HiCLIP analysis of RNA duplexes bound by STAU1 in HEK293 cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500
ORGANISM(S): Homo sapiens
SUBMITTER: Jernej Ule
PROVIDER: E-MTAB-2937 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We performed iCLIP of CPSF-160- GFP as CPSF160 was believed to be the main RNA-binding subunit of CPSF. To this end, we used a stable HeLa cell line that expresses GFP-CPSF160 in a doxycycline (Dox)-dependent manner. After GFP-CPSF160 was induced, we irradiated the cells with UV-C (wavelength=254nm) to induce protein-RNA crosslinks and immunoprecipitated the complex with a GFP antibody.
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Project description:Monosome and disome profiling was performed on Flag-STAU1 Flp-In 293 T-REx to study the causes of ribosomal collisions, and whether this may be modulated by the presence/absence of Staufen-1. Cells were treated with either an siRNA targeting STAU1 transcript (4x samples) or a control siRNA (2x samples). Two of the four samples treated with the STAU1 siRNA had siRNA-resistant STAU1 mRNA expression induced by doxycycline (rescue). Sequencing libraries from monosome and disome fractions were generated in parallel from the same samples. Note that unique molecular identifiers/random barcodes (UMIs/RBCs) were included in the sequencing experiment. Each UMI has been moved to the fastq read name of each read. For example \\"xxxxxxrbc:AGCCAAT\\" in the read name signifies that the given read had a UMI of \\"AGCCAAT\\". Using these UMIs, PCR duplicates can be removed with UMI-Tools following read alignment.
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2021-10-05 | E-MTAB-10830 | 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 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: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.
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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.
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