Project description:Characterization of AGO2 bound short RNAs from nuclear extracts of Jurkat cell line. Nuclei were isolated from cells; a fraction of Nuclear lysates was harvested and sequenced as the input samples. The rest of nuclear lysates were Immunoprecipitated using AGO2 monoclonal antibodies or Isotype matched IgG. Following immunoprecipitation, RNA was extracted from AGO2 or control IgG IP for short RNA sequencing. Two independent biological replicates were carried out.
Project description:Characterization of AGO2 bound short RNAs from nuclear extracts of HeLaS3 cell line. Nuclei were isolated from HeLaS3 cells; a fraction of Nuclear lysates was harvsted and sequenced as the 'input' samples. The rest of nuclear lysates were Immunoprecipitated using AGO2 monoclonal antibodies or Isotype matched IgG. Following immunoprecipitation, RNA was extracted from AGO2 or control IgG IP for short RNA sequencing. Two independent biological replicates were carried out.
Project description:DICER_Ex5 cells were created by disrupting exon 5 of the human Dicer gene using an AAV targeting construct, thereby interrupting a well conserved segment of the N-terminal helicase domain while sparing the RNase III domains. In DICER_Ex5 cells, Dicer is expressed at a level lower than the wild type. This experiment started with RIP-anti-Ago2 pull-down, followed by high throughput sequencing analysis in wild type and Dicer-hypomorphic HCT116 cell lines.
Project description:Among the four Argonaute family members in mammals, only AGO2 protein retains endonuclease activity, and facilitates cleavage of target RNAs base-pairing with highly complementary guide RNAs. Despite the deeply conserved catalytic activity, only a small number of targets have been reported to extensively base pair with cognate miRNAs to be cleaved by AGO2. Here, we analyzed AGO2-bound RNAs by CrossLinking ImmunoPrecipitation (CLIP) of genetically modified cells that express epitope-tagged AGO2 from the native genomic locus. We found that HMGA2 mRNA is cleaved by AGO2 loaded with let-7 and miR-21. In contrast to the generally accepted notion, the base-pairing from the seed region to the cleavage site, rather than perfect or near perfect complementarity, was required for cleavage of the target mRNA in cells. Non-templated addition of nucleotides at the 3’ end of the cleaved RNA was observed, further supporting the AGO2-mediated cleavage. Based on the observation that the limited complementarity is the minimum requirement for cleavage, we found that AGO2-mediated cleavage of targets is more common than previously thought. Our result may explain the vital role of endonuclease activity in controlling miRNA-mediated gene regulation. Overall design: CLIP-seq of FLAG-AGO2 by using an anti-FLAG-antibody (Wako) and IgG controls.
Project description:Our genome wide analyses of microRNA expression profiles involve the hybridization of fluorescently labeled RNA samples to custom made, DNA microarrays based on the GAPSII coated slides. We describe the differences in genome wide miRNA expressions between wild type 293 T mammalian cells and Ago2 transformed stable cell lines. Keywords: Expression Comparing one control and three Ago2-overexpressing stable cell lines (A, B, C). One replicate per array.