Human gastric cancer cells SGC7901-NM RIP-Chip result: has-miR-625 lentivirus infected vs. control
Ontology highlight
ABSTRACT: Transcriptional profiling of RIP products of human gastric cancer cells SGC7901-NM comparing control with SGC7901-NM infected with has-miR-625 lentivirus Stable transfected cell lines, SGC7901-NM-has-miR-625 vs. SGC7901-NM-NC, after RNA-binding protein immunoprecipitation with Ago2 antibody, the experimental group (Ago2) vs. the control group (input) per array.
Project description:The aim of this study was to investigate the effect of hsa-miR-143-3p on global gene expression in T-cell acute lymphoblastic leukemia (T-ALL) and to unravel its direct target genes. For this purpose we selected Jurkat T-ALL cell line characterized by low expression of this miRNA. The cells were transduced either with pCDH-CMV-MCS-EF1_GFP_puro empty vector (System Biosciences) or pCDH-CMV-MCS-EF1_GFP pre-mir-143 vector, each variant in three biological replicates. Total RNA was isolated from each samples. Additionally, RISC complexes containing miRNAs bound to their target mRNAs were isolated by AGO2 immunoprecipitation and the obtained fraction of RISC-bound RNA was isolated from each sample. All 12 libraries (6 total RNA samples and 6 AGO2-RIP samples) were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 60M paired reads/sample.
Project description:We established a neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high throughput sequencing (AGO2-RIP-seq) to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this technique, we identified more than two thousand miRNA target genes in hippocampal neurons, regulating essential neuronal features such as axon guidance and transcription. Furthermore, we found that stable inhibition of the highly expressed miR-124 in hippocampal neurons led to significant changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. Our data suggest that target redundancies are common among microRNA families. Together, these findings greatly enhance our understanding of the mechanisms and dynamics through which miRNAs regulate their target genes in neurons. Analysis of the miRNA targetome in hippocampal neurons after inhibition of 2 different miRNAs. AAV5 injections into the hippocampus of adult C57BL/6 mice producing either of the following under a synapsin promoter: GFP only (Samples beginning with 'GFP124…' or 'GFP125…'), GFP-miR124sp (Samples beginning with 'miR124…'), GFP-miR125sp (Samples beginning with 'miR125…'), GFP-AGO2-miR292sponge (samples ending with '…292'), GFP-AGO2-miR124sponge (samples ending with '…124'), GFP-AGO2-miR125sponge (samples ending with '…125'). All other samples were sham-injected.
Project description:We report that microRNAs strongly regulate targets of exceptionally high affinity and that such targets can be identified upon microRNA silencing in Argonaute 2 ribonucleoprotein immunoprecipitation (Ago2-RIP) experiments
Project description:The reduced sperm count observed in Ago2 cKO mice implies that AGO2 has non-redundant functions in the male germ line. Because AGO2 is a key protein in the RNA interference (RNAi) pathway, we first postulated that AGO2 loss disrupts normal transcriptional and translational dynamics of target mRNAs relevant to sperm maturation. To address this hypothesis, we took advantage of the apparently normal spermatogenesis in Ago2 cKO mice, which allowed us to purify matched meiotic and post-meiotic germ cells from Ago2 cKO and wild type controls. We examined changes in the transcriptome and proteome of these two spermatogenic stages in Ago2 cKO relative to control mice using RNA-seq and quantitative mass spectrometry (MS). To further examine if the changes in mRNA and protein levels detected in Ago2 cKO germ cells was due to a loss of regulation by the canonical AGO2-miRNA pathway, we characterized AGO2 protein interactors by AGO2 immunoprecipitation-mass spectrometry (IP-MS) in cytoplasmic and nuclear fractions of post-meiotic cells
Project description:Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis This study constructed and sequenced two independent small RNA libraries from the upper uninoculated leaves of (i) WT Arabidopsis plants 14 d after mock inoculation and of (ii) rdr1 rdr6 plants 14 d after infection with Fny CMV-Δ2b, and one library each from the upper uninoculated leaves of (iii) WT, (iv) rdr1, and (v) rdr6 plants 14 d after infection with TuMV-GFP. Coimmunoprecipitation with FLAG- and HA-specific antibodies was used to obtain (vi,vii) AGO1 and (viii,ix) AGO2 complexes, respectively, from the FLAG-AGO1/ago1-36 and HA-AGO2 plants 14 d after infection with Fny CMV-Δ2b for extracting total loaded small RNAs for the construction and sequencing of small RNA libraries.
Project description:We deciphered Ago2:RNA interactions in post-mortem human heart tissues using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Profiling AGO2-associated RNAs in cardiac tissues obtained from 6 donors with heart diseases of differing etiologies
Project description:Compared gene enrichment in Ago2-RNA immunopreciptant between wildtype and Mir140-null chondrocytes Identified Mir140 targets that are most exclusively regulated by Mir140 Biological duplicate. Total RNA and Ago2-immunoprecipitated RNA were prepared from the same primary chondrocyte culture. Chondrocytes were isolated from newborn mouse rib and cultured overnight in 10%FCS-containing medium. Total RNA and Ago2-immunoprecipitated RNA were amplified using Ovation pico, and subjected to microarray analysis (HT-mouse 430 PM). Two sets of experiments (two samples from wildtype mice for total RNA and Ago2 immunoprecipitated RNA, and two samples from Mir140-null mice for total RNA and Ago2 immunoprecipitated RNA) were performed (a total of 8 chips)
Project description:We show that target mRNAs trigger the non-templated addition of nucleotides (mainly adenosines and uridines) and shortening of their cognate miRNAs in primary hippocampal neurons. The induced effect is observable both in total RNA and in Ago2-associated RNA, demonstrating that the process is initiated in Ago2 bound miRNAs. Illumina Small RNA sequencing was performed on rat hippocampal neurons infected with different target mRNAs in different conditions. The data includes 3 independent experiments: 1. Hippocampal neurons (DIV15) 6 days after transducing them with target mRNAs (driven by the Synapsin promoter) against different miRNAs. Samples are titled "Syn-Target". Samples transduced with targets containing extensive complementarity are labeled "Ext" and those with Seed complementarity are labeled "Seed". 2. Hippocampal neurons (DIV16) 7 days after transducing them with an inducible target mRNA against miR-132, and inducing for different times with Doxycycline. Samples are titled "Time-course" 3. Hippocampal neurons (DIV16) 7 days after co-transducing them with FLAG/HA-Ago2 plus an inducible target mRNA against miR-132, and inducing them for different times with Doxycycline. Samples are titled "Time-course_Ago2"
Project description:Transgenic Arabidopsis plants (AGO2::HA:AGO2) were treated with either mock (10 mM MgCl2) or Pseudomonas syringae pv. tomato (Pst) expressing avrRpt2 (R2) at a concentration of 2 x 107 cfu/ml for 14 hours. sRNAs associated with AGO1 and AGO2 were co-immunoprecipitated using antibodies against either AGO1 (AGO1-IP), or HA (hemagglutinin) (AGO2-IP). As controls, we also gel-purified the 18-28 nt fraction of the total RNAs from an AGO2 mutant (ago2-1). The co-immunoprecipitated or gel-purified RNAs were cloned and sequenced by Illumina deep sequencing. Examination of AGO-associated sRNAs in pathogen-treated or control plants