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
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.
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.
Project description:RNA-DNA duplexes have been extensively catalogued in interphase cells, but relatively little is known about the genomic loci of these duplexes in mitotic cells. We compared RNA-DNA duplexes in DLD1 cells arrested in mitosis to libraries previously published in asynchronous HEK293T cells prepared with the same protocols, and processed with the same bioinformatic pipeline. We also observed that Aurora B inhibitors increased the immunofluorescence signal for RNA-DNA duplexes in mitotic cells, so we tested the effect of Aurora B inhibition on genomic enrichment of RNA-DNA duplexes.
Project description:We present a novel transcriptome-wide experimental approach based on sequencing RNA fragments longer than 18 nts in order to detect accessible RNA duplexes
Project description:Alternative splicing patterns have diverged rapidly during vertebrate evolution, yet the mechanisms and functional consequences of species-dependent splice isoform differences are not well understood. Through an integrative analysis of genome-wide predictions of highly stable RNA duplexes, alternative splicing profiles, and proximity ligation-detection of RNA-RNA interactions in vivo, we observe evidence that the majority of long-range intronic RNA duplexes are mediated by inverted Alu-repeat elements, and that these structures are associated with widespread exon skipping in primates. Our results further reveal evidence that Alu-derived RNA duplexes have contributed to divergence in alternative splicing patterns during mammalian evolution.
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.
Project description:We report the genomic occupancy of the BAHD1 protein in HEK293 cells over-expressing the BAHD1 gene (HEK293-HPT-BAHD1) We used Native ChIP-seq to identify DNA regions bound to BAHD1 (native ChIP involves use of native chromatin before precipitation of immune complexes, without formaldehyde crosslinking of proteins with nucleic acids)
