. To characterize STAU1-dependent mRNA transport and localization on the spindle, we used RNAseq analysis to identify spindle-associated mRNAs on purified spindles of wild-type and STAU1-KO CRISPR cell lines. Our datasets identify 161 protein-coding transcripts that are less abundant on the mitotic spindle of STAU1-KO cells compared to WT, and 660 that are more abundant. Altogether, these data demonstrate that STAU1 controls the transport and the localization of specific sub-populations of mRNAs to the mitotic spindle of cancer cells and suggest that at least some spindle-localized mRNAs undergo local translation during mitosis.
2019-10-05 | GSE138441 | GEO
Project description:Purpose: We performed RNA-Immunoprecipitation in Tandem (RIPiT) experiments against human Staufen1 (Stau1) to identify its precise RNA binding sites in a transcriptome-wide manner. To monitor the consequences of Stau1 binding in terms of target mRNA levels and ribosome occupancy, we modified the levels of endogenous Stau1 in cells by siRNA or overexpression and performed RNA-sequencing and ribosome-footprinting experiments. Staufen1 (Stau1) is a double-stranded RNA (dsRNA) binding protein implicated in mRNA transport, regulation of translation, mRNA decay and stress granule homeostasis. Here we combined RNA-Immunoprecipitation in Tandem (RIPiT) with RNase footprinting, formaldehyde crosslinking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1 binding sites transcriptome-wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3'UTRs or in "strongly distal" 3'UTRs extending far beyond the canonical polyadenylation signal. Stau1 also interacts with both actively translating ribosomes and with mRNA coding sequences (CDS) and 3'UTRs in proportion to their GC-content and internal secondary structure-forming propensity. On mRNAs with high CDS GC-content, higher Stau1 levels lead to greater ribosome densities, suggesting a general role for Stau1 in modulating the ability of ribosomes to elongate through secondary structures located in CDS regions.
2013-11-25 | GSE52447 | GEO
Project description:Purpose: We performed RNA-Immunoprecipitation in Tandem (RIPiT) experiments against human Staufen1 (Stau1) to identify its precise RNA binding sites in a transcriptome-wide manner. To monitor the consequences of Stau1 binding in terms of target mRNA levels and ribosome occupancy, we modified the levels of endogenous Stau1 in cells by siRNA or overexpression and performed RNA-sequencing and ribosome-footprinting experiments. Staufen1 (Stau1) is a double-stranded RNA (dsRNA) binding protein implicated in mRNA transport, regulation of translation, mRNA decay and stress granule homeostasis. Here we combined RNA-Immunoprecipitation in Tandem (RIPiT) with RNase footprinting, formaldehyde crosslinking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1 binding sites transcriptome-wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3'UTRs or in "strongly distal" 3'UTRs extending far beyond the canonical polyadenylation signal. Stau1 also interacts with both actively translating ribosomes and with mRNA coding sequences (CDS) and 3'UTRs in proportion to their GC-content and internal secondary structure-forming propensity. On mRNAs with high CDS GC-content, higher Stau1 levels lead to greater ribosome densities, suggesting a general role for Stau1 in modulating the ability of ribosomes to elongate through secondary structures located in CDS regions. We used HEK293 cells expressing near endogenous levels of wild-type Flag-Stau1 (65KDa isoform with an N-Terminal Flag tag). As a control we used a mutant version of Stau1 that is not functional for dsRNA binding. Formaldehyde crosslinking experiments and RNase footprinting experiments were done in two biological replicates. All RNASeq, Ribosome footprinting and PAS-Seq were done in two biological replicates.
2013-11-25 | E-GEOD-52447 | biostudies-arrayexpress
Project description:Two human rhabdomyosarcoma cell lines (RD, RH36, RH30, and RH18) and one none-transformed skeletal muscle cell line (HSMM-C3) cell line were studied for the impact of Staufen1 depletion on their protein expression profile. Proteomics was performed for control and Staufen1 knockdown cells (n=3). For this, we generated STAU1 knockdown cell lines using a mixture of three independent STAU1-targeting lentiviral-shRNA constructs. cells were cultured for 72 hours and then protein extraction was performed as follow: Cells were lysed in 1X RIPA buffer (20 mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM Na2- EDTA, 1mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na3 VO4, 1µg/ml leupeptin). Protein concentration was tested according to the BCA assay protocol (Thermofisher Scientific) and samples were used in proteomics experiment.
2023-10-22 | PXD042156 | Pride
Project description:In human cells, Staufen1 is double-stranded RNA-binding protein involved in several cellular functions including mRNA localization, translation and decay. We used a genome wide approach to identify and compare the mRNA targets of mammalian Staufen1. The mRNA content of Staufen1 mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with a Stau1-HA expressor. Our results indicate that 7% of the cellular RNAs expressed in HEK293T cells are found in Stau1-containing mRNPs. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes and catalytic activity. Keywords: Immunoprecipitation, Staufen, microarray
2008-01-18 | GSE8438 | GEO
Project description:In human cells, Staufen1 is double-stranded RNA-binding protein involved in several cellular functions including mRNA localization, translation and decay. We used a genome wide approach to identify and compare the mRNA targets of mammalian Staufen1. The mRNA content of Staufen1 mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with a Stau1-HA expressor. Our results indicate that 7% of the cellular RNAs expressed in HEK293T cells are found in Stau1-containing mRNPs. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes and catalytic activity. Experiment Overall Design: HEK293 cells were transiantly transfected with plasmids coding for Staufen1-HA. Cell extracts were immunoprecipitated using anti-HA antibodies and co-immunoprecipitated mRNAs were purified and used to hybridize microarrays. As control, cell extract from mock transfected cells were used.
2008-06-16 | E-GEOD-8438 | biostudies-arrayexpress
Project description:It is currently unknown how extensively the double-stranded RNA binding protein Staufen (Stau)1 is utilized by mammalian cells to regulate gene expression. To date, Stau1 binding to the 3’ untranslated region (3’UTR) of ARF1 mRNA has been shown to target ARF1 mRNA for Stau1-mediated mRNA decay (SMD). ARF1 SMD depends on translation and recruitment of the nonsense-mediated mRNA decay factor Upf1 to the ARF1 3’UTR by Stau1. Here, we use microarray analyses to examine changes in the abundance of cellular mRNAs that occur when Stau1 is depleted. Results indicate that 1.1% and 1.0% of the 11,569 HeLa-cell transcripts that were analyzed are, respectively, upregulated and downregulated at least two-fold in three independently performed experiments. Additionally, we localize the Stau1 binding site to the 3’UTR of four mRNAs that we define as natural SMD targets. Together, these and substantiating results suggest that Stau1 influences the expression of a wide variety of physiologic transcripts and metabolic pathways. Keywords: Staufen1-mediated mRNA decay; Stau1 downregulation by siRNA.
2007-12-26 | GSE6679 | GEO