Monosome and disome profiling of Flag-STAU1 Flp-In 293 T-REx cells
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ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Homo sapiens
SUBMITTER: Jernej Ule
PROVIDER: E-MTAB-9669 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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 3â untranslated region (3âUTR) 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 3âUTR 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 3âUTR 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. Experiment Overall Design: We report the results of three independently performed microarray analyses that examined changes in the abundance of transcripts from HeLa-cell genes upon Stau1 depletion. HeLa cells were transiently transfected with either a nonspecific Control small interfering (si)RNA or Stau1 siRNA. Stau1 siRNA reduced the level of cellular Stau1 to as little as 4% of normal, where normal is defined as the level in the presence of Control siRNA (data not shown). RNA from three independently performed transfections was separately hybridized to microarrays.
2008-06-15 | E-GEOD-6679 | biostudies-arrayexpress
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:Purpose: To analyze the sensitivity and specificity of the AmpFISH method, we sequenced the NIH3T3 cell line via UMI-RNAseq experiments. Methods:NIH3T3 cells were grown in DMEM (Dulbecco’s Modified Eagle Medium, Gibco) supplemented with 10% FBS (Fetal Bovine Serum, Sigma), 50U/ml Penicillin and 50 mg/ml streptomycin (Gibco,cat.no.15070) at 37℃ with 5% CO2. Cells were treated with 0.25% trypsin solution (HyClone, No.SH42605.01) when they reached ~106 cells/ml. Then, the cells were washed with 1X PBS, and then mixed with 1ml TRIzol solution (ThermoFish, No.15596029), and snap-frozen with dry ice. Total RNA was qualitatively and quantitatively evaluated as follows: (1) the RNA sample was initially qualitatively evaluated using 1% agarose gel electrophoresis for possible contamination and degradation; (2) RNA purity and concentration were then examined using NanoPhotometer spectrophotometer; (3) RNA integrity and quantity were finally measured using RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system. After library preparation and pooling of different samples, the samples were subjected to Illumina sequencing. The libraries were sequenced using the Illumina NovaSeq 6000 Platform for 6G raw data and generated 150nt pair-end reads. UMI sequences on each read were identified by UMI-tools (1.0.0), and reads with UMIs were used for the subsequent analysis. To identify the duplicated reads, UMIs were initially removed from the UMI reads, and the remaining parts of each read were mapped to the reference genome using Hisat2. Reads that mapped to the same location on the reference genome were identified as duplicated reads. Then, the UMIs on each read were recalled, and the duplicated reads with the same UMI were identified as non-natural duplications, which were subsequently removed from the processed data. HTSeq v0.6.1 was used to count the read numbers mapped to each gene. Then, the FPKM of each gene was calculated based on the length of the gene, and the read count was mapped to the gene. Conclusions:AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows
2021-08-11 | GSE181685 | GEO
Project description:Single-cell transcriptomics, reliant on the incorporation of barcodes and unique molecular identifiers (UMIs) into captured polyA+ mRNA, faces a significant challenge due to synthesis errors in oligonucleotide capture sequences. These inaccuracies, which are especially problematic in long-read sequencing, impair the precise identification of sequences and result in inaccuracies in UMI deduplication. To mitigate this issue, we have modified the oligonucleotide capture design, which integrates an interposed anchor between the barcode and UMI, and a 'V' base anchor adjacent to the polyA capture region. This configuration is devised to ensure compatibility with both short and long-read sequencing technologies, facilitating improved UMI recovery and enhanced feature detection, thereby improving the efficacy of droplet-based sequencing methods.
2024-04-09 | GSE263458 | GEO
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:hiCLIP protocol was followed to isolate RNA bound to 3XFLAG-STAU1 protein and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction, and are specified below for each sequencing run. Random barcodes were also incoperated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina paired-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3
2015-03-02 | E-MTAB-2937 | biostudies-arrayexpress
Project description:Ribosome profiling was prepared in parallel with RNA-seq, using the same protocol for cDNA library preparation.
2015-03-01 | E-MTAB-2941 | biostudies-arrayexpress
Project description:RNAseq was prepared in parallel with ribosome profiling, using the same protocol for cDNA library preparation.
2015-03-01 | E-MTAB-2940 | biostudies-arrayexpress
Project description:Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7kb lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high mRNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a suite of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25 nucleotide “TINCR box” motif which is strongly enriched in interacting mRNAs \and required for TINCR binding. A high-throughput screen to analyze TINCR binding capacity to ~9,400 human recombinant proteins revealed direct binding of TINCR RNA to the Staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay (SMD), however, lacked differentiation impacts. Instead, the TINCR/STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through inducible lncRNA binding to differentiation mRNAs to ensure their expression. Gene expression analysis: To establish a gene expression signature for regenerated human epidermis depleted of TINCR, as well as STAU1, total RNA was isolated in biologic duplicate from organotypic cultures of cells with a non-targeting siRNA, or an siRNA to the gene of interest. This RNA was processed and then hybridized to Affymetrix HG-U133 2.0 Plus arrays.
2012-10-10 | E-GEOD-40122 | biostudies-arrayexpress
Project description:Many library preparation methods are available for gene expression quantification. Here, we sequenced and analysed Universal Human Reference RNA (UHRR) prepared using Smart-Seq2, TruSeq (public data) and a protocol using unique molecular identifiers (UMIs) that all include the ERCC spike-in mRNAs to investigate the effects of amplification bias on expression quantification. UHRR 10 and 12 replicates for Smart-seq2 and UMI-seq library preparation methods, respectively.
2016-05-13 | E-GEOD-75823 | biostudies-arrayexpress