Mapping microRNA-target interactions in cell lines with Spy3-AGO2 Pull-down [mESC SAP-seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE289643 | GEO | 2025/04/02

REPOSITORIES: GEO