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Transcription profiling of human HEK-293 cells to identify Dicer and Argonaute regulated gene expression

ABSTRACT: HEK-293-derived cell lines depleted of Dicer or one of four Argonaute proteins were generated. Microarray analysis of these knock down cell lines and various control cell lines was performed to address how large the proportion of genes is that are regulated by RNA silencing in an individual cell type. Comparison of different arrays revealed commonly (up) regulated genes. The highest overlap was observed in Ago2 and Dicer knockdown cells. Experiment Overall Design: three control cell lines (T-Rex, shRNA, empty vector), two induvidual Argonaute 1 and 4 (Ago1kd, Ago4kd) knock down cell lines, one Argonaute 2 and 3 (Ago2kd, Ago3kd) and two induvidual Dicer knock down cell lines were analyzed as biological replicates

ORGANISM(S): Homo sapiens

SUBMITTER: Daniela Schmitter

PROVIDER: E-GEOD-4246 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells.

Schmitter Daniela D Filkowski Jody J Sewer Alain A Pillai Ramesh S RS Oakeley Edward J EJ Zavolan Mihaela M Svoboda Petr P Filipowicz Witold W

Nucleic acids research 20060913 17

RNA interference and the microRNA (miRNA) pathway can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes hundreds of miRNAs that can post-transcriptionally repress thousands of genes. Using reporter constructs, we observed that degradation of mRNAs bearing sites imperfectly complementary to the endogenous let-7 miRNA is considerably stronger in human HEK293 than HeLa cells. The degradation did not result from the Ago2-mediated endonucleolytic clea ...[more]

PMID: 16971455

Similar Datasets

Analysis of transcripts regulated by Dicer and Argonaute proteins in human HEK-293 cells

Project description:HEK-293-derived cell lines depleted of Dicer or one of four Argonaute proteins were generated. Microarray analysis of these knock down cell lines and various control cell lines was performed to address how large the proportion of genes is that are regulated by RNA silencing in an individual cell type. Comparison of different arrays revealed commonly (up) regulated genes. The highest overlap was observed in Ago2 and Dicer knockdown cells. Keywords: different timepoints of induction of stable knock down cell lines compared to various control cell lines

2006-09-01 | GSE4246 | GEO

Analysis of transcripts in PNA-A(15) oligo treated human HEK-293 cells

Project description:Mononucleotide A and T repeats are abundant in human genome. Many of A repeats are bound by Argonaute proteins (AGOs). To evaluate the role of AGOs and A repeats in gene regulation, HEK293 cells were treated with 8-amino-3,6-dioxaoctanoic acid added peptide nucleic acid (PNA) AAAAAAAAAAAAAAA oligo (OO-A(15)). Matched two sets of two individual HEK293 cells were transfected with OO-A(15) and scramble OO-PNA oligo, and were analyzed as biological duplicates. Cells were grown in DMEM (Gibco-BRL) according to the manufacturer’s protocol. OO-A(15) and scramble oligo were dilute with dH2O to reach 10μM. Cells were transfected in 6-well plates, seed 6 x 105 cells per well with 50 nM OO-A(15) and scramble oligo using TransIT-siQUEST transfection reagent (Mirus). The transfected cell lines were cultured for 48 h post-transfection and harvested total RNA by Trizol reagent (Invitrogen). All RNA integrity assays were performed and hybridized on beadchip according to the protocol.

2013-09-03 | E-GEOD-43185 | biostudies-arrayexpress

A variety of Dicer substrates in human and C. elegans (HEK RNA-seq)

Project description:The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer binding sites. We find known and hundreds of novel miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside the miRNA pathway. Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells. Drosha knockdown and mock transfections were used as controls. Knockdown was performed with two independent siRNAs each. In total 5 samples.

2014-11-20 | E-GEOD-63475 | biostudies-arrayexpress

Argonaute proteins couple chromatin silencing to alternative splicing (exon array)

Project description:While Argonaute (AGO) proteins play a major role in transcriptional gene silencing (TGS) in many organisms, their role in the nucleus of somatic mammalian cells remains elusive. Here, we have purified AGO1 and AGO2 chromatin-embedded complexes, and found these proteins associated with previously described partners, but also with chromatin modifiers and, rather unexpectedly, with different splicing factors. Using the CD44 gene as a model for alternative splicing, we show that both AGO1 and AGO2 are required for Protein Kinase C (PKC)-dependent variant exon inclusion. AGO proteins facilitate the spliceosome recruitment and modulate the elongation rate of RNA polymerase II (RNAPII). The recruitment of AGO proteins to CD44 transcribed region is dependent on both the endonuclease Dicer and the chromodomain-containing protein HP1g, and results in locally increased levels of histone H3 lysine 9 (H3K9) methylation on variant exons. Genome wide analysis of splicing in either AGO2 or Dicer null cells showed that the two proteins have similar effects on many splicing events. Finally, sRNAs associated with nuclear AGO2 are mostly in sense orientation relative to protein-coding genes, supporting a role for intragenic antisense non-coding RNAs in the recruitment AGO and splicing factors. Together, our data demonstrate for the first time that the endogenous RNAi pathway is involved in alternative splicing decisions, unravelling a new model in which AGO proteins couple RNAPII elongation and chromatin modification. Study of AGO2 or Dicer knock-out on gene expression and splicing regulation in MEF cells Transcriptome analysis of AGO2 and Dicer null MEF cells on GeneChipM-BM-. Mouse Exon 1.0 ST Arrays (Affymetrix). Dicer null MEF cells and wild-type MEF cells were from M. Otsuka. AGO2 null MEF cells were from A. Tarakhovsky. Experiment has been done in experimental triplicates. 9 Total samples were analyzed.

2012-09-10 | E-GEOD-39746 | biostudies-arrayexpress

Analysis of transcripts in PNA-A(15) oligo treated human HEK-293 cells

2013-09-03 | GSE43185 | GEO

Argonaute 2 binds directly to tRNA genes and promotes gene repression in cis

Project description:To further our understanding of the RNAi machinery within the human nucleus, we analyzed the chromatin and RNA binding of Argonaute 2 (AGO2) within human cancer cell lines. Our data indicated that AGO2 binds directly to nascent tRNA and 5S rRNA, and to the genomic loci from which these RNAs are transcribed, in a small RNA- and DICER-independent manner. AGO2 chromatin binding was not observed at non-TFIIIC-dependent RNA polymerase (Pol) III genes or at extra-TFIIIC (ETC) sites, indicating that the interaction is specific for TFIIIC-dependent Pol III genes. A genome-wide analysis indicated that loss of AGO2 caused a global increase in the mRNA expression level among genes that flank AGO2-bound tRNA genes. This effect was shown to be distinct from that of the disruption of DICER, DROSHA, or CTCF. We propose that AGO2 binding to tRNA genes has a novel and important regulatory role in human cells.

2015-05-17 | GSE68813 | GEO

Argonaute Surveillance and Dicer-Independent priRNAs TriggerRNAi and Heterochromatin Formation

Project description:The assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associate with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on the random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats. small RNA profiling in wild type S. pombe cells and in 12 mutant cells

2010-02-23 | E-GEOD-19734 | biostudies-arrayexpress

Argonaute and Trimmer generate Dicer-independent priRNAs and nucleate RNAi

Project description:RNA interference is involved in silencing of transposable and repetitive elements. How these elements are initially recognized by RNAi is a fundamental and unanswered question. We previously identified a class of Dicer-independent small RNAs, called primal small RNAs (priRNAs), in fission yeast. The mechanism by which Dicer-independent small RNAs are generated is not clear for any species. Here we reconstitute priRNA biogenesis in vitro and demonstrate that priRNAs can nucleate RNAi in vivo. We identify 3'-5' exonuclease Trimmer and show that Argonaute, loaded with longer RNA precursors, recruits Trimmer to generate the new 3' end. Next, we show that antisense priRNAs accumulate in rrp6M-NM-^T cells and nucleate RNAi at subset of protein coding genes in a Trimmer- and priRNA-dependent manner. Thus, Rrp6-mediated degradation of antisense transcripts and priRNA precursors protects the genome from spurious RNAi. Our results suggest that Argonaute association with random RNA degradation products triggers RNAi in a process of transcriptome surveillance. small RNA profiling in wild type S. pombe cells and in mutant cells

2014-04-21 | E-GEOD-38636 | biostudies-arrayexpress

Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (HEK RNA-Seq)

Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells. Drosha knockdown and mock transfections were used as controls. In total 3 samples.

2014-11-20 | E-GEOD-55326 | biostudies-arrayexpress

Cholesterol down-regulates BK channels stably expressed in HEK 293 cells.

Project description:Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca(2+)-activated K(+) (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the ?-subunit hKCa1.1 and the auxiliary ?1-subunit or in hKCa1.1-HEK 293 cells expressing only the ?-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-?-cyclodextrin (M?CD), and enriched with cholesterol-saturated M?CD (M?CD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the ?1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by M?CD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary ?1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using M?CD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary ?1-subunit.

| S-EPMC3832390 | biostudies-literature

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