AGO2 bound short RNAs in Jurkat cell line
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR375872.fastq.gz | Fastqsanger.gz | |||
| ERR375873.fastq.gz | Fastqsanger.gz | |||
| ERR375874.fastq.gz | Fastqsanger.gz |
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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.
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Project description:Design of antiviral AGO2-dependent short hairpin RNAs
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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.
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Project description:Ago1-bound short RNAs in HeLaS3 cell line
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Project description:Characterization of AGO1 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 AGO1 monoclonal antibodies or Isotype matched IgG. Following immunoprecipitation, RNA was extracted from AGO1 or control IgG IP for short RNA sequencing. Two independent biological replicates were carried out.
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Project description:The increasing emergence and re-emergence of RNA virus outbreaks underlines the urgent need to develop effective antivirals. RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by small interference RNAs (siRNAs) or short hairpin RNAs (shRNAs), which exhibit significant promise for antiviral therapy. AGO2-dependent shRNA (agshRNA) generates a single-stranded guide RNA effector and presents significant advantages over traditional siRNA and shRNA. In this study, we applied a logistic regression algorithm to a previously published chemically siRNA efficacy dataset and built a machine learning-based algorithm with high predictive power. Using this algorithm, we designed siRNA sequences targeting diverse RNA viruses, including human enterovirus A71 (EV71), Zika virus (ZIKV), dengue virus 2 (DENV2), mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and developed them into agshRNAs according to the rule of agshRNA design. These agshRNAs inhibited viral replication in infected cells and their efficiencies of antiviral effects displayed a consistent trend with the score ranking of siRNA sequences predicted by the algorithm. Using the agshRNA targeting EV71 as an example, we showed that the anti-EV71 effect of agshRNA was more potent compared with the corresponding siRNA and shRNA. Moreover, the antiviral effect of agshRNA is dependent on AGO2-processed guide RNA, which can load into the RISC. We also confirmed the antiviral effect of agshRNA in vivo. Together, this work develop a novel approach that combines machine learning-based algorithm and agshRNA design to custom design antiviral agshRNAs with high efficiency.
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Project description:In order to identify miR-21 targets by a biochemical high-throughput method, we immunopurified RISC Complex and associated mRNAs in both control and miR-21 overexpressing Jurkat cells. Following transduction of miR-21 encoding construct (or a cognate control vector) in Jurkat cells, we used microarray technology to profile Total RNA, AGO2 Immunopurified RNA and control IgG purified RNA.
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