Mapping microRNA-target interactions in rare cell types with Spy3-AGO2 Pull-down
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ABSTRACT:
PROVIDER: PRJNA1017897 | ENA |
REPOSITORIES: ENA
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Project description:To illuminate the molecular mechanisms driving neuronal differentiation we generated a mouse line amenable to mapping miRNA-target interactions in rare cell types. Biochemical approaches to purify AGO2-miRNA-target complexes have successfully mapped MTIs in abundant populations of neurons. However, due to their technical complexity and high background, these approaches are not suitable for mapping interactions in rare cell populations such the many neuronal subtypes that compose the mammalian brain. We therefore generated a mouse line with a conditional SpyTag3, which is small and offers near-infinite affinity for pull-downs, in the endogenous Ago2 gene. We then developed a method Spy3-AGO2 pull-down and sequencing (SAPseq), which we have used to accurately map miRNA-target interactions in developing Purkinje cells, a rare population of cells in the cerebellum.
2025-04-02 | GSE243368 | GEO
Project description:To illuminate the molecular mechanisms driving neuronal differentiation we generated a mouse line amenable to mapping miRNA-target interactions in rare cell types. Biochemical approaches to purify AGO2-miRNA-target complexes have successfully mapped MTIs in abundant populations of neurons. However, due to their technical complexity and high background, these approaches are not suitable for mapping interactions in rare cell populations such the many neuronal subtypes that compose the mammalian brain. We therefore generated a mouse line with a conditional SpyTag3, which is small and offers near-infinite affinity for pull-downs, in the endogenous Ago2 gene. We then developed a method Spy3-AGO2 pull-down and sequencing (SAPseq), which we first benchmarked for in vivo use using cortical pyramidal neurons, an abundant population.
2025-04-02 | GSE289640 | GEO
Project description:Mapping microRNA-target interactions in abundant cell types with Spy3-AGO2 Pull-down [SAP-seq]
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Project description:Mapping microRNA-target interactions in abundant cell types with Spy3-AGO2 Pull-down [CLIP-seq]
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Project description:Mapping microRNA-target interactions in cell lines with Spy3-AGO2 Pull-down [mESC SAP-seq]
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Project description:Studies have demonstrated that T6B can selectively de-repress miRNA targets in non-neuronal cells. However, since neurons may have a different expression profile of miRNA pathway genes as well as other binding partners of TNRC6, we sought to validate the intended function of in neurons. The first step was to identify bona fide miRNA targets in PNs, we performed AGO2 crosslinking and immunoprecipitation followed by sequencing (CLIPseq).
2025-04-02 | GSE289638 | GEO
Project description:Mapping of microRNA (miRNA) targets using AGO2 CLIPseq and HEAPseq has provided major insight into the function of miRNAs in various systems. HEAPseq is a powerful method because it allows for cell type-specific mapping of miRNA targets and relies on a mouse line harboring a conditional HaloTag in the endogeous Ago2 locus. However, the homozygous mice are not viable, suggesting that the insertion of the large (>1 kb) HaloTag conditional cassette in the Ago2 gene promoter region, or appending the HaloTag domain to the AGO2 protein, is deleterious to some aspects of AGO2 function. To overcome this limitation, we created tagged AGO2 mouse and mESC lines designed for minimal perturbation of miRISC function. We used a cleavable SpyTag3, which rapidly forms a covalent bond with its ligand SpyCatcher3 and is ten times smaller than HaloTag (3 vs 33 kDa)36. To minimize any perturbation on AGO2 expression and protein structure, constitutive Spy3-AGO2 mice and mESC lines were created by inserting the SpyTag3 coding sequence into exon 2 of the Ago2 gene, which is separated from the promoter region by a 38 kb intron and encodes a small unstructured region of AGO2 – the only part of the protein sequence that is not perfectly conserved between mice and humans. We then developed a method SAPseq, based on HEAPseq and benchmarked it first using mESCs.
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Project description:RNA pull-down assay.<br>For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified.<br><br>RNA purification and amplification:<br><br>RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual.<br><br>Microarray analysis:<br>The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. <br>Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.
2012-06-30 | E-MEXP-3057 | biostudies-arrayexpress
Project description:The aim of the study is to identify miRNA targets in Hodgkin lymphoma cell lines. By immunoprecipitation of wild type Ago2, it is expected to pull down the Ago2 associated gene transcripts. Through microarray analysis, the Ago2 associated gene transcripts identified are expected to be miRNA targets. Keywords: Ribonucleoprotein Immunoprecipitation - Gene Chip (RIP-Chip)
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Project description:RNA isolated from MLE12 cells was subjected to a pull down assay with biotinilated miR-154 to enrich for miR-154 target mRNAs
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