Exon-Intron circular RNAs regulate transcription in the nucleus
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Ge Shan
PROVIDER: E-GEOD-64443 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Purpose: We are using the illumina sequencing to compare the false positive and true positive circular RNA findings to confine the method to detect the true circular RNAs Methods: The testis whole transcriptome profiling was generated from 4-week mouse testis using illumina Nextseq, duplicated. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: our data suggest that circular RNAs identified based on junction sequences in the RNA-seq reads, especially those from Illumina Hiseq sequencing, mostly result from template-switching events during reverse transcription by MMLV-derived reverse transcriptases. It is critical to employ reverse transcriptases lacking terminal transferase activity (e.g., MonsterScript) to construct sequencing library or perform RT-PCR for identification and quantification of true circular RNAs. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. The wild type mouse testis RNAs were constructed NGS library by two different enzyme, then parallel sequenced in illumina Nextseq
2016-07-05 | E-GEOD-83992 | biostudies-arrayexpress
Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.
2015-12-11 | E-GEOD-62171 | biostudies-arrayexpress
Project description:DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense transcriptional status of both RNA Pol I and Pol II to control transcriptional and post-transcriptional steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of protein-coding and noncoding RNAs. Although broad, these molecular interactions, both at the chromatin and at the RNA level, exhibit a remarkable specificity for the ribosomal pathway. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs and, as a functional component of the snoRNA ribonucleoprotein (snoRNP) complex, promotes modification of rRNA. In the nucleoplasm, DDX21 is incorporated into the 7SK snRNP complex, which facilitates DDX21 association with promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of P-TEFb from the 7SK snRNP, enhancing productive Pol II elongation. Altogether, we present a unifying mechanism for the coordinated regulation of ribosomal genes across nuclear compartments, and provide first evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control. Examination of DDX21 chromatin association and DDX21 RNA interacting partners in HEK293 cells
2014-11-17 | E-GEOD-56802 | biostudies-arrayexpress
Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.
2020-05-22 | PXD016618 | Pride
Project description:Promoter-proximal RNA polymerase II (Pol II) pausing is implicated in the regulation of gene transcription. However, the mechanisms of pausing including its dynamics during transcriptional responses remain to be fully understood. We performed global analysis of short capped RNAs and Pol II Chromatin Immunoprecipitation sequencing in MCF-7 breast cancer cells to map Pol II pausing across the genome, and used permanganate footprinting to specifically follow pausing during transcriptional activation of several genes involved in the Epithelial to Mesenchymal Transition (EMT). We find that the gene for EMT master regulator Snail (SNAI1), but not Slug (SNAI2), shows evidence of Pol II pausing before activation. Transcriptional activation of the paused SNAI1 gene is accompanied by a further increase in Pol II pausing signal whereas activation of non-paused SNAI2 gene results in the acquisition of a typical pausing signature. The increase in pausing signal reflects increased transcription initiation without changes in Pol II pausing. Activation of the heat shock HSP70 gene involves pausing release that speeds up Pol II turnover, but does not change pausing location. We suggest that Pol II pausing is retained during transcriptional activation and can further undergo regulated release in a signal-specific manner. Untreated MCF-7 cells were analyzed for the distribution of Pol II using ChIP-sequencing with Anti-Pol II N-20 antibody (two independent biological replicates, A, B), and for the distribution of paused RNA polymerase II by sequencing of short capped RNAs (scRNAs) prepared from nuclei (three independent biological replicates, 1-3). All samples were sequenced on a MiSeq instrument in paired-end format
2015-03-27 | E-GEOD-67041 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE31485: CTCF promotes RNA pol II pausing and links DNA methylation to alternative splicing [ChIP-Seq] GSE31486: CTCF promotes RNA pol II pausing and links DNA methylation to alternative splicing [RNA-Seq] Refer to individual Series
2011-10-05 | E-GEOD-31278 | biostudies-arrayexpress
Project description:The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide association of Pol V with chromatin is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents the first genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution. For wild type plants (ecotype Columbia) and nrpe1 mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. In addition whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of wild type plants carrying a C-terminally epitope tagged (2XFLAG) NRPE1, as well as the NRPE1-FLAG construct in drd1, dms3, and rdm1 mutant backgrounds.
2012-08-05 | E-GEOD-39247 | biostudies-arrayexpress
Project description:RNA-directed DNA methylation (RdDM) is a transcriptional silencing mechanism mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1a (TOP1a) was able to de-repress LUCL by reducing its DNA methylation and H3K9 dimethylation (H3K9me2) levels. Further studies with Arabidopsis top1a mutants showed that TOP1a promotes RdDM by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at transposable elements (TEs). 5 small RNA libraries were sequenced
2014-07-08 | E-GEOD-50719 | biostudies-arrayexpress
Project description:We report genome-wide detection of long noncoding RNA (lncRNA) generate by Pol V in transcriptional gene silencing in Arabidopsis thaliana. We further show that most of these transcripts are bound by AGO4. RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) RNA immunoprecipitation with an anti-AGO4 antibody was performed in one biological replicate and included Col-0, nrpe1, and ago4-1; RNA immunoprecipitation with an anti-NRPE1 (largest subunit of Pol V) was performed in two replicates; replicate 1 included Col-0 and nrpe1, replicated two included Col-0, nrpe1, ago4-1, and idn2-1
2016-10-24 | E-GEOD-70290 | biostudies-arrayexpress
Project description:Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of the Arabidopsis RDR2 gene. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wildtype maize and in the isogenic mop1-1 loss-of-function mutant using Illumina’s sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24 nt (nucleotide) endogenous heterochromatic short-interfering siRNAs were dramatically reduced resulting in an enrichment of miRNAs and trans-acting siRNAs (ta-siRNAs). In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic ~22 nt class of small RNAs. These data suggest that maize, unlike Arabidopsis, has a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24 nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced. This library was derived from maize variety K55 wild-type and mop1 mutant. Immature ears were harvested from plants grown outdoors in Tuscon, AZ. The material was harvested from plants 68 days after germination, with the floral tissue including young tassels 2-3 cm long. Total RNA was isolated using a standard TRIZOL-based method and the small RNA libraries were generated as described by Lu et al. (Methods 2007, 43:110), followed by sequencing with Illumina's SBS method. The adapter sequences were removed, and the abundance of each distinct small RNA was determined. Raw data are unavailable from Illumina
2008-09-30 | E-GEOD-12173 | biostudies-arrayexpress